JP6004290B2 - Transgenic chickens with inactivated endogenous loci - Google Patents

Description

  Transgenic animals offer the potential for significant progress in the continued production of beneficial pharmaceuticals such as antibodies. However, the production of transgenic animals has many technical obstacles, only a few of which have been overcome. The ability to incorporate a genetic modification encoding an exogenous protein into another species of DNA requires several different techniques that must be developed for each species. One approach to altering an animal's genetic and physical characteristics is to introduce cells into the animal's recipient embryo. These cells have the ability to contribute to the tissues of the animals born from the recipient embryo and contribute to the genome of the transgenic offspring of the resulting animals.

  In some cases, cells can be engineered with a transgene that contains DNA encoding an exogenous product such as a protein or antibody. The transgene includes a blueprint for protein production and is sufficient to contain coding and regulatory elements capable of protein expression in animal tissue made from the insertion of cells into the recipient embryo. In some situations, it is desirable that expression be ubiquitous to occur in all tissue types. However, in many situations where beneficial proteins are desired, such as recovery of beneficial antibodies, expression must be restricted to certain specific tissue types that facilitate recovery of the expressed protein. For example, in cows, protein expression in milk allows the protein to be easily recovered simply by collecting milk and separating exogenous proteins. In chickens, mass production of antibodies in egg white also provides an attractive vehicle for the expression and recovery of beneficial proteins. Furthermore, if tissue-specific expression is specific to the chick oviduct, the expression has certain specific desirable chemical properties that increase the therapeutic utility of the antibody when used in the treatment of human patients. An antibody is obtained. Thus, one particularly attractive area of research and commercial development is to selectively express antibodies in either egg white or egg yolk to facilitate isolation and recovery of proteins with desirable chemical properties. A genetically engineered chicken.

  For the production of exogenous antibodies, avian biological systems offer many advantages, including efficient farming, rapid growth, and economic production. Furthermore, avian eggs impart an ideal biological design for both large-scale antibody synthesis and ease of product isolation and recovery. Furthermore, as described below, in the context of the present invention, for example, the advantages of a transgenic chicken expression system compared to vertebrate, plant, or bacterial cell lines are readily demonstrated and inherent advantages for large amounts of antibody products. It can be applied to bring about chemical properties. The purpose of producing transgenic chickens has been pursued by scientists for many years. This goal has been reached in other species (such as mice, cattle, and pigs), but no transgenic chickens have been created other than through the use of retroviral or direct injection technologies, It suffers from inherent limitations in the size and / or lack of expression of transgenes that can be introduced into the DNA of transgenic animals. Moreover, viral vectors do not follow applications that require site-specific changes to the genome, such as those provided by homologous recombination.

  Furthermore, in some situations, the animal's own endogenous gene can interfere with the production of beneficial proteins that result from introducing genetic constructs specifically designed to express such proteins. Under these circumstances, the ideal solution is to inactivate the animal's endogenous genes. Unfortunately, due to the unique challenges in genetic engineering in chickens, transgenic chickens with site-specific modifications that result in inactivation of endogenous loci have not been described. Still further, the introduction of site-specific gene inactivation produces animals that lack endogenous gene function, and such animals differ with complementary specific genetic modifications introduced into their genome. Can be bred with animals. For example, animal families lacking specific genes could be established, and breeding could be combined with animals containing specific genes for humans. In this case, the inactivation of the endogenous gene as well as the genomic modification introduced for the production of a specific animal expression type or for the production of the protein encoded by the insertion of the endogenous gene. An animal group with both will be created. Such animals do not currently exist, because viral vectors do not allow the ability to select for endogenous site-specific targeting or integration events. Thus, viral vectors do not provide a mechanism by which activation of the endogenous locus can be achieved.

  When cell cultures are sufficiently stable to allow large transgenes to be integrated into the cell's genome or to introduce site-specific changes into the genome, tissue-specific expression of any protein can be achieved. The encoding transgene can be passaged into the transgenic organism by several different techniques depending on the target cell and the specific construct used as the transgene. The same technique can be used to perpetuate organisms that have an inactivated endogenous gene. The whole genome can be transferred by cell hybridization, intact chromosomes can be transferred by micronuclear cells, subchromosome segments can be transferred by chromosome-mediated gene transfer, and kilo chromosomes can be transferred by DNA-mediated gene transfer. A DNA fragment in the base range can be transferred (Non-patent Document 1). Intact chromosomes can be transferred by micronucleus cell-mediated chromosome transfer (MMCT) (MMCT) (Non-Patent Document 2). Specific design or gene inactivation of any such transgene carrying an exogenous gene also takes into account the content of the exogenous gene, the nature of any gene inactivation, and the phenotypic characteristics obtained in the animal Must.

  Unless the transgene is carefully designed, insertion of a transgene that inactivates the endogenous locus or allows tissue-specific expression can threaten cellular pluripotency. Thus, a suitable cell line can be used to inactivate a cell with a gene construct that is large and complex enough to inactivate the gene or, if necessary, include all elements necessary for tissue-specific and high levels of expression. When transfected, it must be stable during culture and maintain pluripotency. In the resulting transgenic animals, the transgene can optionally be expressed in specific individual tissue types that are designed to express the transgene. Depending on the gene content of the transgene, the transgene cannot be expressed in other tissues if the survival rate of the animal or the advantageous chemistry of the resulting protein is compromised.

  Chicken primordial germ cells have been genetically modified using retroviral vectors within several hours after isolation from stage 11-15 embryos (Non-patent Document 3). However, the resulting modifications are randomly incorporated and the size of the transgene is generally limited to less than about 15 kb, usually less than 10 kb, and most commonly less than 8 kb, allowing site-specific changes in the genome to Cannot be made, and transferred cells cannot be selected to identify site-specific modifications to the exclusion of random integration. No stable genetic modification has been reported previously that requires the insertion of exogenous DNA greater than 15 kb into the cultured avian PGC genome.

  Any limitation on the size or site specificity of any DNA transgene or construct that can be stably introduced into long-term PGC cell cultures is important in the ability to achieve beneficial genetic modification of the PGC genome in the culture Then restrict the types of genetic modifications that can be passaged through the germ line to the recipient embryo's progeny. For example, introduction of exogenous DNA encoding a protein into the genome of an inactivated vector or transgenic chicken is a highly desirable genetic modification. If such transgenic chicken populations can be generated, a large amount of beneficial protein can be expressed in chickens and recovered in eggs. Bird eggs provide an ideal reservoir for bioactive proteins and provide a convenient environment in which proteins can be isolated. Avian animals are also attractive candidates for a wide variety of transgenic technologies. However, the application of the full range of mammalian transgenic techniques to avian species has not been successful due to the lack of a population of cultured cells that can introduce genetic modifications and transmit them to the germline. In a recent paper, Sang et al., “Can maintain PGCs in culture and grow for as long as necessary to identify gene targeting events without losing the ability to migrate to the developing gonad after transfer. The possibility is low ”(Non-Patent Document 4). Thus, to date, genetically transfected PGCs have not been made and the transmission of genetic modifications introduced into avian PGCs to mature living animals has not been demonstrated.

  Primordial germ cells (PGCs) are sperm and egg precursors that are isolated from somatic tissues at an early stage of development in most animals. In accordance with the present invention, chicken PGCs are isolated, cultured and genetically modified while maintaining germline involvement. Furthermore, PGCs are induced to differentiate into embryonic germ cells (EGC) and are similar to chicken embryonic stem cells (ESC) in terms of their involvement in somatic tissues. These PGCs are involved in somatic tissues and germline and provide a unique source for genomic genetic modification in chickens.

  Production of transgenic animals, particularly mice, is important for elucidation of mammalian gene functions. Traditional approaches are the random integration of the transgene into the genome or the targeted insertion of the transgene into a specific locus by homologous recombination.

  There are two disadvantages to random insertion of the transgene. The first major disadvantage is that many genes perform essential functions at various stages of development, and the elimination of transcription of these genes often causes embryo death. Embryonic death can be prevented using, for example, specific site recombination such as Cre-loxP or FIp-FRT under the control of a promoter that provides tissue-specific and developmental stage-regulated gene expression. In these cases, site-specific recombination is usually used to inactivate genes at separate cells and / or at different times during development in the context of normal animals (referred to as conditional genetic modification). used. For example, the Cre-loxP system is used to specifically inactivate the insulin receptor gene in β-cells, creating an insulin secretion deficiency similar to that in type 2 diabetes (Non-Patent Document 5). . Initially, the null allele of DNA polymerase β was shown to be embryonic lethal when homozygous. In order to analyze possible requirements for antigen receptor gene placement, a conditional knockout technique was used to create a deficiency of DNA polymerase β in T-cells (6).

  Both random and targeted insertion of the transgene is plagued by the inability to excise a positive selection cassette from the transgene in the transgenic animal. The presence of the selection cassette causes several problems, such as disruption of gene expression at nearby loci due to strong transcriptional control elements often present in the selection cassette (Non-Patent Document 7; Non-Patent Document 8). Removal of the positive selection cassette can be achieved using site-specific recombinant enzymes under the control of a tissue-specific promoter.

  Cre is a recombination enzyme that catalyzes recombination between two loxP sites, a 34 base pair DNA element. When the two loxP sites are integrated into the genome in the same direction, recombination catalyzed by Cre excises the intervening DNA. The loxP site can be incorporated into the transgene before being randomly inserted into the genome, or the loxP site can be inserted into the genome at the correct location using a targeting vector. After excision of the intervening DNA, adjacent loxP sites are converted to a single loxP site. The loxP site of the mutant produces a product that is not fully recognized after Cre excision. Another member of the lambda integrase superfamily, FIp recombinase, a site-specific recombination enzyme, shares the same mechanism of DNA recombination as Cre recombinase. Like Cre, FIp recombinase recombines DNA at two defined 34 base pair target sites (FRT sites). After excision of the intervening DNA, the adjacent FRT site is also converted to a single FRT site.

  One application of conditional knockout is the expression of lethal products in cells that are to be removed in specific tissues at the exact stage of development. For example, Non-Patent Document 9 used the Cre-loxP system to express a diphtheria toxin A fragment specifically in muscle cells to study skeletal muscle development in mice. The ligand-regulated form of Cre was developed with the goal of adding a transient control of the Cre-loxP system that allows precise induction of genetic changes in late embryogenesis and / or adult tissues in vitro or in vivo. .

  Chromosome rearrangement is a major cause of inherited disease and fetal death and has been implicated in cancer progression and maintenance (Non-Patent Document 10; Non-Patent Document 11). Chromosomal translocations often result in abnormal gene fusions, so that tumor-specific mRNAs and proteins are attractive targets for gene therapy. Thus, the ability to manipulate chromosomal rearrangements at specific cleavage sites using site-specific recombinant enzymes has been used to create mouse models of human disease. For example, translocations corresponding to human rearrangements t (8:21) (q22; q22) and t (9:11) (p22q23) were successfully induced in mice to model acute leukemia ( Non-patent document 12; Non-patent document 13). Random chromosomal deletions can occur by inserting loxP sites at random locations in the genome and then expressing Cre recombinase (Non-Patent Document 14).

  Conditional genetic modification has been a powerful tool for manipulating gene expression during cell line specifications, and analysis of cell fate has contributed to an understanding of normal development. The Cre-loxP system has been used to genetically activate murine lineage tracers, for example, to define adult fate of Engrailed 2-expressing cells that occur in midbrain-hindbrain contractions (non- Patent Document 15). This approach included two mouse strains that were mated. One Cre recombinase mouse expresses Cre under the control of the Engrailed 2- (En-2) genomic regulatory fragment towards expression in the fetal midbrain-hindbrain contraction region, and the indicator / reporter mouse Provides a permanent record of this event by having a transgene “indicating” that recombination has occurred and turning it into an inheritable lineage marker. The indicator strain has a transcription / translation-loxP-stop cassette loxP-stop driven by regulatory sequences from the widely expressed chick β-actin gene. By crossing En2-Cre mice and indicator mice, the double recombinants carry a copy of each transgene. Only cells that expressed Cre under the En2 regulatory element were reconstructed between the reporter constructs loxP and then excised the arrest to allow lacZ expression. Because Cre-mediated excision is cytogenetic, Mark cells and all their progeny expressed lacZ at a later stage, even after Cre is no longer expressed. Thus, staining for LacZ in the brains of mature double transgenic animals revealed the progeny of all cells that transiently expressed Cre in the course of midbrain-hindbrain contraction development.

Klobutcher, L.M. A. and F. H. Ruddle, Annu. Rev. Biochem. , 50: 533-554, 1981. Fournier, R .; E. and F. H. Ruddle, Proc. Natl. Acad. Sci. U. S. A. 74: 319-323, 1977. Vick et al. (1993) Proc. R. Soc. London. B 251, 179-182 Prospects for Transgenesis in the Chick, Mechanisms of Development, 121, 1179-1186, (2004) Kulkarni et al., 1999 Tissue-specific knockout of the insulin receptor in pancreatic beta cells creates an insulative secre- ate defect asymmetric to thirty. Cell 96: 329-39 Gu et al., 1994. Selection of a DNA polymerase beta gene segment in T cells using cell type-specific gene targeting. Science 265: 103-106 Lerner et al., 1993 CD3 zeta / eta / theta locus is colloidal with and transferred antisense to the gene encoding the transcription factor Oct-1. J Immunol. 151: 3152-62 Ohno et al., 1994 Targeted description of the CD3 et al locus causes high maturity in mice: modulation of Oct-1 transcription on the opacity. EMBO J.M. 13: 1157-65 Grieshammer et al., 1998 Muscular-specific cell ablative conditional up-regenerated DNA recombination in transgenic mice to mass spinal and cranone. Dev Biol. 197: 234-47 Ramirez-Solis et al., 1995 Chrome engineering engineering in mice. Nature 378: 720-4 Rabbitte et al., 2001 Mouse models of human chromosomal translocations and procedures to cancer therapy. Blood Cells Mol Dis. 27: 249-59 Buchholz et al., 2000 Alteration of Cre recombinase site specificity by substrate-linked protein evolution. Nat Biotechnol. 19: 1047-52 Collins et al., 2000 Inter-chromosomal recombination of M11 and Af9 genes moderated by cre-loxP in mouse development. EMBO Rep. 1: 127-32 Zhu et al., 2007. Efficient generation of random chromosome deletions, Biotechniques 42, 572-575 Zinyk et al., 1998 Fate mapping of the mouse midbrain-hindbrain constriction using a site-specific recombination system. Curr Biol. 8: 665-8

SUMMARY OF THE INVENTION The present invention creates a technology that allows genetic engineering of transgenic chickens and birds, as well as transgenic chickens with inactivated endogenous loci resulting from homologous integration of targeting constructs into primordial germ cells. A long-term culture of PGC used to These transgenic chickens have a transgene integrated into the chicken primordial germ cell genome by homologous recombination resulting in gene inactivation by deletion of at least a portion of the endogenous locus. The present invention includes transgene constructs, which are stable cultures of primordial germ cells carrying the transgene, often referred to as knockout vectors, targeting vectors, knockout constructs, etc., where endogenous gene inactivation Transgenes designed for stable integration into the primordial germ cell genome maintained in culture for a sufficient amount of time to accomplish the recombination event and to select for transfected cells It is.

  The invention also includes primordial germ cells and the resulting transgenic chicken, whose genome has been modified by inactivating the endogenous locus, including but not limited to one of the genes required for endogenous gene expression. It contains a site-specific deletion of the part. For all of the foregoing embodiments, the present invention also includes a transgenic chicken obtained from site-specific modification of the endogenous genome. The invention also relates to antibodies produced in chickens that have advantageous chemical properties that enhance their therapeutic utility in certain applications. Because antibodies produced in chickens have a different pattern of chemical modification compared to antibodies produced in vertebrate, plant, or bacterial cell lines, the aim is to bind toxins to target tissues, such as tumors As the target tissue is treated with increased therapeutic effectiveness. In one embodiment, a long-term culture of PCG is engineered with a specially designed genetic construct to introduce genetic alterations in the bird, which includes transgene insertions that result in tissue-specific expression of exogenous proteins. . To facilitate the expression of exogenous proteins, either by manipulation of inactivated loci in the same pluripotent cell, or by manipulation of separate groups of transgenic chickens with inactivated endogenous loci A transgenic chicken carrying a combination of exogenous DNA encoding protein expression is combined with a transgenic chicken having an inactivated endogenous locus for subsequent breeding in birds with the inserted transgene. , Providing a unique and advantageous group of animals that express exogenous proteins.

  Transgenic chickens with inactivated endogenous loci are also used for gene expression studies and to select unique gene functions that are not possible without the ability to inactivate selected endogenous loci Provide useful animal models. Similarly, inactivation of the endogenous chicken locus can result in specific portions of the endogenous immunoglobulin gene locus (V, D, to prevent immunoglobulin gene remodeling and to inactivate endogenous antibody expression). Or the J region). As a result, one embodiment of the present invention substantially reduces endogenous immunoglobulin gene expression and endogenous immunoglobulin protein production resulting from site-specific genetic modification at selected portions of the endogenous chicken immunoglobulin locus. Includes missing transgenic chickens. In a preferred embodiment, the transgene is constructed for target inactivation of both light and heavy chains encoding endogenous immunoglobulin production. The transgenic birds of the present invention are also capable of expressing transgene-derived antibodies in the oviduct and the antibodies are deposited in large quantities in the egg. In a preferred embodiment, the exogenous antibody protein is endogenous so that the native human antibody is expressed in the chick oviduct in the absence of endogenous avian antibody production, thereby creating the ability to recover only human antibodies from the egg. It is encoded by a human DNA sequence that is expressed in the background lacking antibody production.

  The present invention includes a flock that exhibits tissue-specific expression of antibodies, a transgene construct that allows for exogenous antibody expression, an isolated composition of antibodies produced in chickens and having specially defined chemical properties, As well as related methods for the production of birds, the production of antibodies, and their therapeutic use in humans. The present invention uses a long-term progenitor cell culture and special techniques to produce a chimeric or transgenic bird derived from a long-term PGC cell culture, where the progeny of the cultured cells are stably integrated To include the gene, the genome of the PGC has a stably integrated transgene that expresses the exogenous protein. When introduced into a host bird embryo, the modified donor cells produce birds that express the transgene in specific selected somatic tissues of the resulting animal by the procedure described below.

  The present invention also includes compositions of exogenous proteins that are expressed in transgenic chickens and have certain desirable chemical properties compared to vertebrate, plant or bacterial cell lines. Specifically, these proteins, particularly antibodies, have low concentrations of fucose, galactose, N-acetylneuraminic acid, N-glycolylneuraminic acid and high concentrations of mannose. Antibodies that have some or all of these properties exhibit increased therapeutic utility when administered to humans. Specifically, these antibody compositions exhibit enhanced antibody dependent cytotoxic activity (ADCC). Accordingly, the methods of the present invention include enhancing the therapeutic utility of an antibody composition based on the ADCC effect by using a transgenic chicken to express the antibody in the transgenic chicken.

  The present invention also provides a transgenic chicken that expresses an exogenous antibody having the advantageous chemistry defined herein in an oviduct tissue such that the exogenous antibody is enriched in egg white in a defined amount. Including. In one preferred embodiment, the exogenous protein is a human sequence monoclonal antibody encoded by a transgene construct incorporated into the genome of the transgenic bird. The polynucleotide sequence encoding the human monoclonal antibody is specifically constructed for expression in the oviduct and within a transgene containing appropriate promoter and regulatory sequences to facilitate tissue-specific expression. include.

  The present invention also relates to long-term cultures of avian primordial germ cells (PGCs), and some further inventions relate to the growth of avian PGCs and the viability of the cultures for 40 days, 60 days, 80 days, It is made possible by the production of long-term cultures that can be extended by multiple passages to extend for 100 days or more. The PGCs of the present invention grow in long-term cultures and produce germline chimeras when injected into recipient embryos.

  The present invention also relates to the introduction of genetic material into the PGC genome to obtain desirable results. In one embodiment, the gene construct surrounded by the HS4 element is incorporated into the PGC of the invention to ensure production of the transgene product. In another embodiment, genetic recombination is performed using integrase for direct insertion of the construct into a repetitive element of the chicken genome. In another embodiment, DNA encoding a selectable marker is inserted into the chicken genomic region to prevent gene product production.

Conditional mutations occurred in chicken cells, but unlike mice from which transgenic lines expressing Cre were generated, under the control of promoters that are ubiquitous, tissue specific, or developmentally regulated. No transgenic chicken lines expressing Cre recombinase were generated. In mouse cells, transient expression of Cre recombinase (Arakir et al., 1997 Efficiency of recombination by Cretransient expression in embryonic stem cells: comparison of reversible cells 97 (Jo et al., 2001 Epigenetic regulation of gene structure and function with a cell-permable Cre recombinase. Nat Biotechnol. 19: 929-33) was excised between loxP sites. In the chicken DT40 cell line, however, transient expression of the Cre recombinase failed to remove DNA between the loxP sites (Fukagawa et al., 1999 The chicken HPRT gene: a counter selectable marker for the DT40 cell). line.Nucleic Acids Research 27, 1966-1969). The Cre transgene was then incorporated into the genome of DT40 cells to achieve excision of DNA sequences between loxP and / or mutant loxP sites (Fukagawa et al., 1999 The chicken HPRT gene: a counter selectable marker for the DT40 cell line.Nucleic Acids Research
27,1966-1969; Arakawa, et al., 2001 Mutant loxP vectors for selectable marker recycle and conditional knockouts BMC biotechnology 1,7-14; Dhar et al., 2001 DNA repair studies: experimental evidence in support of chicken DT40 cell line as a unique model. J. et al. Environ Pathol Toxicol Oncol 20, 273-83; Kanayama et al., 2005, Reversible switching of immunohybridization machinery in a chicken cell. Biochem. Biophys. Res. Commun. 327, 70-75).

  The ability to cause conditional mutations in chickens is advantageous. For example, chickens substantially derived from ES cells can be generated using an apoptosis-inducing gene under the control of a ubiquitous promoter that is silent due to the presence of a stop codon adjacent to the loxP site. When this strain of chicken is crossed with a bird strain carrying a gene encoding Cre recombinase under the control of a promoter expressed in the light region, the embryo does not develop. If embryonic stem cells are injected into an embryo simultaneously with the expression of an apoptosis-inducing gene, the embryo can be derived substantially from embryonic stem cells.

In another application, a transgene that includes a sequence encoding a selectable marker can be flanked by loxP sites. Transgenic birds carrying these transgenes can be bred with birds that express Cre recombinase under the control of a germline-expressed promoter. Birds created from this mating will hatch after excision of the selectable marker.
In a preferred embodiment of the present invention, for example, the following is provided:
(Item 1)
A transgenic chicken comprising a deletion of a selected portion of an endogenous locus.
(Item 2)
The transgenic chicken according to item 1, wherein the disrupted endogenous gene is an immunoglobulin locus.
(Item 3)
Item 3. The transgenic chicken according to item 2, wherein the disrupted endogenous gene is an immunoglobulin heavy chain locus.
(Item 4)
Item 3. The transgenic chicken according to item 2, wherein the disrupted endogenous gene is an immunoglobulin light chain locus.
(Item 5)
The transgenic chicken according to item 2, wherein the disrupted endogenous gene comprises detection of a gene segment selected from the group consisting of a V-gene segment, a J-gene segment, and a C-gene segment, and combinations thereof. .
(Item 6)
The transgenic chicken of item 1, wherein the disrupted endogenous locus comprises a targeting construct.
(Item 7)
The transgenic chicken according to item 1, wherein the targeting construct inserts a stop codon, an att-p site, a nonsense sequence or a combination thereof into the endogenous locus.
(Item 8)
The transgenic chicken according to item 6, wherein the targeting construct is composed of two homologous regions for an endogenous locus and a selectable marker located between the homologous regions.
(Item 9)
3. The transgenic chicken of item 2, comprising functional disruption of both the heavy chain endogenous immunoglobulin heavy chain gene and the endogenous immunoglobulin light chain gene.
(Item 10)
The transgenic chicken according to item 1, wherein the deleted portion of the endogenous locus comprises at least 10 kb.
(Item 11)
A method for producing a transgenic chicken, wherein the targeting construct is incorporated into a chicken primordial germ cell such that the targeting construct destroys at least a portion of the endogenous gene by stable integration into the genome of the cell, and the primordial germ cell is incorporated into the chicken. A method comprising inserting into an embryo and hatching a transgenic chicken from the embryo, wherein the chicken comprises a functional disruption of an endogenous gene.
(Item 12)
Item 12. The method according to Item 11, wherein the endogenous gene is an immunoglobulin gene.
(Item 13)
Item 13. The method according to Item 12, wherein the immunoglobulin gene is a light chain gene.
(Item 14)
Item 13. The method according to Item 12, wherein the immunoglobulin gene is a heavy chain gene.
(Item 15)
13. The method of item 12, wherein the targeting construct deletes a segment selected from the group consisting of a V-gene segment, a J-gene segment, and a C-gene segment, and combinations thereof.
(Item 16)
12. The method of item 11, wherein the targeting construct inserts a stop codon, nonsense sequence, att-p site or a combination thereof.
(Item 17)
Item 12. The method according to Item 11, wherein the targeting construct is composed of at least two homologous regions with respect to an endogenous gene.
(Item 18)
18. The method of item 17, wherein the integration of the targeting construct into the cell genome places a selectable marker located between two homologous regions between the targeting construct and the endogenous gene.
(Item 19)
12. The method of item 11, wherein the targeting construct is composed of a positive selection marker.
(Item 20)
12. A method according to item 11, wherein the targeting construct is composed of a negative selection marker.

FIG. 1A shows PGCs maintained in culture for 54 days. It should be noted that the cells are not attached and remain round. Arrows indicate some dividing cells visible in this culture. In FIG. 1B, long-term PGC cell cultures are shown to be stable when maintained in culture for at least 136 days. These cells are cultured on the feeder layer of irradiated STO cells. Gene expression measured by RT-PCR of germ cell markers CVH (Vasa) and Dazl. Cells were placed in culture for 32 days. Lane 1 shows the expression of both CVH and Dazl in PGC aliquots. The second sample in lane 2 was measured in the absence of actin and did not contain sufficient mRNA. CES cells were also analyzed; actin was expressed, but cES cells did not express CVH, only Dazl was weakly expressed. Western analysis of number 13 and number 16 sample PGC cultures maintained in culture for 166 days. Testis was used as a positive control and liver was used as a negative control. Rabbit anti-chicken CVH IgG was used as the primary antibody. Telomere repeat amplification protocol (TRAP) assay. Cell extracts of two different PGC cell lines (# 13 and # 16) at different dilutions were maintained in culture for 146 days. The positive control consisted of a transformed human kidney cell line 293, and the negative control was only lysis buffer with no template added. In the PGC and positive control lanes, repetitive sequences are observed, indicating the presence of telomerase. FIG. 5A shows PGC-derived cEG cells maintained in culture. FIG. 5B shows chicken embryonic stem cells. Note small cells, large nuclei (light gray), and distinct nucleoli in both cell types. Southern analysis of the cx-neo transgene in primordial germ cell (PGC) lines. FACS analysis of DT40 cells (negative control population), EG cells, ES cells, and PGCs stained with chicken tube homolog (CVH) and antibodies to 1B3. DT40 cells, ES cells, and EG cells were negative for both markers, but most PGCs stained for both CVH and 1B3. The cell lines used were PGC102; ES439 and EG455. Southern analysis of the HS4-β-actin-neo transgene in two primordial germ cell PGC lines. Southern analysis of HS4 β-actin-eGFP-β-actin-puro transgene in primordial germ cell (PGC) strain TP103. Plasmid control DNA was linearized with NotI. The internal fragment was released by digesting the DNA with KpnI. Both TP103 and the plasmid released the same size fragment. Digestion of TP103 genomic DNA with NcoI, MfeI, and SphI should show a larger band compared to the corresponding lane of digested plasmid DNA. In the lane of MfeI digested TP103 genomic DNA, no bands that could be due to bands that are too large are seen. In the lane representing NcoI and SphI digestion, the fragment was released into TP103 genomic DNA substantially larger than the fragment released into plasmid DNA, indicating that the transgene is incorporated into the genome of the TP103 cell line. Indicates. G-09 karyotype indicating that all chromosomes are diploid. In one copy of GGA2, the majority of the p-arm is lost or translocated to another chromosome. The other copy of CGA2 is normal. The cell is ZZ (male). Section of testis on the 18th day of development stained with DAPI. GFP positive germ cells are clearly seen in the seminiferous tubules. The DAPI staining panel shows a section through the seminiferous tubule of the E18 testis. GFP expressing cells are located in the seminiferous tubule and stained with anti-CVH antibody. Chimera-derived transgenic offspring carrying PGCs stably transfected with the β-actin-GFP transgene grow between stage X (EG & K) and stage 34 (H & H). All of the tissues shown in these photographs show GFP expression. Tissue from a chimera carrying PGCs stably transfected with a β-actin-GFP transgene prepared for histological examination. Blue DAPI staining reveals the presence of nuclei, and green fluorescence demonstrates that all of the tissues express the GFP transgene. Southern blot analysis showing that the clone-derived transfected PGC line contributes to the germline of the chimeric chicken and can differentiate into EG cells. Upper panel: genomic DNA from PGC transfected with HS4 bactin-eGFP-bactin-puro construct, genomic DNA from three embryos from chimeric male chickens made with transfected PGC, and transfected Genomic DNA from the resulting PGC-derived EG cells was digested with restriction enzymes to detect the internal fragment (KpnI) and ligation point fragment (NcoI, AflII) of the transgene insertion. Digested DNA was separated on a 0.7% agarose gel, blotted to a nylon membrane, and probed with a radiolabeled eGFP sequence. The size of the hybridizing fragment was identical in PGC, EC cells, and two embryos that showed green fluorescence (GFP + embryo). A third non-fluorescent embryo (WT embryo) showed no hybridization. Lower panel: shows a schematic of the construct and shows the location of the restriction sites and the expected restriction fragment size is shown below. There are two KpnI sites that yield a 5.3 kb fragment. NcoI and AflII cut once within the construct, and the restriction fragment found thereby is a junction fragment that joins adjacent genomic DNA to the construct at the insertion site. FIG. 16A is a diagram of the random integration construct used in this study. Two basic constructs were used: a selectable marker cassette driven by a strong promoter (drug resistance marker and EGFP), and a similar construct adjacent to two sets of HS4 insulators. The promoter used was mouse PGK, chicken β-actin, β-actin + CMV enhancer (CAG) or ERNI. The construct is as follows: a drug selection marker cassette consisting of a promoter driving the expression of the neo or puro resistance gene (top line); addition of the CAG-EGFP gene (second line); isolated drug Selectable marker only (third line); similar isolated selectable marker cassette with addition of EGFP gene (fourth line); and CAG-EGFP CAG with selectable marker adjacent to loxP site and patented gene of interest -Neo construct (box with star). The construct is linearized prior to transfection, resulting in the vector structure shown. FIG. 16B is a diagram of the integrase construct used in this study. In the figure on the left is the attB containing plasmid, where the attB site has been added to the HS4-β-actin-puro construct described above. In the right figure, the plasmid is used to express the CAG promoter derived cell integrase. Both plasmids were transfected as circular DNA. Sequence comparison of attB and attL sequences obtained from transfected PGCs. Shown is the junction between the attB plasmid and the genomic sequence in the PGC clones derived from integrase-mediated transfection. The top line is a wild type attB site with a core TTG, which is usually the underlined recombination crossover point. Below, the attL sequence from the integrase-mediated insert in PGC is shown. To determine where splicing occurred between the attB of the plasmid and the suspected attP site of the genome, the PGC sequence was compared to attB. In the PGC sequence, the attB sequence provided by the plasmid is lower case and the genomic suspect attP sequence is upper case and bold. FIG. 18A is an alignment of PO41-like repeats from the PGC insertion site with the PO41 consensus sequence. PGC flanking sequences from all of the clones inserted in PO41-like repeats were aligned with each other and with the PO41 consensus. The first 21 nucleotides are the attB sequences conferred by the vector (as shown in the sequence comparison above), followed by the genomic flanking sequences from each clone. Nucleotides shared by at least half of the sequence are enclosed in black. B is an alignment of attP and PO41 sequences. 100 bp of the attP site was aligned with 100 bp of PO41, ie about 2.5 copies of 41 bp. The core crossover TTG during attP is indicated by a superscript horizontal line. Targeting chicken IgL gene. In FIG. 20A, the top line is a diagram of a targeting vector for the chicken IgL gene (IgL pKO5). Designed to replace the 2.3 kb J-C region of the IgL gene with a 3.1 kb HS4-ERNI-puro selectable marker adjacent to the HS4 insulator. The two homology arms are 2.3 kb and 6.3 kb in length. At the 3 'end, the β-actin-EGFP gene allows screening of puro-resistant clones to enrich green fluorescence for the target clone. The dotted line at the end is the pKO vector backbone (Stratagene). The middle line is a diagram of the wild-type allele of the germline composition of the IgL gene with a single variable region gene (V), a junction region gene (J) and a constant region gene (C). The restriction sites used for Southern analysis of the target clone are shown ((S, SacI; B, BstEII), and the wild type fragment size is shown below it with a double arrowhead. The structure of the mutant allele, where the J and C regions have been deleted and replaced with HS4-ERNI-puro, a restriction map is shown, and the size of the mutant fragment is shown below. Both probes used in the Southern analysis are outside the targeting vector and their position is indicated, scale bar = 1 kb, Figure 20B is a Southern blot analysis of 4 clones. Mycin resistant clones were analyzed, two of which (clone 1 and 2) were not green and two of them Clones 3 and 4) were green.In the left panel, genomic DNA from the PGC clone was digested with SacI, hybridized with probe A and analyzed for targeting on the 5 ′ site of the IgL gene. In the right panel, the DNA was digested with BstEII and hybridized with probe B for targeting of the 3 ′ site of the IgL gene Clone 2 contains a fragment of the expected size for the heterozygous target clone. Indicated. ERNI-puro PCR, a marker for inactivation of the immunoglobulin light chain gene in semen from GO roosters generated in PGCs carrying IgL knockout. Ten ng of genomic DNA prepared from semen samples was used in PCR for the ERNI-puro selectable marker present in the IgL knockout allele. As a control, a primer for the endogenous chicken β-actin gene was included. As a positive control for ERNI-puro PCR, PGC with IgL knockout allele was used. ALDH3A2 expression in BN birds. The upper panel is on RNA isolated from two homozygous BN birds (BN / BN), one heterozygous BN bird (BN / +) and one wild type bird (+ / +). And shows RT-PCR for ALDH3A2. In addition, two negative controls are shown for RT-reaction (-RT-control) negative control, positive genome control, and PCR reaction (-control PCR). The 544 and 680 bp bands indicate the presence of aldehyde dehydrogenase mRNA with or without an unspliced intron between exons 5 and 6, respectively. The 597 bp band in the lower panel confirms the presence of RNA in all samples. RT-PCR showed that ADH was expressed in heterozygous BN birds but not in homozygous BN birds, indicating that the transgene insertion stopped transcription of this gene. Sequence of RT-PCR product from wild type allele in aldehyde dehydrogenase 3 family member A2 transcription. Products A and B are derived from transcripts that do not have or have 136 bp of unspliced introns between exons 5 and 6, respectively. A chicken carrying a UbC-loxP-stop-loxP-Reaper transgene. A. Southern blot analysis of three UbC-loxP-stop-loxP-Reaper transgenic lines (6-03, 6-51 and 9-51). Genomic DNA samples from G1 birds and UbC-loxP-stop-loxP-Reaper vectors were digested with SpHI or BclI. The digested DNA was separated on a 0.7% agarose gel, blotted on a nylon membrane, and hybridized with a radiolabeled Reaper specific probe to identify the ligated fragment. The size of the hybridizing fragment is larger for genomic DNA than for the vector, indicating that the transgene has been integrated. B. Schematic of UbC-loxP-stop-loxP-Reaper construct. The transgene is composed of a UbC-promoter and a loxP-stop-loxP-Reaper transgene. The SV40 polyadenylation signal (SV40) and blasticidin resistance cassette (bsd) were inserted 3 'to the UbC-loxP-stop-loxP transgene. The construct was adjacent to the 5 'and 3' LTR. The location of the restriction enzyme site is indicated and the expected restriction size is indicated. A chicken carrying the pLenti-ERNI-Cre transgene. A. Southern blot analysis of 8 ERNI-Cre strains. Genomic DNA samples were digested with BglII. Digested DNA was separated on a 0.7% agarose gel, blotted on a nylon membrane and probed with radiolabeled Cre. The size of the hybridizing fragment was the expected 4.6 kb. B. Schematic of ERNI-Cre transgene. The transgene consists of the ERNI promoter and the Cre transgene. The SV40 polyadenylation signal (SV40) and blasticidin resistance cassette (bsd) were inserted 3 'to the ERNI-Cre transgene. The construct was adjacent to the 5 'and 3' LTR. The location of the BglII restriction enzyme site is indicated and the expected restriction size is indicated. FACS sorting of GFP positive and GFP negative cells from the Doc-2 cell line. Two types of cells were generated by transfecting the Doc-2 cell line with a circular plasmid containing the ERNI-Cre transgene that expresses Cre recombinase in PGC. GFP-negative cells are the result of excision of sequences between LoxP sites on the docking site vector carrying the CX-eGFP gene. Southern analysis of two chicks showing integration of the 10,652 bp transgene into the Doc-1 strain and chicken of PGC. Genomic DNA from two chicks derived from the breeding of G0 chimeras generated with the DOCl PGC strain (P-marked lanes) and DOCl PGC (Cl and C2 marked lanes) was digested with BglII or EcoRI. The digest was fractionated on an agarose gel, transferred to a nylon membrane and hybridized with a radiolabeled EGFP sequence. This analysis detects a ligation fragment containing a docking site vector bound to an adjacent genomic sequence at the vector integration site. The size of these linking fragments varies depending on the integration site and is diagnosed for each transgene insertion event in PGCs. In this case, the BglII fragment is about 12 kb and the EcoRI fragment is larger than 12 kb. The fragment size is identical for PGCs and chicks derived from them, indicating that these chicks were derived from PGCs carrying the DOC1 insert. Southern blot assay for Cre-mediated recombination of the Reaper transgene with 10 different strains of pLenti-ERNI-Cre transgenic chicken. A. Southern blot analysis of DNA is a Southern blot for DNA isolated from brain (b) and muscle (m) from double transgenic embryos carrying one copy of the Cre transgene and one copy of the loxP transgene. analysis. Genomic DNA was digested with SacI. The digested DNA was separated on a 0.7% agarose gel, blotted on a nylon membrane, and hybridized to a probe consisting of the Reaper gene and the lentiviral vector backbone (blasticidin gene and SV40 sequence). This probe hybridizes equally to both the full length and recombinant loxP-Reaper transgene. The ratio of full-length (non-recombinant) band intensity to the recombinant transgene represents the activity of the Cre strain. B. Overview of Cre-mediated recombination of loxP-Reaper transgene. The full length loxP-Reaper transgene contains a 1.4 kb sequence that is called the stop cassette and is flanked by loxP sites in the same direction. Recombination between two loxP sites results in excision of the 1.4 kb intervening sequence from the chromosome, leaving behind one loxP site. After excision, the recombined loxP-Reaper transgene is reduced in size by 1.4 kb. The location of the probe and SacI restriction enzyme site is indicated, and the expected restriction size is indicated. Recombination of three different Reaper loxP cassette transgenes (6-03, 6-51 and 9-51) by the Cre4 strain. A: loxP for a transgenic embryo carrying only one copy of the loxP-Reaper transgene (R) or one copy of the Cre4 transgene and three different loxP-Reaper strains (6-03, 6-51 and 9-51) Southern blot analysis of double transgenic embryos carrying 1 copy of the Reaper transgene (C + R). Genomic DNA was digested with SacI. The digested DNA is separated on a 0.7% agarose gel, blotted onto a nylon membrane, and hybridized to a radiolabeled probe consisting of the Reaper gene and the lentiviral vector backbone (blasticidin gene and SV40 sequence). did. The size of the hybridizing fragment was as expected at 2.8 kb for the full length (non-recombinant) loxP-Reaper fragment and 1.4 kb for the recombinant loxP-Reaper fragment. B: Overview of Cre-mediated recombination of loxP-Reaper transgene. The full length loxP-Reaper transgene contains a 1.4 kb sequence (referred to as a STOP cassette) and is flanked by loxP sites in the same direction. Recombination between two loxP sites results in a 1.4 kb intervening sequence excision from the chromosome, leaving one loxP site. After excision, the recombined loxP-Reaper transgene is reduced in size by 1.4 kb. The location of the probe and SacI restriction enzyme site is indicated, and the expected restriction size is indicated. Southern analysis of Doc2 cell lines positive for uncut GFP (lane 1) and Doc2 cells lacking the cx-GFP-cx-neo sequence (lane 2). Cells were sorted by FACS analysis for the expression of green fluorescence. Genomic DNA from two cell populations was prepared, digested with HindIII restriction enzyme, and the DNA hybridized to a radiolabeled sequence from the puromycin resistance gene. A 5521 bp predicted fragment was present in GFP positive (non-excised cells) cells and a 1262 bp predicted fragment was present in excised cells. This result indicates that Cre-lox recombination results in the deletion of the CX-EGFP-CX-neo sequence present between the two loxP sites in the docking site construct integrated in DOC2 cells. Illustration of IgL pKO5B targeting vector. The top line shows the construction of the targeting vector IgL pKO5B. This line shows the vector construct for the 5 'homology region containing the loxP and attP sites, and the 3' homology region. The second line shows the relationship of the targeting vector to the chicken wild type IgL allele. The third line shows the mutant allele created by J and C gene deletion or gene disruption. Southern blot analysis showing deletion of the IgL locus in the KO-07 knockout clone. Left panel: DNA obtained from 5 clone homologous regions when DNA from 5 clone PGC strains transfected with IgL pKO5B was digested with SacI and probed with a 0.5 kb SacI-BstEII fragment. Hybridization. The wild type IgL locus is about 10 kb and the mutant fragment with the targeted deletion is about 4 kb. Right panel: hybridization obtained for the 3 'homology region using DNA from the same five clones. Genomic DNA was digested with BstEII and hybridized with a 3 '1.7 kb Nsil-Mfel fragment (which is outside the targeting vector). Southern blot analysis showing that IgL knockout was transmitted to 5 of 7 chicken embryos (embryos 2, 3, 4, 6 and 7). Embryos 1 and 5 were wild type embryos that inherited the wild type IgL allele from the heterozygous KO-07 knockout PGC.

DETAILED DESCRIPTION OF THE INVENTION As used herein, the term “chick embryo stem (cES) cells” refers to the morphology of ES cells, and stage X (EG & K) embryos (in mouse blastocysts). Means cells that contribute to somatic tissue in recipient embryos from the light area of (approximately equal). CES cells share some in vitro properties of mouse ES cells, such as SSEA-1 +, EMA-1 +, and telomerase +. ES cells have the ability to colonize all somatic tissues.

  As used herein, the term “primitive germ cell (PGC)” refers to a cell that indicates the form of PGC and contributes exclusively to the germline in the recipient embryo, where PGC is stage It can be derived from whole blood collected from 12-17 (H & H) embryos. The PGC phenotype is: (1) germline specific genes CVH and Dazl are strongly transcribed in this cell line, (2) cells strongly express CVH protein, (3) stage X and stages 12-17 When injected into (H & H) recipient embryos, the cells do not contribute to somatic tissue, (4) the cells give rise to EG cells (see below), or (5) stages 12-17 ( When injected into H & H) embryos, cells can be established by transmitting the PGC genotype via the germ line (Tajima et al., (1993) Therogenology 40, 509-519; Naito et al., (1994). ) Mol.Reprod.Dev., 39, 153-161; 117, 291-298).

  As used herein, the term “chick embryo germ (cEG) cell” means a cell derived from PGC and similar in function to mouse EG cells. The morphology of cEG cells is similar to that of cES cells, and cEG cells contribute to somatic tissue when injected into stage X (EG & K) recipients.

  As used herein, the term “transgenic” means an animal that encodes a transgene in its somatic and germ cells and can transmit the characteristics conferred by the transgene to its progeny. The term “transgenic” also means an animal that includes inactivation of a selected site-specific gene at an endogenous locus, including but not limited to the deletion of a finite gene segment at an endogenous locus. Use of transgenes or targeting constructs that integrate into the genome of primordial germ cells resulting in inactivation of the gene by literal deletion of the gene, disruption of functionality, stop codon, or nonsense sequence, insertion of attP sites Or deletion through the use of other artifacts that result in functional inactivation of the locus by site-specific genetic modification. Existing retroviral techniques do not allow site-specific modification or selection of transformed cells (ie, sustain long-term culture of PGC cells and manipulate site-specific modifications (eg, gene inactivation)) The term “transgenic” excludes retroviral systems.

  However, animals having site-specific changes that alter the function of the selected gene to give the desired phenotype from the genetic modification are included. These transgenes and the animals derived from them are commonly referred to as “knock-ins”. The transgene can insert a deletion of endogenous DNA of at least 10 tcb, desirably 10-25 'kb, or targeting is further selected depending on the size and composition of the gene. In a preferred embodiment, the transgenic bird lacks any endogenous gene that corresponds to the endogenous gene target for total or partial deletion or other functional disruption.

  In the examples herein, chickens are described, but other bird species such as quail, turkey, pheasant, etc. may be used without undue experimentation and with the methods disclosed herein. Successful implementation can be reasonably predicted to replace a chicken.

  By inserting DNA constructs designed for tissue-specific expression into ES cells in culture, chickens have been created that express beneficial pharmaceuticals (such as monoclonal antibodies) in chicken egg white. Reference: PCT US03 / 25270 WO04 / 015123 (Zhu et al.). An important feasible technique for such animals is that of truly long-term ES cell cultures that can continue to survive long enough for the cloned cell genotype to be manipulated in culture. Making and maintaining.

  However, unlike ES cells, primordial germ cells (PGC) are cultured only on a short-term basis. Once the culture period is extended beyond a short period, these cells lose the ability to contribute exclusively to the germline. Typically, PGCs maintained in culture using current culture techniques do not grow and increase. If it does not grow vigorously, the culture is “terminal” and cannot be maintained indefinitely. Over time, these terminal cell cultures are degraded and the cells lose their inherent PGC morphology and revert to embryonic germ (EG) cells. Embryonic germ cells acquire a different morphology than PGCs and are no longer restricted to the germ line, gaining the ability to contribute to somatic tissues when injected in the early stages of embryonic development. PGCs are sperm and egg precursors in order to introduce a predetermined genotype into the germ line of the recipient embryo, thereby allowing the animal to transmit the desired genotype to the next generation It is known that PGC is very attractive.

  Long-term cultures of PGC have several important benefits, including the maintenance of beneficial gene characteristics of important chicken mating strains that depend on the poultry industry, with or without gene inactivation or exogenous DNA insertion. )I will provide a. Currently, special measures are taken to avoid beneficial mating strains from loss due to accident or disease. These measures require the maintenance of a large number of stock members as breeders and the replication of these breeds at multiple locations around the world. Since conservation of genetic diversity within the mating strain is also important, it is necessary to maintain a large number of beneficial animals stocked for the future. Preserving the genetic characteristics of these beneficial mating strains in PGC cell cultures rather than in live stocked livestock avoids the cost of large stocks of conservative mating populations.

PGC long-term cultures are described by van de Lavoir, MC, Diamond, J. MoI. Leighton, P .; Heyer, B .; Bradshaw, R .; , Mother-Love, C.I. , Kerchner, A .; , Hooi, L .; Gessaro, T .; Swanberg, S .; , Delany, M .; , And Etches, R .; J. et al. (2006). Germline transmission of genetically modified primary germ cells. Nature
441, 766-769.

  Production of genetically engineered chickens using PGC introduces genetic modifications into the PGC genotype, isolates rare cells with genetic modifications, and introduces them into recipient embryos for analysis and generation of G0 chimeras Requires expansion of genetically modified cell populations for Extensive genetic engineering techniques for target cells in culture are well known. However, one major difficulty is to introduce a genetic modification in order to change the PGC genotype in the culture, and for the appropriate period of time and the transfected cells to select for successfully transformed cells. This means that the survival of the culture must be maintained for a long time while growing and proliferating in the culture.

  Successfully transformed cells that can proliferate are distinguished by their ability to generate large numbers of cells (eg, 104-107 cells) within days to weeks of clonal or nearly clonal induction. Founder cells are rare cells that carry the desired genetic modification. Typically, these cells are generated in culture at a frequency of 10-4 to 10-7 after application of well-known genetic modification technologies (eg, lipofection or electroporation). Thus, the production of PGCs in culture provides the cells with space and nutrients to generate sufficient numbers of cells to grow and select rare genetically modified cells in culture. It is necessary to be subcultured.

  In order to provide such a population, the culture conditions allow cells to grow from individual genetically modified cells to a colony of 104-107 cells used for in vitro genetic analysis and chimera production. Must be strong enough for it. These engineered PGCs will contribute exclusively to the initial population of spermatogonia or oocytes (ie, sperm and eggs) at maturity in the resulting animal. In such resulting animals, the entire somatic tissue will be derived from the recipient embryo and the germline will receive contributions from both the donor cell and the recipient embryo. Because of the combined contribution to the germline, these animals are known as “germline chimeras”. Depending on the extent of the chimera, the germline chimeric progeny are derived from either donor cells or recipient embryos.

  The chicken germline begins as cells derived from the upper blastoderm of stage X (EG & K) embryos and enters the early lower blastoderm (Kagami et al. (1997) Mol Reprod Dev 48, 501-510; Pettitte (2002) J Pourtry Sci 39, 205-228). As the lower blastoderm progresses forward, primordial germ cells advance into the reproductive crescent, which can be identified as a giant glycogen-loaded cell. The earliest identification of cells in the germ line according to these morphological criteria was stage 4 using the staged classification established by Hamburger and Hamilton, (1951) J Morph 88, 49-92, approximately 8 hours after the start of incubation. ). Primordial germ cells are present in the reproductive crescent ring from stage 4 (H & H) until they migrate through the vasculature during stages 12-17 (H & H). At this point, the primordial germ cells are a small population of about 200 cells. From the vasculature, primordial germ cells migrate into the genital ridge and become integrated into the ovary or testis as the gonadal differentiates (Swift, (1914) Am. J. Anat. 15, 483-516; Meyer, (1964). Dev Biol. 10, 154-190; Fujimoto et al. (1976) Anat. Rec. 185, 139-154).

  In all species that have been tested so far, primordial germ cells have not proliferated in long-term cultures without differentiation into EG cells. Long term culture is required to produce a sufficient number of cells to introduce genetic modification or inactivation by conventional electroporation or lipofection protocols. Typically, these protocols require 105-107 cells, so that all cell division occurs (1) synchronously and (2) produces two live daughter cells. Assuming the production of these cells from one precursor requires a 17-24 doubling. The introduction of a genetic modification into the cell genome is a rare event and typically occurs once in 1 × 10 4 to 1 × 10 6 cells. After genetic modification, the cell must be able to establish a colony from one cell that carries and / or expresses the genetic modification. Colonies must be obtained in sufficient numbers by expanding to a population of 105-107 cells that can be analyzed by PCR or Southern analysis to assess transgene reliability, Injection into recipient embryos at stages 13-15 (H & H). Therefore, an additional 17-24 cell divisions are required to produce a cell population and a total of 34-58 doublings are required to produce a genetically modified cell population. Assuming a cell cycle of 24 hours, a minimum of 34 days, generally 58 days, is generally required to produce genetically modified primordial germ cells for injection into stage 13-15 (H & H) recipient embryos. Culture is required. The injected cells must then be able to colonize the germ line, form functional gametes and develop into new individuals after fertilization.

  PGCs maintained in the cultures described herein maintain a characteristic PGC form while maintained in the culture. PGC morphology can be observed by direct observation and cell growth in the culture is assessed by common techniques to ensure that the cells grow in the culture. A proliferating cell culture is defined as non-terminal and it is observed that the later time of the two different time points has a higher number of cells in the culture. It will be appreciated that the PGCs in the cultures of the invention can have 1 × 10 5 or more cells in any particular culture, and this number increases over time. Thus, the present invention includes growing PGC cultures that contain a large number of cells after days, weeks or months compared to earlier points in the life of the culture. Ideally, the culture contains at least 1 × 10 5 cells and can be seen to have a greater number of cells after any growth period of the culture. Furthermore, it is recognized that PGC is the dominant species in culture, which takes into account the minimal contribution made by non-chicken feeder cells, so that the growth component of the cell culture is derived from other chickens The cell is substantially excluded and consists essentially of chicken primordial germ cells.

  The cultures also exhibit growth characteristics by passage that can separate samples or aliquots of cells from existing cultures and show strong growth when placed in a new culture medium. By definition, the ability to pass a cell culture indicates that the cell culture grows and proliferates and is not terminal. Furthermore, the cells of the present invention demonstrate the ability to create germline chimeras after several passages and maintain PGC morphology. As described herein, this growth is an essential feature of any cell culture suitable for stable integration of exogenous DNA sequences.

  PGCs can be obtained by any known technique and can be grown under the culture conditions described herein. However, it is preferred that whole blood be removed from the stage 15 embryo and placed directly into the culture medium described below. This approach differs from other approaches described in the literature where the PGC is subjected to processing and separation steps prior to entering the medium. The robust differential growth between whole blood-derived PGCs and other cells that may initially coexist in the medium provides a large population of PGCs in the cultures described herein. Thus, PGCs derived directly from whole blood can grow into large cell concentrates in culture and can be passaged an unlimited number of times, with PGCs in culture essentially growing and proliferating. It shows robust growth and proliferation that is only cells.

  One aspect of the present invention is the production of a large number (including more than 3, more than 4, more than 5, 10, 15, and 20) germline chimeric animals, all of which are in the germline. Have genetically identical PGC-derived cells. Another aspect of the invention is the creation of a germline chimeric population having age-different within populations and having genetically identical PGC-derived cells in its germline, which creates a germline chimera. Reflects the use of the same long-term cell culture for. The age difference exceeds the currently available ability to culture primordial germ cells for long periods, which can be as long as 190 days without freezing. Thus, the present invention provides for inheriting in the germline that is different in age beyond 40, 60, 80, 100, 190, etc., or any other integer value therebetween, without freezing the cells. Two or more germline chimeras having identical PGC-derived cells. The present invention also provides a sexually mature germline chimera having genetically identical PGC-derived cells in its germline with the presence of non-terminal PGC cultures used to make these germline chimeras. From these, additional germline chimeras can be generated.

  Since PGCs can be maintained in culture in a very stable manner, the ability to cryopreserve and thaw cells to produce progeny defined by the PGC phenotype maintained in the culture. A long-term storage method for producing a germline chimera can also be obtained.

  The ability to produce multiple germline chimeras also provides the ability to transmit PGC-derived genotypes to germline chimera progeny. Accordingly, the present invention includes both populations of germline chimeras that have genetically identical PGC-derived cells that have inactive endogenous gene loci in the germline, the genotype and phenotype of which are in culture. Also included are progeny of germline chimeras that are completely determined by the genotype of PGCs grown in Incorporation of the PGC-derived knockout phenotype in the germline was observed. Thus, the present invention includes germline chimera progeny generated by transmission of the primordial germ cell genotype containing the inactivated endogenous locus to the germline. Accordingly, the present invention relates to a primordial germ cell culture comprising PGCs consisting of site-specific gene inactivation, a germline chimera having the same primordial germ cell as part of its germline, and a germ having a knockout genotype and phenotype Includes the presence of each progeny of the lineage chimera.

  The ratio of donor-derived PGC to recipient-derived PGC in the recipient embryo can be varied to aid germline colonization in the PGC-derived chimera. In developing chicken and quail embryos, exposure to busulfan greatly reduces or disappears the primordial germ cell population as it moves from the reproductive crescent to the genital ridge (Reynaud (1977a)). Bull Soc.Zool.Francaise 102,417-429; Reynaud (1981) Arch Anat.Micro.Morph.Exp.70, 251-258; Aige-Gil and Simkiss (1991) Res.Vet.Sci.44, 139. ). If busulfan is injected into the yolk after 24-30 hours of incubation, primordial germ cells are reintroduced into the vasculature after 50-55 hours of incubation and the germline is repopulated with donor-derived primordial germ cells, Donor-derived gametes are produced (Vick et al. (1993) J. Reprod. Fert. 98, 637-641; Bresler et al. (1994) Brit. Poultry Sci. 35 241-247).

  The method of the present invention comprises obtaining PGCs from chickens (eg, from whole blood of stage 15 embryos), placing PGCs in culture, manipulating inactivation of endogenous gene loci, Shows growing PGCs to increase their number, allowing multiple passages, creating germline chimeras from engineered long-term cultures of PGCs, and PGC-engineered gene inactivation Obtaining offspring of germline chimeras having genotype and phenotype. The method of the present invention also inserts an inactivated gene or “knock-out” gene into a PGC population in culture to stably transduce an endogenous locus that has been inactivated or functionally disrupted. Creating a transfected PGC, selecting cells from this population carrying a stably integrated transgene, and injecting a genetically modified cell carrying a stably integrated transgene into a recipient embryo A step of generating an embryo in a germline chimera containing a germline-inactivated locus, a step of breeding the germline chimera until sexual maturity, and mating the germline chimera into transgenic offspring Wherein gene inactivation is derived from cultured PGCs. Genetic modifications introduced into the PGC to achieve gene inactivation include, but are not limited to, random integration of the transgene into the genome, transgene inserted into the promoter region of the gene, repeats in the genome Insertion of transgenes into elements, site-specific changes to genomes introduced using integrase, site-specific changes to genomes introduced by homologous recombination, and others that are lox sites or substrates for site-specific recombination It contains a conditional mutation introduced into the genome by excising DNA adjacent to the sequence.

  As described below, according to the present invention, the chicken PGC cell line is derived from blood collected from stage 14-16 (H & H) embryos and has a large round shape (FIG. 1). These cells are confirmed to be chicken PGCs by their morphology after long-term culture and the ability to produce PGC-derived progeny. Furthermore, PGC cultures express germline specific genes Dazl and CVH (FIG. 2) and CVH protein is produced by cells in the culture (FIG. 3). PGCs in culture also express telomerase (FIG. 4), indicating that they have an immortal phenotype. Furthermore, PGCs give rise to embryonic germ (EG) cells in appropriate culture conditions (Figure 5). By similarity, mouse and human PGCs give rise to EG cells when treated in a similar manner. Mouse EG cells contribute to somatic tissue, and chicken EG cells also contribute to somatic tissue, as shown by the pigmentation of chimeric black feathers. Chicken PGC has been genetically modified as shown by Southern analysis (Figure 6). In this embodiment, the CX promoter is stably integrated into the PGC genome and is used to facilitate the expression of a gene encoding an aminoglycoside phosphotransferase (APH) that is integrated together into the PGC genome. Thus, resistance to neomycin added to the culture medium is imparted, and genetically modified PGCs are selected.

Example 1. Induction of chicken PGC cultures
2-5 μL of blood collected from the terminal venous sinus of stage 14-17 (H & H) embryos was added to medium (stem cell factor (SCF; 6 ng / ml or 60 ng / ml), human recombinant fibroblast growth factor (hrFGF; 4 ng). / Ml or 40 ng / ml), 10% fetal calf serum, and 80% KO-DMEM conditioned medium). Preferably, 1-3 μL was taken from the vasculature of stage 15-16 (H & H) embryos. Irradiated STO cells were seeded at a concentration of 3 × 10 4 cells / cm 2 in the wells of a 96-well plate.

  KO-DMEM conditioned medium supplemented with 10% fetal bovine serum, 1% pen / strep, 2 mM glutamine, 1 mM pyruvate, 1 × nucleoside, 1 × non-essential amino acid, and 0.1 mM β-mercaptoethanol, 5% Prepared by 3 day growth of BRL cells to confluence in DMEM with fetal calf serum. After 24 hours, the medium was removed and a new batch of medium was conditioned for 3 days. This was repeated three times to produce a PGC culture medium by combining three batches.

  After about 180 days of culture, one of the PGC strains was grown in a medium consisting of 40% KO-DMEM conditioned medium, 7.5% fetal calf serum, and 2.5% chicken serum. Under these conditions, the doubling time of PGC was about 24-36 hours.

  The main cell type at the start of culture was fetal erythrocytes. The main cell type within 3 weeks was the PGC cell type. Two PGC cell lines were derived from 18 cultures initiated from each embryo.

  The PGC strain is cultured for 9 months, remains round and remains unattached (FIGS. 1A and B). PGCs were successfully thawed after cryopreservation in CO2-independent medium containing 10% serum and 10% DMSO.

(Example 2. Cultured PGC expresses CVH and Dazl)
CVH is a chicken homologue of the germline-specific gene VASA in Drosophila, whose expression is restricted to cells within the chicken germline and is expressed by approximately 200 cells in the reproductive crescent (Tsunekawa, N , Naito, M., Sakai, Y., Nishida, T. & Noce, T. Isolation of chicken vasa homolog gene and tracing the origin of primary cells, 27. V. 41, V. e. Necessary for proper functioning of male germline, loss of CVH function causes male mice to cause infertility (Tanaka, SS, et al., The mouse homolog of Drosophila Vasa is required for the development of male germ cells. Genes Dev 14, 841-53. (2000) Dazl is expressed in frogs (Hoston, D. W. and L. cit. a germ plasma-localized RNA, in the differentiation of prime germ cells in Xenopus. Development 127, 447-56, 2000), Ahoroth (Johnson, A.D. T. Expression of axolotl DAZL RNA, marker of germ plasma: widespread manual RNA and onset of expression in germ cells, the gond. Dev Biol 234, 402-15, 2001), mouse (Ssns. H. J. & de Roijj, D. G. Nature of the spermatogenetic arrest in Dazl-/-mice.Biol Reprod 65, 771-776, 2001), rat (Hamra, F. K. et al., Production ration of production. lentilal transmission of male erm-line stem cells. Proc Natl Acad Sci USA 99,14931-6,2002), and humans (Lifschitz-Mercer, B. et al., Absence of RBM expression as a marker of intra11. -1120, 2000). Dazl deletion results in spermatogenesis deficiency in transgenic mice (Reijo, R, et al., Diverse genomics in humans causative by Y chromosome deletion 10-Netetinga novel RNAp binding RNA-bind. .

  After 32 days, PGCs were washed with PBS, pelleted, and mRNA was isolated from tissue samples using the Oligotex Direct mRNA kit (Qiagen). CDNA was then synthesized from 9 μl of mRNA using the SuperScript RT-PCR system for First-Strand cDNA synthesis (Invitrogen). 2 μl of cDNA was used in subsequent PCR reactions. The primer sequences derived from the CVH sequence (accession number AB004836), Dazl sequence (accession number AY211387), or β-actin sequence (accession number NM — 205518) were as follows.

Primers V-1 and V-2 were used to amplify a 751 bp fragment from the CVH transcript. Primers Dazl-1 and Dazl-2 were used to amplify a 536 bp fragment from the Dazl transcript. Primers Act-RT-1 and Act-RT-R were used to amplify a 597 bp fragment from the endogenous chicken β-actin transcript. PCR reactions were performed using AmpliTaq Gold (Applied Biosystems) according to the manufacturer's instructions (Figure 2).

(Example 3. PGC expresses CVH protein)
Protein was extracted from freshly isolated PGCs using the T-Per tissue protein extraction kit (Pierce). Protein was extracted from the cells by lysis of the cells in 66 mM EDTA, 10 mM, Tris (pH 7.4) supplemented with 1% NP4O, 0.4% deoxycholate. Samples were run on a 4-15% Tris-HCL ready gel (Bio-Rad). After transfer to the membrane, Western blots were performed using the Super Signal West Pico chemiluminescent substrate kit (Pierce) as described. Rabbit anti-CVH antibody was used as the primary antibody (1: 300) and HRP-conjugated goat anti-rabbit IgG antibody (Pierce, 1: 100,000) was used as the secondary antibody (FIG. 3).

(Example 4. Cultured PGC expresses telomerase)
Primordial germ cells were pelleted, washed with PBS and then frozen at −80 ° C. until analysis. Cell extracts were prepared and detected based on the telomere repeat amplification protocol (TRAP) (Kim, N., et al., Specific association of human telomerase activity with immortal cells and cancer. Science 266, 2011-2014, 1994e). Analysis was performed according to the manufacturer's instructions using the Serologicals Corporation. FIG.

(Example 5. Embryonic germ (EG) cells can be derived from PGC cultures)
Chicken EG cells were derived from PGC by allowing the cells to adhere to the plate, removing FGF, SCF, and chicken serum, and culturing the cells under the same conditions used for ES cell culture (van de Lavoir et al., 2006 High Grade Somatic Chimeras from Chicken Embryonic Stem Cells, Mechanisms of Development 12,31-41; van de Lavoir and Mather-Love (2006) Chicken Embryonic Stem Cells; Culture and Chimera Production, Methods in Enzymology, during printing ). The morphology of cEG cells is very similar to that of cES cells (FIGS. 5A, B). When cEG cells are injected into stage X (EG & K) embryos, cEG cells colonize somatic tissues and create chimeras that appear to be identical to chimeras created using cES cells as young birds Have the ability. Chicken EG cells are observed in both the newly induced transgenic PGC strain and the clonal derived transgenic PGC strain. Southern analysis of GFP-positive PGC-derived EG cells proved that the EG cells originated from PGC (FIG. 15).

Example 6. Cultured male PGCs produce functional gametes in roosters
Male primordial germ cell lines were derived from each Barred Rock embryo. After establishment of the line, the cells were injected into stage 13-15 (H & H) embryos. Phenotypically, hatched chickens were similar to white leghorns. Males were raised to sexual maturity and mated with Barred Rock hens (Table 1). Black offspring show germline transmission of injected PGCs. Rooster germline transmission rates varied from less than 1% to 86% (Table 1).

PGCs can also be injected into the subembryonic space of stage X embryos. After 209 days in culture, 1000 or 5000 PGCs were injected into the irradiated embryos. Hatched male chicks were grown to sexual maturity and mated to test germline transmission. Germline transmission was observed in 3 out of 4 roosters tested, the frequency of which varied from 0.15 to 0.45%. This indicates that PGC can colonize the germline when injected prior to gastrulation. Germline transmission of male PGCs was not observed in 1,625 offspring from 14 female chimeras.

(Example 7. Cultured female PGCs produce functional gametes in hens)
Female PGCs derived from Barred Rock embryos cultured for 66 days were injected into white leghorn embryos at stages 13 to 16 (H & H), and all chicks hatched had white leghorn phenotype. The hens were raised until sexual maturity and mated with Barred Rock roosters. Female PGCs were transmitted through female chimeras, with a frequency of up to 69% (Table 2).

Female PGCs were also injected into male recipient white leghorn embryos. Male chimeras were raised until sexual maturity and mated with Barred Rock hens. Germline transmission of female PGCs was not observed in 506 offspring of 3 test roosters.

(Example 8. PGC-derived progeny are reproductively normal)
Three male and four female non-transgenic PGC-derived progeny were mated to each other. Between 53% and 100% of eggs fertilized (Table 3), and between 79% and 100% of fertilized eggs, embryos formed hatched embryos (Table 3), which means that PGC-derived progeny can reproduce Is normal.

Example 9
Primordial germ cells were isolated from stage 14-17 embryos and shown to contribute to the germline (see: Examples 1-8). In this case, the PGC circulates through the vascular system. Prior to vasculature formation, PGCs were located in the reproductive crescent ring (in front of the embryo body). The precursors of reproductive crescent ring PGCs are not well understood, but it is usually believed that PGCs are derived from cells in the light region of stage X (Eyal-Giladi and Kochav) embryos (Petitete, J. et al. N. 2002. The Avian germline and Strategies for the Production of Transgenic Chickens.Journal of Power Science 39, 205-228). While in stage X embryos, PGCs cannot be identified using the classic morphological criteria used for their identification in the reproductive crescent. Surprisingly, disposition of dispersed cells from stage X Barred Rock embryos has been shown to result in PGCs and contribute to the germline. We proved this principle by collecting blastoderm individually, grinding them with a Pasteur pipette and mechanically dispersing them. Cells were washed and plated into 48-well plates previously seeded with irradiated BRL cells containing the media described in Example 1. Cultures were passaged for an initial period of 6-10 days after seeding. Subsequently, passage was dependent on the concentration of PGC present. Two male cell lines (PGC-A12 and PGC-B11) were established and injected into the recipient embryos as described in Example 6 after 45 and 36 days of culture, respectively. Five male chimeras were generated from each cell line. As shown in Table 4, the Barred Rock phenotype could be cultured in a medium provided with cells destined to become functional PGCs transmitted by the germ line of 3 out of 10 males. Prove that.

Example 10. PGC sensitivity to neomycin and puromycin.
To establish the concentration of puromycin and neomycin required for growth of cells expressing antibiotic resistance under the control of the highly expressed CX-promoter (in all tissues), the sensitivity of PGC to neomycin and puromycin was determined. . These experiments demonstrated that neomycin at a concentration of 300 μg / ml was required for 10 days to eliminate all untransfected cells. Puromycin at a concentration of 0.5 μg / ml was sufficient to eliminate PGC within 7-10 days.

(Example 11. Genetic modification of PGC)
20 μg (20 μl) of NotI linearized cx-neo transgene (see FIG. 6) was added to the 5.8 × 10 6 PGC population cultured for 167 days. Cells and DNA were resuspended in 800 μl electroporation buffer and 8 square wave pulses of 672 volts and 100 seconds duration were applied. After 10 minutes, the cells were resuspended in culture medium and aliquoted into 24-well plates. Two days after electroporation, 300 μg of neomycin per ml of medium was added to select cells expressing the cx-neo transgene. Cells were kept under selection for 19 days. After 19 days, cells were removed from selection and expanded for analysis. Holding the PGC ratio at 300 μg / ml for an additional 31 days proved that PGC was functionally resistant to antibiotics.

  With reference to FIG. 6, for plasmid control, cx-neo plasmid DNA was linearized with NotI and then digested with EcoRI or BamHI, a slightly smaller fragment (5 kb) than the intact plasmid found with HindIII digestion. Was produced. An internal fragment of the approximately 2 kb cx-neo plasmid was released by digestion with StyI or NcoI. A larger internal fragment of approximately 2.6 kb was released by digestion with EcoRI and KpnI. Digestion of genomic DNA from the PGC strain with EcoRI, BamHI, and HindIII revealed a band exceeding 6 kb, indicating that the cx-neo transgene was integrated into the PGC genome. The internal fragment revealed in plasmid DNA after digestion with KpnI with StyI, NcoI, and EcoRI is also present in the genomic DNA of PGC and is integrated into the PGC genome without change in the cx-neo transgene. It shows that. Additional elements can be incorporated using conventional transgene construction techniques, examples of which are exogenous DNA encoding regulatory elements, tissue-specific promoters and proteins.

  As noted above, the ability of PGCs to genetically modify to produce transgenic animals has been demonstrated only in a very small number of species. Similar genetic manipulations can be performed in chicken PGCs by reference to genetic manipulations performed using mouse ES cells. In mice, chromosomal transfer into embryonic stem (mES) cells for individual use of homologous recombination and subsequent production of chimeric and transgenic offspring is well known. A powerful technique for site-specific homologous recombination or gene targeting has been developed (Thomas, KR and MR Capecchi, Cell 51: 503-512, 1987; Waldman, AS, Crit. Rev. Oncol. Hematol. 12: 49-64, 1992). Insertion of cloned DNA (Jakobovits, A., Curr. Biol. 4: 761-763, 1994) and manipulation and selection of chromosomal fragments by Cre-loxP system technology (Smith, AJ et al., Nat. Genet. 9: Ramirez-Soris, R. et al., Nature 378: 720-724, 1995; U.S. Pat. Nos. 4,959,317; 6,130,364; 6,130,364. No. 6,091,001; see No. 5,985,614) can be used for genetic manipulation to produce stable gene chimeras and gene transfer into mES cells.

  The genome of primordial germ cells is generally considered to be quiescent and thus chromatin can be highly enriched. Extensive testing with conventional electroporation protocols suggests that special methods are required to introduce genetic modifications into the genome of PGC. As described below, the transgene can be surrounded by an insulator element derived from the chicken β-globin locus to enhance expression. Clones that can be grown by inclusion of the β-globin insulator element are routinely produced and analyzed and injected into the recipient embryo.

  Conventional promoters used to cause expression of antibiotic (eg, neomycin, puromycin, hygromycin, his-D, blasticidin, zeocin, and gpt) resistance genes are ubiquitously expressed. Typically, the promoter is derived from a “housekeeping” gene such as β-actin, CMV, or ubiquitin. While constitutive promoters are useful because they are typically expressed at high levels in whole cells, they continue to be expressed in most, if not all, tissues throughout the life of a chicken. In general, expression should be limited only to the tissues and developmental stages that require expression. For the selection of primordial germ cells, the period that requires expression is the period that exists in vitro when antibiotics are present in the medium. It is preferred that the expression of the selectable marker (ie, antibiotic resistance gene) is terminated once the cell is inserted into the embryo. To limit the expression of antibiotic resistance genes, the “early response to neural induction” (ERNI) promoter is used. ERNI is a gene that is selectively expressed in early stages of development (eg, Stage X (EG & K)) and in culture, and thus uses this promoter to drive the expression of antibiotic resistance genes, A PGC carrying the genetic modification is selected. Since only ERNI is expressed at an early stage of development, genes that confer antibiotic resistance are not expressed in mature animals.

Example 12. Homogenous PGC cell culture homogeneity
To determine the homogeneity of PGC cultures after long-term culture, ES, EG, DT40 (chicken B cell line), and PGC were stained with anti-CVH, antibodies against chicken tube homologs, and 1B3 antibody (Halfter, W., Schurer, B., Hasselhorn, HM, Christ, B., Gimpel, E., and Eperlein, H. H., An ovo mucin-like protein on the surface of migratory. rat.Development 122,915-15.1996)). CVH antibody expression is restricted to germ cells, and thus anti-CVH antibodies are reliable markers of germ cells. The 1B3 antigen recognizes ovomucin-like proteins that are present on the surface of chicken PGCs during gonadal migration and colonization.

  Cells were washed with CMF / 2% FBS, fixed with 4% paraformaldehyde for 5 minutes, and washed again. 0.1% Triton X-100 was permeated for 1-2 minutes into cell aliquots to be stained for vessels. Primary antibody was added for 20 minutes and cells were washed twice and incubated for 15 minutes with secondary antibodies (CVH and Alexa 488 anti-rabbit IgG for control and Alexa 488 anti-rabbit IgM for 1B3). As a control, aliquots of cells were stained with secondary antibody alone. After two additional washes, cells for FACS analysis were prepared.

  With respect to FIG. 7, DT40 cells, ES cells, and EG cells all show background when stained with CVH and 1B3 antibodies. However, PGCs are even more intensely stained with both CVH and 1B3 antibodies. There is a subpopulation of PGCs that do not stain with either CVH or 1B3, indicating that the subpopulation of cells does not exhibit a PGC phenotype. Using 4 transfected cell lines (G-09, P84, P97 / 6, and P97 / 33) from 2 parental PGC and PGC13 parental cell lines, using vascular and 1B3 antibodies (PGC13 and 102) And tested. All show the same pattern, indicating that different PGC cultures contain cells that express the PGC phenotype at the same high rate.

(Example 13: Genetic modification of primordial germ cells)
Electroporation using the circular CX-GFP plasmid revealed that the transient transfection rate of PGC varied between 1-30%. Using eight 100 μs and 800 V square wave pulses, we obtained PGC cells carrying the CX-neo construct and named G-09. See FIG. Southern blot analysis was used to assess the incorporation of the construct. However, the isolation of this stably transfected strain was a non-reproducible event. With the exception of G-09, after electroporation of 17 × 10 7 PGCs using linearized constructs in 37 transfection experiments using both square wave and exponentially decaying pulses, PGCs stably translocated. It wasn't perfect. In each of these experiments, the PGC number varied from 1 × 10 6 to 10 × 10 6. The following promoter widely used in ES cell studies in mice, chickens, and humans was tested: CX promoter, also called CAG (Niwa, H., Yamamura, K., and Miyazaki, J., Efficient selection for high- expression transfectants with a novel eukaryotic vector. Gene 108, 193-9.1991), chicken β-actin promoter including CMV enhancer, PGK promoter, MC1 promoter, and Ubc promoter. These promoters did not increase transfection efficiency. Insulators were used with integrated constructs to allow expression of selectable markers and clonal induction of genetically modified cell lines.

  An insulator is a DNA sequence that separates an active chromatin domain from an inactive chromatin domain and sequesters the gene from the activation effect of adjacent enhancers or the silencing effect of adjacent aggregated chromatin. In chickens, the 5 ′ HS4 insulator present 5 ′ of the β-globin locus has been well characterized by Felsenfeld and collaborators (Burgess-Beusse, B., Farrell, C., Gaszner, M.). , Litt, M., Mutskov, V., Recilas-Targa, F., Simpson, M., West, A., and Felsenfeld, G. (2002)). The insulation of genes from external enhancers and silencing chromatin. Proc. Natl. Acad. Sci. USA 99 Suppl. 4, 16433-7. This insulator protects the β-globin locus from the constitutively condensed upstream region of chromatin. We have used the chicken β-actin promoter that causes neomycin resistance using the chicken β-globin 5 ′ HS4 sequence as both 5 ′ and 3 ′ insulators of the chicken β-actin-neo cassette. Assembled the transgene.

  The 250 bp core sequence of sensitive site 4 from the chicken β-globin locus was PCR amplified using the following primer set:

The PCR product was cloned into pGEM-T and sequenced. A tandem duplication of the HS4 site was made by digesting HS4 in the pGEM clone with BamHI and BglII to release the insert and digesting with BglII to linearize the vector. The HS4 fragment was ligated into a vector containing a copy of the HS4 insulator. Clones were screened and clones were selected in which the two HS4 copies were in the same orientation. This is called 2 × HS4.

(Example 14: Bulk selection using HS4β-actin-neo)
β-actin neo was obtained from Buerstedde (clone 574) and transferred to pBluescript. 2 × HS4 was then cloned at both the 5 ′ and 3 ′ ends of β-actin neo to produce HS4-β-actin neo. Using this construct, 8 transfections were performed. For each transfection, 5 × 10 6 PGCs were resuspended in 400 μl electroporation buffer (Specialty Media) and 20 μg linearized DNA was added. One exponential decay (ED) pulse (200 V, 900-1100 μF) or 8 square wave (SW) pulses (250-350 V, 100 μs) were applied. After transfection, the cells were allowed to grow for several days, after which neomycin selection agent (300 μg / ml) was added. Each fraction was grown as a pool. Resistant cells were isolated from 5-8 fractions.

  Southern analysis was performed on two pools of transfected cells (FIG. 8). 2 μg genomic DNA from PGC cell lines P84 and P85 and 20 pg plasmid (HS4-β-actin neo) were digested. Digests were run on a 0.7% gel, transferred to nylon membranes in 10 × SSC overnight by capillary action, and probed with radiolabeled neo gene sequences in Rapid Hyb (Amersham) for 2 hours. After washing, the blot was exposed to film at −80 ° C. overnight. With respect to FIG. 8, lane 1 is P84, lane 2 is P85, and lane 3 is a plasmid. For plasmid control, HS4-β-actin neo plasmid DNA was linearized with NotI. To obtain a 2.3 Kb internal fragment, PGC DNA and the linearized plasmid were digested with BamHI. Both P84 and P85 show an internal fragment with a size of 2.3 Kb. A larger internal fragment of approximately 2.6 kb was released by digestion with HindIII. Furthermore, this internal fragment is present in both P84 and P85 digests. Digestion of P84 and P85 genomic DNA with EcoRI and BglII should reveal bands above 2.9 Kb when the transgene is integrated into the genome. In P84, no junction point fragment is observed, indicating that P84 is a composite of several different clones. In P85, there is a 4.5-5 Kb junction point fragment in the EcoRI digest, and there is a 5 Kb junction fragment in the BglII digest, which indicates that P85 is integrated into the genome and the culture is In other words, it is composed of one clone. This example demonstrates the utility of an insulator as a preferred element of a construct for reliable expression of a selectable marker in primordial germ cells.

(Example 15: Clone induction of genetically modified PGC)
The following examples show that genetically modified strains of primordial germ cells can be derived clonally.

  First, β-actin-eGFP was prepared. The eGFP gene is released from CX-eGFP-CX-puro using XmnI and KpnI, β-actin is released from HS4-β-actin puro using EcoRI and XmnI, and the two as a three-way ligation It was cloned into pBluescript digested with EcoRI and KpnI to produce β-actin EGFP. BamHI and KpnI were then used to release β-actin eGFP (blunted using T4 DNA polymerase) and cloned into HS4-β-actin puro digested with BglII and EcoRV.

  This construct was used for 5 transfections. For each transfection, 5 × 10 6 PGCs were resuspended in 400 μl electroporation buffer (Specialty Media) and 20 μg linearized DNA was added. An ED pulse (150 to 200 V, 900 μF) or a SW (350 V, 8 pulses, 100 μsec) pulse was applied. After transfection, the cells were each plated in 48 wells and allowed to grow for several days before the selection agent (0.5 μg / ml) was added. A total of 5 clones were observed in 4 out of 5 transfections. One clone TP103 was analyzed by Southern (Figure 9). Referring to FIG. 11, plasmid control DNA was linearized with NotI. The internal fragment was released by digestion of the DNA with KpnI. Fragments of the same size were released in both TP103 and the plasmid. Digestion of TP103 genomic DNA with NcoI, MfeI, and SphI should reveal a larger band than the corresponding lane of digested plasmid DNA. No band was observed in the lane of MfeI digested TP103 genomic DNA, which may be due to the very large band. In the lane showing NcoI and SphI digestion, a fragment substantially larger than the fragment released in plasmid DNA was released in TP103 genomic DNA, indicating that the transgene was integrated into the genome of the TP103 cell line.

Clone induction of HS4-β-actin-puro First, β-actin puro was obtained by purifying CX-EGFP-CX-puro (XmnI-EcoRI), β-actin neo-derived β-actin in pBS (described above) ) (Sal-XmnI), and pBluescript (SalI-EcoRI). Next, β-actin puro was cloned into pBS containing two copies of 2 × HS4 by ligation of BamHI digested β-actin puro to BamHI / SAP treated 2 × HS4 vector.

  Three transfections were performed using this construct. For each transfection, 4-5 × 10 6 PGCs were resuspended in 400 μl of electroporation buffer (Specialty Media) and 20 μg of linearized DNA was added. An ED pulse of 200 V and 900 μF was applied. After transfection, the cells were each plated in 48 wells and allowed to grow for several days before the selection agent (0.5 μg / ml) was added. No clones were observed after two transfections. Two colonies were isolated from the third transfection.

Cloning induction of HS4-cx-eGFP-cx-Puro Transfections were performed 3 times using HS4-cx-eGFP-cx-Puro. 5 × 10 6 PGCs were resuspended in 400 μl of electroporation buffer (Specialty Media) and 20 μg of linearized DNA was added. A SW pulse of 350 V and 100 μs was applied 8 times to each transfection. After transfection, the cells were plated in 48 wells and allowed to grow for several days before puromycin selection agent (0.5 μg / ml) was added. A total of 16 clones were isolated from two transfections.

Cx-neo clonal induction The PGC13 cell line was electroporated using a plasmid carrying the cx-neo selectable marker. After exposure to neomycin, a cell line (G-09) that was resistant to neomycin was induced. The karyotype of this cell line was determined and all cells lacked the p-arm of chromosome 2 (Table 5 and FIG. 10). These data demonstrate that G-09 is clonally derived from PGC carrying a signature deletion in the p-arm of chromosome 2.

(Example 16: Tissue-specific expression of a selection marker in PGC)
The gene ERNI is expressed from the preprimitive streak stage of the chick embryo and is an early response gene to signals from Hensen nodules (Street, A., Berliner, AJ, Papanayouou, C., Sirulnik, A.,). and Stem, CD (2000) .Initiation of neural induction by FGF signaling before gastration. Nature 406, 74-8). Furthermore, ERNI is expressed in chicken ES cells (Acloque, H., Risson, V., Birot, A., Kunita, R., Pain, B., and Samarat, J. (2001). Identification of af. new gene family specifically expressed in chicken embryonic stem cells and early embryo. Mech Dev 103, 79-91). The ERNI gene (also called cENS-1) has a unique structure in which, in addition to the unique 5 ′ and 3 ′ UTR sequences, one long open reading frame is flanked by 486 bp tandem repeats. Based on the idea that this structure is reminiscent of a retroviral LTR-like structure, Acloque et al. (2001) assayed different parts of the cDNA sequence for promoter / enhancer activity, and the unique sequence region in the 3′UTR was the promoter. I found it to work. PCR primers were designed essentially as described in Acloque et al. (2001) to amplify the 822 bp fragment of the 3′UTR of the ERNI gene. After amplification of the ERNI sequence, they were cloned upstream of the neomycin resistance gene using the SV40 poly A site to create ERNI-neo (1.8 kb). The 2 × HS4 insulator was then cloned on either side of the ERNI-neo selectable marker cassette.

  Transfections were performed twice using HS4-Emi-neo. 5 × 10 6 PGCs were resuspended in 400 μl of electroporation buffer (Specialty Media) and 20 μg of linearized DNA was added. In the first transfection, a 175 V, 900 μF ED pulse was applied once, and in the second transfection, a 100 μsec and 350 V SW pulse was applied 8 times. After transfection, the cells were plated in 48 wells and allowed to grow for several days before adding a neomycin selective agent (300 μg / ml). Five colonies were isolated with the first transfection (ED pulse) and 11 colonies were isolated with the second transfection (SW pulse).

  Isolation of stably transfected clones indicates that ERNI is expressed in PGC and can be used as a tissue specific promoter.

Example 17: Contribution of transfected PGC to germline
PGCs were transfected with HS4-β actin-GFP and injected into the vasculature of stage 13-15 (H & H) embryos. On day 18, the gonadal was removed, fixed, sectioned and stained with CVH antibody to identify germ cells. The stained sections were then analyzed for the presence of GFP positive cells in the gonadal. GFP positive germ cells were found in both male (Figure 11) and female gonads. A histological preparation study of these embryonic brain, myocardium, and liver showed only four green cells on one slide. These data demonstrate that a small number of cultured PGCs were found ectopic, but most cultured PGCs preferentially colonize the germline.

  To determine that the GFP positive cells are germ cells, the sections were stained with anti-CVH antibody. As can be seen in FIG. 12, GFP positive cells are also stained for CVH protein, indicating that the GFP positive cells are germ cells.

  With respect to FIG. 12, GFP positive cells are present in this section, and the DAPI / GFP panel shows that these GFP positive cells are present in the seminiferous tubules. When germ cells are stained with anti-CVH antibodies, they show a strongly red-stained ring that outlines the germ cell cytoplasm. The DAPI / CVH panel shows that these cells are present in the seminiferous tubule. The last panel shows that GFP positive cells are also stained for CVH and that the seminiferous tubules contain GFP negative CVH positive germ cells.

Example 18. Germline transmission of genetically modified PGCs
A Barred Rock PGC transfected with one of the following transgenes: β-actin-neo, β-actin-eGFP-β-actin-puro, or cx-eGFP-cx-puro, stage 13-14 (H & H) embryos Injected into the vasculature. Chicken chicks were hatched, roosters were grown to sexual maturity, and mated with Barred Rock hens to determine germline transmission of the transgene. All black offspring were derived from PGCs and these were tested for the presence of the transgene (Table 6). Germline transmission rate was calculated by dividing the number of black chickens by the total number of chickens scored for feather color (Table 6).

(Example 19. Transgene is inherited according to Mendel's law)
Black progeny from crosses of chimeric roosters carrying a Barred Rock PGC genetically modified to contain one of β-actin-neo, β-actin-GFP, or cx-GFP were analyzed for the presence of the transgene. As shown in Table 7, the transgene is inherited in approximately 50% of PGC progeny and exhibits Mendelian inheritance.

(Example 20: Transgene expression of a transgene in a progeny of a chimera carrying a genetically modified PGC)
Chimeras carrying PGCs with β-actin-GFP stably integrated into the genome were crossed with wild-type hens and the embryos were scored for GFP expression. An example of expression in the embryo is shown in FIG. 13, which shows that GFP is expressed in all tissues of the transgenic progeny up to stage 34 (H & H) of development. In older animals, tissues were prepared for histological experiments using frozen sections. 1-2 week old chickens chicks pancreas, skin, lung, brain, ovary (not shown), kidney, bladder (not shown), the duodenum, breast, heart, liver, spleen, stomach and testis tissue, It is demonstrated that expression after hatching remains ubiquitous in the animal (FIG. 14).

(Example 21: HS4 containing a transgene is inserted into the promoter region of a chicken genome)
To state whether the construct containing HS4 was preferentially inserted into a specific region of the genome that avoids silencing and allows expression of the selectable marker, we will use a clonal transfected PGC strain. The transgene insertion site was confirmed. Genomic DNA was extracted from the transfected PGC strain and digested with either a restriction enzyme that does not cut the transgene or a restriction enzyme that once cuts the HS4 element. The DNA was self-ligated and transformed into E. coli. Cells were plated on ampicillin plates and colonies containing the amp gene from a plasmid linked to a genomic sequence flanking vector were isolated.

  The plasmid was purified and sequenced from 31 PGC strains transfected with the HS4-construct. We performed a BLAT (UCSC Chicken Genome Browser Gateway) and BLAST (NCBI) search to create a map of each genomic location of the insert. Of note, 25 of the 31 HS4 containing constructs are inserted into CpG islands, and it is commonly found near the promoter region (especially the housekeeping gene region). Of the insertions at CpG islands, the genes were found to be associated with most of the known genes defined by EST (Table 8) or most of the new genes (23/25). CpG islands often expanded from the first exon to the first intron, from several hundred base pairs upstream of the transcription start, and inserts were found in all of these regions. There was no bias in the transcription direction of the vector compared to the endogenous gene. Many of these genes are predicted to be expressed in PGCs based on their known function as housekeeping genes (eg, isocitrate dehydrogenase, aldehyde dehydrogenase, and mitochondrial solute carrier). Seven of the inserts were among the new genes as defined by EST. Five of these ESTs were originally cloned from the gonad or PGC library, suggesting that these genes can also be expressed in our PGC cell lines. Three of the gene inserts were not in the CpG island but rather in the more distal intron. Five of the inserts were in regions without any apparent gene. Three of these inserts were in close proximity to the LINE or satellite repeats.

(Example 22: Efficient integration with phiC31 integrase)
We used the phiC31 integrase system, which catalyzes site-specific recombination between the attB and attP sites to insert exogenous DNA into the chicken genome. Recombination between the phiC31 attB and attP sites is irreversible, so the insertion of a circular construct with an attB site into the genome is stable and does not form a loop, even in the continued presence of an integrase . Xenopus (Allen and Weeks 2005 Nature Methods 2,975-9.Transgenic Xenopus laevis embryos can be generated using phiC31 integrase), mouse (Olivares et al., 2002 Nature Biotechnology 20,1124-8.Site-specific genomic integration produces therapeutic Factor IX levels in mice; Belteki et al., 2003 Nature Biotechnology 21,321-4.Site-specific cassette exchange and germline transmission. ion with mouse ES cells expressing phiC31 integral) and human cells (Groth et al., 2000 Proc Natl Acad Sci USA USA, 97, 5995-6000. Aphage integrated strains effective strains). 21, 3926-34. In Site-specific genomic integration in mammalian cells medium by phagephiC31 integral), phiC31 integrase is a protein that has not modified an attB-containing plasmid. Adjust the incorporation into beam, these species genome has been proved to show that including a false attP site with sufficient sequence homology and attP sites of bacterial recognized by integrase. It has also been shown that the inserted plasmid must possess an attB site rather than an attP site for effective integration (Belteki et al., 2003; Thyagarajan 2001). The attB site was added to the sequestered HS4β-actin EGFP β-actin puro (HS4 BGBP) construct, resulting in attB HS4 BGBP. Referring to the left panel of FIG. 16B, the integrase construct used in this study is shown to contain an att-B containing plasmid with an att-B site added to the HS4β-actin EGFPβ-actin puro construct. Referring to the right panel of FIG. 16B, the plasmid used to express integrase in CAG promoter-derived cells is shown. Two versions of the integrase have been created, one with the SV40 nuclear localization signal and one without it. attB HS4 BGBP and CAG-integrase plasmid DNAs were cotransfected as circular plasmids into PGCs. Colony formation from 0.3 colonies per 106 cells with 20 ug linear DNA to 5-10 colonies per 106 cells with only 5 ug DNA compared to non-integrase linearized HS4 BGBP A large increase was observed, representing a 20-fold increase in colonies per DNA. The NLS version of the integrase produced slightly fewer colonies compared to the non-NLS version. These data suggested that the integrase can recognize a pseudo attP site in the chicken genome and it can be used for efficient and stable transfection with PGCs.

(Example 23: Identification of insertion site for integrase clone)
The increase in stable transfection efficiency using a plasmid containing integrase and attB suggested that the chicken genome contained a pseudo-attP site that could be recognized by phiC31 integrase. Southern blot analysis of genomic DNA from five independent PGC clones was performed to prove that the attB HS4 BGBP plasmid was integrated via integrase-mediated reactions rather than by random disruption of the vector, An intact full-length transgene was then observed in each case and had a structure consistent with integration via the attB site (data not shown). To further characterize recombination breakpoints at the nucleotide level, to identify pseudo-attP sites, and to identify the chromosomal location of insertions, vectors from 12 of our integrase PGC strains The junction between the genome insertion site was cloned and sequenced. Plasmid rescue was performed as described above for non-integrase strains. We have observed a dramatic decrease in the cloning efficiency of the ligation fragment; the number of E. coli colonies obtained is an average of 69 colonies per transformation for the non-integrase PGC strain. There were 3.1 colonies from the integrase-mediated PGC strain per conversion. The reason for this is unknown, but one possibility is that it was more difficult to digest the repetitive DNA adjacent to the integrase clone (see below) with restriction enzymes. Plasmid DNA was purified from colonies, sequenced, and attL and flanking sequences were determined (see Table 9 below). Since genomic DNA was digested with an enzyme that cleaves within the transgene, only adjacent genomic DNA adjacent to the amp gene on the vector backbone could be identified in this way; the other site of the transgene insertion. Adjacent DNA was not analyzed.

  Referring to FIG. 17, the junction point between the attB plasmid and the genomic sequence in the PGC clone derived from integrase-mediated transfection is shown. The top line is a wild type attB site with a core TTG, which is usually the underlined recombination crossover point (SEQ ID NO: 9), below which is an integrase-mediated The attL sequence from the insertion is shown (SEQ ID NO: 10-21). The PGC sequence was compared with attB (SEQ ID NO: 9) to determine where splicing occurred between the plasmid attB and the pseudo-attP site of the genome. In the PGC sequence, the attB sequence provided by the plasmid is shown in lower case and the genomic pseudo attP sequence is shown in upper case and bold.

  An attL sequence consisting of approximately half of the attB sequence on the plasmid was seen in each case associated with a pseudo attP site in the genome, suggesting that the integrase mediated the recombination reaction. Recombination between plasmid-derived attB and the genome was not accurate and usually did not occur at the core TTG nucleotide of attB. A map of each genomic location of the insert was created by BLAT and BLAST searches. Of note, 7 out of 11 insertions that could be mapped occurred with repetitive DNA sequences. Using Repeat Masker Web Server (Institute for Systems Biology) and ClustalW sequence alignments, repeats were analyzed and found to be able to be classified as PO41 repeats (Wicker et al.) With previously confirmed sequences. With respect to FIG. 18A, a sequence comparison of PO41-like repeats from the PGC insertion site and PO41 consensus sequence (SEQ ID NO: 29) is shown. PGC flanking sequences from all of the clones inserted in PO41-like repeats were sequenced with each other using the PO41 consensus (Wicker et al., 2004). The first 20 nucleotides were attB sequences conferred by the vector (as shown in the alignment above), followed by genomic flanking sequences from each clone. Nucleotides that share more than half of the sequence are enclosed in black boxes. Sequence alignment showed that two of the PGC strains (2-47 SEQ ID NO: 23 and 18-5-36-2 SEQ ID NO: 22) carry the attB HS4 BGBP insert at the same genomic site. The two insertions are independent, as shown by the fact that the nucleotide sequence of the attB pseudo attP junction differs between the two. The other sequence (18-3-12 SEQ ID NO: 24) was the same as the first two except for the 20 bp insert. None of the PO41 repeats identified in the insert are exact copies of the consensus sequence, and the level of sequence homology between the PO41 consensus and the insertion site repeat is 47% (18-3-43) to 77% The range of nucleotide homology was (1-30). With respect to FIG. 18B, sequence comparison of the 100 bp PO41 consensus repeat with 100 bp attP showed about 46% sequence homology, more than that observed for the pseudo-attP site in human cells (Thyagarajan 2001). it was high. PO41 repeats are thought to exist at 259 positions in the genome (Wicker et al.), Each position consisting of several kilobases of 41 bp repeat units. Some of the flanking genomic fragments ranged in size from 10 to 12 kb; both were repetitive when the two ends of these fragments were sequenced, which means that the overall Suggested that the size was large. Thus, the PO41 sequence represents a large and preferred target in the chicken genome for phiC31 integrase.

  The remaining four integrase-mediated inserts were among the unique DNA sequences. One of the sequences (19-1-1 SEQ ID NO: 12) is in the intergenic region of the unique sequence of chromosome 21 and one (1-41 SEQ ID NO: 10) is the Wilm tumor of chromosome 5 ( WT1) inserted into the promoter region of a chicken homologous molecular species of the gene. One sequence (5-7 SEQ ID NO: 11) was in multiple locations on chromosome 1 and could represent a local gene family or low copy number repeat. One sequence (18-4-11 SEQ ID NO: 13) did not match the existing sequence in the chicken genome or general “non-redundant database”, but thus the region of the genome that has not yet been sequenced Seems to be. One last insert (2-38 SEQ ID NO: 21) gave only a very short sequence consisting of a pseudo attP site, which could not be identified in the database.

(Example 23: Identification of genetically modified chromosome)
We focused on the chromosome where the insertion occurred for those strains that contained an insertion into the unique sequence that could be assigned a position. Among 28 independent insertions into the PGC, we found insertions into 17 different chromosomes (Tables 8 and 9) out of 38 chicken karyotype chromosomes. Approximately half of the insert is in the macrochromosome (chromosomes 1-6; 13 insertions) and the other half is in the microchromosome (chromosomes 7-38; 14 insertions) The thing is in the Z chromosome. The ratio of insertions into the macrochromosome and microchromosome is proportional to their physical contribution to the genome, indicating no region bias for recombination.

(Example 24: Gene targeting)
The targeting vector was designed to replace the J and C regions of the immunoglobulin light chain gene with the HS4 ERNI-puro selection cassette when inserted into the endogenous locus by homologous recombination (FIG. 19). As noted above, the ERNI promoter (causing the puromycin cassette) is specifically expressed in early embryos (Acloque et al., 2001, supra) and produces drug-resistant colonies in PGCs with a frequency similar to other promoters. Expected to be. Referring to FIG. 20, the top line is a diagram of a targeting vector for the chicken IgL gene (IgL KO5). It is designed to replace the 2.3 kb J-C region of the IgL gene with a 3.1 kb HS4 ERNI-puro selectable marker (shown as I and HS4 insulators). The two homology arms are 2.3 kB and 6.3 kB in length. At the 3 ′ end, the β-actin EGFP gene allows screening of puro resistant clones to enrich green fluorescence for the target clone. The dotted line at the end is the pKO vector backbone (Stratagene). The middle line is a diagram of the wild type allele of the germline composition of an IgL gene with a single variable region gene (V), a junction region gene (J) and a constant region gene (C). The restriction sites (S, SacI; B, BstEII) used for Southern analysis of the target clone are illustrated, and the wild type fragment size is shown below it with a double arrowhead. On the lower line is the structure of the mutant allele, where the J and C regions are deleted and replaced with HS4 ERNI-puro. A restriction map is shown below which shows the size of the mutant fragment. Both probes used in the Southern analysis are outside the targeting vector and indicate their location.

  Four clones were isolated from 21 transfections using a total of 1.05 × 10 8 cells and 210 μg of linearized DNA. Two of the clones expressed GFP, indicating that they were randomly integrated in the genome and retained the GFP gene. Southern blot analysis of 4 clones using probes from both sides of the integration showed that one of the non-green clones (clone 2) was heterozygous for the target mutation. With reference to FIG. 20, four puromycin resistant clones were Southern analyzed. Clones 1 and 2 were not green, while clones 3 and 4 expressed GFP. In the left panel, genomic DNA from the PGC clone was digested with SacI, hybridized with probe A and subjected to targeting analysis on the 5 'side of the IgL gene. In the right panel, DNA was digested with BstEII and hybridized with probe B for 3 'targeting of the IgL gene. Clone 2 showed a heterozygous fragment of the expected size with respect to the target clone.

(Example 25: G0 chimera carrying J-C knockout vector)
PGCs carrying the J-C knockout described in Example 24 were injected into the vasculature of stage 13-15 (H & H) white leghorn recipient embryos. In terms of phenotype, hatched chicks resemble white leghorns. Males were raised until sexual maturity and semen was collected by abdominal massage. And

PCR analysis using is shown in FIG. Referring to FIG. 21, the expected amplified 248 bp size DNA was present in semen from at least two of the G0 chimeras, which indicates that genetically modified primordial germ cells entered the germ line. It shows that.

Example 26: Insertion of β-actin-neo into the aldehyde dehydrogenase locus
PGCs from parental cell line 13 were grown and 5 × 10 6 cells in 400 μl were electroporated with linearized β-actin-neo construct using 198 V and 900 μF exponential decay pulses. A single clone was obtained by plating into 48-1 cm 2 wells. Cells were grown in the presence of neomycin to yield neomycin resistant clones that were transferred to new wells for expansion. Cells were analyzed by Southern analysis to establish stable integration of the transgene, and by sequencing, the construct was integrated into chromosome 19 at the promoter region of the aldehyde dehydrogenase gene (an enzyme involved in aldehyde metabolism).

  PGCs carrying the β-actin-neo construct were grown and injected into recipient embryos at stages 13-16 (H & H). Embryos were hatched and four roosters were grown to sexual maturity and tested for germline transmission. Rooster germline transmission rates were 0, 0, 0.5, and 0.5%, respectively. Heterozygous offspring from one of these roosters were grown to sexual maturity and mated to obtain homozygous offspring.

Five breeding pairs of heterozygous roosters and hens produced a total of 73 chicks, which were evaluated for the presence of the BN transgene. A total of 10 (14%) chicks were wild type, 46 (63%) chicks were heterozygous, and 17 (23%) chicks were homozygous (Table 10). ). This distribution was not significantly different (P> 0.01) from the distribution of 18.25 / 36.5 / 18.25 expected from Mendelian separation (chi-square = 6.55). The proportion of chicks that died near the hatching period was the same between genotypes. These results indicate that the insertion of the BN transgene did not induce a lethal phenotype.

  Homozygous roosters were grown to sexual maturity and tested for fertility. Five roosters were bred with wild-type hens, and fertility, embryo mortality and hatchability were calculated (Table 11). The fertility of the two roosters was relatively low, but the semen production of these birds was weak and therefore the number of sperm per insemination was also low. The fertility of two of the birds was very good (> 90%) and the fertility of one bird was moderate. The hatching rate of fertilized eggs from all birds was within the normal range. Taken together, these data indicate that the reproductive function of BN / BN birds was normal.

Since the BN transgene was integrated into the aldehyde dehydrogenase gene and no effect was seen on the viability of homozygous birds, we evaluated transcription of the aldehyde dehydrogenase message.

  mRNA was determined using the Oligotex Direct mRNA kit (Qiagen), two BN / BN homozygous birds, one BN / + heterozygous bird, and one wild-type bird (+ / +) Blood. The cDNA was then synthesized from 5 ml of RNA using a Thermo-Script RT-PCR system (Invitrogen) for single-stranded cDNA synthesis. 1 ml of cDNA was used in a subsequent PCR reaction using the following primers:

ALDH3A2-3 and A2-4 primers (SEQ ID NOs: 34, 35) were used to amplify 544 bp and 680 bp PCR products for transcription of aldehyde dehydrogenase 3 family member A2. Actin RT-1 and RT-2 primers (SEQ ID NO: 36, 37) were used to amplify a 597 bp PCR product for actin transcription. As shown in FIG. 22, aldehyde dehydrogenase 3 family member A2 transcripts were detected in heterozygous (BN / +) and wild-type birds (+ / +), but homozygous BN birds. In (BN / BN), it was not detected, indicating that insertion of the β-actin-neo transgene caused an insertional knockout of the aldehyde dehydrogenase 3 gene without a morphological phenotype.

  Confirmation that the primer amplified the family member A2 transcript of aldehyde dehydrogenase 3 was obtained by sequencing the 544 bp and 680 bp PCR products. The 544 bp product is entirely contained within the 680 bp PCR product, which also contains a 136 bp unspliced intron between exons 5 and 6 (FIG. 23). Comparison of these sequences with those of the published chicken genome showed that they were identical.

(Example 27: Insertion of β-actin-gfp-bβ-actin-puro into an unknown EST)
PGCs from parental cell line 54 were grown and 5 × 10 6 cells were electroporated with 20 μg of linearized β-actin-GFP-β-actin-puro construct and 48-1 cm 2 A single clone was obtained by plating in a well. Cells were grown in the presence of puromycin resulting in growth of only puromycin resistant clones. Transferred to new wells to expand resistant clones. The cells were analyzed by Southern blot analysis to establish stable integration of the transgene, and by sequencing, the construct was integrated into chromosome 8 of a new gene (EST C0769951).

  PGC constructs were grown and injected into the vasculature of stage 13-16 (H) H recipient embryos. Embryos were hatched and 8 roosters were grown to sexual maturity and tested for germline transmission. Rooster germline transmission rates were 0, 1, 11, 12, 13, 16, 28, and 92%, respectively. Heterozygous offspring from these roosters were grown to sexual maturity and mated to obtain homozygous offspring (Table 12).

Eight breeding pairs of heterozygous roosters and hens produced a total of 298 chicks, which were evaluated for the presence of the BGBP transgene. A total of 90 (30%) chicks were wild-type, 128 (46%) chicks were heterozygous, and 80 (25%) chicks were homozygous. These results are consistent with the expectation of 25% wild-type progeny, 50% heterozygous progeny, and 25% homozygous progeny, indicating that the transgene was inherited with Mendelian function . The majority of homozygous offspring died by 95% of all deaths at or near the time of hatching by 6 weeks of age. These results indicate that the insertion of the BGBP transgene resulted in a gene functional knockout essential for survival.

  To circumvent this problem, it is advantageous to insert the transgene at a predetermined location rather than a random uncontrolled insertion. The ability to insert a transgene at a known location in the genome has further potential advantages over random insertion. A transgene inserted for the purpose of overexpression of a protein product can be inserted where it is known to allow high levels of expression and is not subject to silencing due to heterochromatin invasion. Furthermore, it can be expected that transgene insertions will not cause deleterious effects on animals or cell lines in either a heterozygous state or a homozygous state. It is therefore unnecessary to screen a large number of different random insertions to find one with a high level of expression, and it does not interfere with important endogenous genes.

  (Example 28. Production of a transgenic bird carrying a conditional apoptosis-inducing gene (Reaper)).

Design of Reaper Transgene A Reaper transgene (loxP-stop-loxP-Reaper construct) was generated as follows. Reaper cDNA melanogaster embryo poly (A) + RNA and RAPER F1
It was cloned by RT-PCR using (CAC CAG AAC AAA GTG AAC GA SEQ ID NO: 38) primer and Reaper F2 (TGT TTG ACA AAA AAT TGA TGC) primer (SEQ ID NO: 39). The Reaper cDNA was inserted into the RI site of the CX-backbone generating the CX-Reaper construct. A KpnI site was inserted 3 'of the starter codon Reaper cDNA by site-directed mutagenesis. A 1.5 kb loxP-stop-loxP cassette from pBS302 (Gibco / BRL) was cloned at the KpnI site to generate CX-LoxP-stop-loxP-Reaper. The loxP-stop-loxP-Reaper fragment was inserted into the pENTRB2 clone (Invitrogen) using RI and NotI sites. The loxP-stop-loxP-Reaper fragment was then recombined into pLenti6 / UbC / V5-DEST (Invitrogen's pLenti Gateway vector), which makes a UbC-loxP-stop-loxP-Reaper construct.

Production of transgenic birds using the ViraPower lentivirus expression system To generate a lentivirus carrying the loxP-stop-loxP-Reaper transgene, the ViraPower lentivirus expression system (Invitrogen) was used, and 4.8 x 109 cfu / High titers up to ml were produced. For virus production, 293T cells were co-transfected using lipofectamine with a viperpower packing mix containing the UbC-loxP-stop-loxP-Reaper construct and VSV-G encoding the plasmid. Viral supernatant was collected 24 hours after transfection and concentrated by centrifugation. Viral titer was measured by transducing HT1080 cells with viral supernatant. High titers were obtained to ensure high transduction and germline transmission efficiency.

  To infect chicken embryos with virus, 1.5 ul of concentrated virus solution was injected into the subembryonic space of stage X embryos. After 3 days incubation at 37.5-38 ° C., embryos were transferred to another surrogate shelf and incubated at 37.5-38 ° C., 50% humidity until hatched. A total of 398 embryos were injected with virus carrying the UbC-loxP-stop-loxP-Reaper transgene. 155 birds hatched and the presence and sex of the transgene was analyzed by PCR of DNA isolated from chicken crown tissue. Thirteen male chicks were positive for the UbC-loxP-stop-loxP-Reaper transgene. Also, 10 of them were positive for the UbC-loxP-stop-loxP-Reaper transgene by PCR of DNA isolated from semen. Three males (6-03, 6-51 and 9-51 GO founder males) transmitted the transgene to the next generation. The transmission frequencies for 6-03, 6-51 and 9-51 were 0.32%, 0.26% and 0.16%, respectively (Table 13).

G0 founder males (6-03, 6-51 and 9-51, carrying different inserts) were raised to breeding hens. Those G1 progeny were then analyzed for the presence of the transgene and the site of integration of the transgene. Genomic DNA from individual birds was analyzed by Southern blot analysis. Genomic samples and UbC-loxP-stop-loxP-Reaper vector (control) were digested with SphI or Bc1I. The digested DNA was separated on a 0.7% agarose gel, blotted on a nylon membrane, and probed with a radiolabeled Reaper specific probe to identify the ligation fragment. As shown in FIG. 24, the size of the hybridizing genomic fragment is larger than the control, indicating that the transgene has been integrated. The hybridizing genomic fragments for strains 6-03, 6-51 and 9-51 had different sizes, indicating that 6-03, 6-51 and 9-51 are independent strains. Indicates that there is.

Example 29. Production of a chicken carrying a Cre-recombinant enzyme
Design and assembly of the Cre transgene In order to express Cre recombinase in chickens, a transgene was created in which the Cre gene was placed under the transcriptional control of the chicken ERNI promoter. The ERNI gene (also known as cENS-1) is an early chicken embryo (almost stage X, ie at the stage of a newly born egg, when the embryo is a cell undifferentiated sheet before gastrulation) and It is expressed in nerve tissue. Thus, the Cre transgene is designed to be expressed in early embryos, where it catalyzes recombination of loxP-reaper transgene loxP sites or other loxP-containing transgenes located in the genome. . Since Cre is expressed at an early stage, the resulting developing chicken should carry the recombinant transgene in all germ layers and cells of the body.

  In order to introduce the Cre transgene into the chicken germline, a lentiviral vector approach was taken. The lentiviral transgene was constructed based on the Invitrogen pLenti6-V5 Dest lentiviral vector. The lentiviral vector element of pLenti6-V5 Dest is combined with the ERNI-Cre gene to generate the pLenti-ERNI-Cre construct. A lentivirus was produced and used to infect early embryos, where it was stably integrated into the genome. Approximately 20 transgenic founder birds carrying the pLenti-ERNI-Cre transgene were generated.

  The chicken ERNI promoter was PCR amplified using the following primers:

The 822 bp PCR product was cloned into pGEM T-easy (Promega) and sequenced. The ERNI promoter was then released from the vector by digestion with SacII (subsequently blunted with T4 DNA polymerase) and SpeI. The CMV promoter was removed from the lentiviral vector pLenti6 V5-Dest (Invitrogen) by digestion with ClaI (subsequently blunted with T4 DNA polymerase) and SpeI. The ERNI promoter was then ligated into the pLenti6 V5-Dest lentiviral vector backbone, replacing the ERNI promoter therein with the CMV promoter, resulting in pLenti-ERNI.

  The Cre gene is PCR amplified using the following primers with the SV40 nuclear localization sequence at the N-terminus and convenient restriction sites for cloning (BglII at the 5 ′ end and EcoRI at the 3 ′ end): It was.

The 1040 bp PCR product was digested with BglII and EcoRI and the gel was purified. The shuttle vector pENTR 2B (Invitrogen) was digested with BamHI and EcoRI and the vector backbone was gel purified. The CrePCR product was ligated into pENTR 2B to obtain a clone. The clone was sequenced to determine that the Cre gene was expected and did not acquire any mutation during PCR amplification.

  To recombine the Cre gene into the pLenti-ERNI construct and place it under the transcriptional control of the ERNI promoter, the LR clonase reaction (Invitrogen) was used as a pENTR 2B-Cre clone as a Cre gene source and as a recipient. Of pLenti-ERNI vector. The final construct (pLenti-ERNI-Cre (8408 bp)) was obtained in this way and it was used to produce a lentivirus carrying the ERNI-Cre transgene.

Production of transgenic birds carrying the pLenti-ERNI-Cre transgene The pLenti-ERNI-Cre lentivirus was produced in 293FT cells. For each transfection into 293FT cells to produce lentivirus, 8 million 293FT cells at 75% confluence were transfected with 3 ug of circular pLenti-ERNI-Cre plasmid DNA using Lipofectamine reagent (Invitrogen). Was discharged. Virapower packaging mix (Invitrogen) was used to express the viral proteins necessary to make lentivirus in 293FT cells. Cell culture supernatant containing lentivirus was collected 2 days after transfection, cell debris was removed, and lentiviral particles were concentrated by centrifugation at 48,000 g for 90 minutes. The virus pellet was resuspended in 1/200 of the starting volume of the culture supernatant and 40 ul aliquots were frozen at -80 ° C.

  The infectious titer of each batch of lentiviral stock was measured in HT1080 cells by serially diluting virus stocks 10-4 to 10-8 and adding 1 ul of polybrene to the culture of HT1080. Blasticidin selection (5 ug / ml) was started 2 days after the addition of lentivirus. Since the cells died from blasticidin toxicity, the culture medium was changed every two days. Ten days after initiating blasticidin selection, colonies were stained with crystal violet, counted, and titers calculated. A titer of 108-2x109 was obtained. To infect chicken embryos with virus, 1.5 ul of concentrated virus solution was injected into the subembryonic space of stage X embryos. After 3 days incubation at 37.5-38 ° C., embryos were transferred to another surrogate shelf and incubated at 37.5-38 ° C., 50% humidity until hatched. A total of 310 embryos were injected with the virus carrying the Erni-Cre transgene. Ninety-six chicks hatched and were analyzed for the presence and sex of the transgene by PCR of DNA isolated from chicken crown tissue. Eight male chicks were positive for the Erni-Cre transgene. In addition, 13 males were positive for the Erni-Cre transgene by PCR of DNA isolated from semen. Of the 6 males tested, 4 transmitted the Erni-Cre transgene to the next generation. Germline transmission frequency for Erni-Cre G0 roosters varied between 0.24 and 1.32% (Table 14).

Transgenic chickens carrying the pLenti-ERNI-Cre transgene Southern blot analysis was used to demonstrate that the pLenti-ERNI-Cre transgene was fully integrated into the chicken genome. Genomic DNA from a chicken that was first identified by PCR to carry a Cre transgene is extracted and cleaves the viral 5 'and 3' LTR sequences so that a full-length complete transgene is observed. Digested with enzyme (BglII). Birds with independent transgene insertions were selected for analysis. Genomic DNA was digested with BglII enzyme, transferred to nylon membrane and probed with radiolabeled Cre gene. FIG. 25 shows representative Southern blotting of 8 ERNI-Cre strains. Of the 11 strains tested, 10 strains contained the expected 4.6 kb ERNI-Cre transgene band, indicating complete integration into the genome. One strain showed a smaller band, indicating transgene deletion or rearrangement.

(Example 30. Establishment of cell line capable of site-specific integration of transgene)
There are two main ways to insert external DNA at a predetermined location in the host genome: homologous integration (gene targeting) or site-specific recombination to a recognition site such as attP. Homologous recombination is inefficient in most vertebrate cell types and usually requires selection of many clones to identify one or a few clones with the desired insertion. Site-specific recombination is a highly reproducible and highly efficient process that can be used to insert external DNA into a predetermined site without selecting a large number of clones. Site-specific insertion relies on the use of phiC31 integrase to insert an attB-containing construct into a unique attP or pseudo-attP site in the genome. In order to use a genuine attP site located in the genome as a docking site, the attP site must be placed in a preferred, predetermined position. The attP site is placed in such a preferred position in the genome by random insertion or homologous recombination. If the recognition site is entered into the genome by random insertion, the insertion position must be confirmed to ensure that the important gene has not been destroyed. The recognition site placed in the genome is then used as a “docking site” for insertion of the transgene using phiC31 integrase.

  To select for integrase-mediated transgene insertion, particularly at the docking site, a selectable marker system is used to select the correct insertion. The docking site is designed so that the attP site is flanked by a drug selectable marker (eg, puromycin resistance gene) without a promoter. Cells that possess docking sites are thus sensitive to drug selection using puromycin. The transgene inserted into the docking site contains a promoter adjacent to its attB site, but does not contain a selectable marker. Insertion of the transgene into the docking site places a promoter upstream of the selectable marker and activates its transcription, conferring puromycin resistance. Insertion of the transgene at other locations in the genome does not lead to drug resistance, and such insertion is eliminated by drug selection.

  The attP docking site construct consists of an attP site placed adjacent to a promoterless drug selectable marker (eg, puromycin resistance, etc.). Since the puromycin resistance gene is not expressed, another selectable marker (such as the β-actin promoter that causes the neomycin selectable marker) must also be included. An EGFP gene can also be included. Each adjacent side of these elements is two copies of a β-globin HS4 insulator to sequester the construct from neighboring chromatin. The loxP site is placed adjacent to these elements for future removal of the β-actin neo and EGFP parts of the construct. All of these parts of the construct are used as a vehicle for delivery to the authentic attP site into the genome. The order of the DNA elements is as follows: HS4; attP; promoterless puromycin resistance gene; loxP; β-actin or CAG promoter; EGFP; β-actin or CAG promoter; neomycin resistance gene; HS4; (PBluescript). The construct is linearized, transfected into cultured PGCs, and drug resistant colonies are obtained. These colonies are expanded for further analysis.

  Since it is important to know where the docking site in the genome is located, the chromosomal insertion site of the docking site construct of each clone is determined. Flanking genomic DNA is obtained, sequenced, and compared to the chicken genome database. It can be seen that most clones are inserted into CpG islands, which are usually genomic regions associated with promoter regions of genes, particularly housekeeping genes or ubiquitous genes. Furthermore, most inserts are determined to be in the promoter region of the gene, the first exon of the gene, or the first intron. Thus, many of the insertions are predicted to impair the function of these genes (see Example 29; Table 8). These genes are either known genes based on the expressed sequence tag (EST) sequence or predicted genes. Preferred cell lines are those that do not appear to disrupt genes such as DOC1 or DOC33.

  The CAG-EGFP-CAG-neo part of the docking site can be deleted by Cre-lox recombination. After Cre-lox recombination, all that remains at the docking site is the HS4 insulator, the attP site, and the promoterless puro gene. This is the number of external proteins produced by the cells and transgenic chickens, which can affect chicken health, especially if expressed ubiquitously from strong promoters such as CAG or β-actin. Decrease. Cre-lox recombination can be performed in cell culture by transient transfection of a docking site clone with a circular Cre expression vector. After several days, the culture is monitored for loss of EGFP expression due to excision of the CAG-EGFP gene. About 50% of the cells no longer express EGFP, and these cells can be sorted by flow cytometry to purify them (FIG. 26).

  Alternatively, Cre recombination can be performed by crossing a transgenic chicken carrying a docking site construct with a chicken (Cre4 bird) carrying an ERNI-Cre transgene.

  When transgenic chickens with these docking site integrations are generated, the resulting homozygous chickens are healthy and fertile despite having an insert in the gene. An example of such a strain is the TP85 strain (also referred to as BN; see Example 26), which is an insertion into the gene encoding aldehyde dehydrogenase 3 family member A2 on chicken chromosome 19. The construct was an HS4-blocked β-actin neotransgene and was inserted into the promoter region within approximately 10 bp of the transcription start point of the gene. Birds that are homozygous for the insert are healthy and fertile.

  However, in some cases, insertion can cause adverse effects (eg, developmental disorders, anatomical or physiological disorders, infertility, etc.). See Example 27. It is therefore important to confirm the insertion of a randomly inserted docking site to confirm that it does not cause any adverse effects in the animal.

(Example 31: Establishment of a docking cell line consisting of more than 10.5 kb of expressed DNA)
The Doc-1 cell line carries a transgene consisting of HS4; attP; promoterless puromycin resistance gene; loxP; CAG promoter; EGFP; CAG promoter; neomycin resistance gene; HS4 (see Example 29). The construct was linearized and transfected into cultured PGCs to obtain drug resistant colonies. These colonies were expanded for further analysis. The Doc-1 cell line was injected into recipient embryos and hatched G0 chimeric chicks. Roosters were grown to sexual maturity and their sperm were analyzed by FACS analysis for the presence of GFP positive semen. Two roosters were selected for breeding and their germline transmission rates were 3% and 8%. Blood was collected from GFP-positive chicks and analyzed by Southern analysis to confirm the presence of the docking site.

(Example 32: Target insertion of docking site)
In order to avoid placing an authentic attP site on the CpG island of the gene, it is possible to use gene targeting to place the attP site at a predetermined site in the chicken genome. A region of the genome is selected, homology arms are prepared by highly accurate PCR or genomic cloning in a plasmid vector, and a targeting vector is assembled using a selectable marker for transfection or selection of PGC clones.

(Example 33: Site-specific insertion into a cell line carrying a docking site)
For insertion into the docking site, a circular construct containing the attB site is constructed. The attB site-containing construct is similar to that used in the above example, the important difference being the absence of a selectable marker. Instead, a promoter (eg, an ERNI promoter, etc.) is placed adjacent to the attB site so that upon incorporation into the docking site, the promoter is placed at a location that causes expression of the selectable marker at the docking site. Is done.

  The promoter attB backbone is used to select for insertion into the puro docking site without the attP-promoter. The attB construct carries other genes of interest such as, for example, tissue specific promoters that cause expression of genes encoding pharmaceutical proteins such as antibodies.

  The functionality of the docking site and the efficiency of integration at the docking site was tested in PGC cell lines containing the docking site. 5 × 10 6 cells were cotransfected with 0.5 μg of the construct containing Erni-attB and 0.5 μg of the circular construct expressing integrase. After electroporation, the cells were replated into 48-1 cm 2 wells to obtain single colonies. Colonies were observed in 42 out of 48 wells.

PCR revealed that the ERNI-attB construct was correctly integrated into the docking site by amplification of the attL site generated by recombination of attB with attP. One primer is in the ERNI sequence, one primer is in the puromycin sequence, and amplification can only occur if the ERNI promoter is incorporated upstream of the puromycin gene. Three primer sets were used and all gave positive results:
ERNI-37F + puro-8R product size 152bp
ERNI-133F + puro-8R product size 248 bp
ERNI-342F + puro-83R product size 522 bp

Four independent clones from ERNI-attB transfected into DOC2 cells were tested by PCR and all four showed the correct size amplification product with the three primer sets. The PCR product generated with Erni-133F (SEQ ID NO: 32) + puro-8R (SEQ ID NO: 33) primer was cloned and sequenced to prove that the PCR product was correct. The sequence matched the expected attL sequence (a combination of attB and attP) and included the expected partial ERNI and puromycin sequences. An integrase-mediated recombination crossover event thus occurred at the correct core nucleotide at the attB and attP sites and was demonstrated as authentic.

(Example 34: Cre efficiently recombines LoxP sites in chicken embryos)
Ten Cre strains with the complete pLenti-ERNI-Cre transgene (and one strain with a rearranged ERNI-Cre transgene) were tested for Cre recombinase activity. The ERNI promoter was expected to cause high level expression of Cre recombinase in early embryos, but if the transgene was accidentally integrated into an undesired region of the genome (a phenomenon known as “positional effects”) The transgene cannot be expressed. Therefore, it was important to measure the activity of Cre recombinase in all of our Cre strains in order to select one or more strains with the desired level of activity.

  To measure our Cre transgene activity levels, we catalyze recombination of loxP-Reaper transgenes in double transgenic embryos carrying one copy of the Cre transgene and one copy of the loxP transgene. Cre's ability to analyze was analyzed by Southern blot. The loxP-Reaper transgene contains a 1.4 kb sequence, called the STOP cassette, and is flanked by loxP sites in the same direction. Recombination between the two loxP sites results in a 1.4 kb intervening sequence excision from the chromosome, leaving behind a single loxP site. The intervening sequence is then lost because it is no longer linked to the chromosome. After excision, the loxP-Reaper transgene is reduced in size by 1.4 kb. Southern blot analysis evaluation has been developed, where a reduction in the size of the loxP-Reaper transgene is used to measure Cre recombinase activity. When hybridized with a probe consisting of the Reaper gene and the lentiviral vector backbone (blasticidin gene and SV40 sequence), digestion with the restriction enzyme SacI results in an approximately 2.8 kb full length (non-recombinant) loxP-Reaper fragment. Produce. Upon Cre-mediated recombination and excision of the 1.4 kb STOP sequence, the size of the SacI fragment is reduced to about 1.4 kb when hybridized with the same probe. The probe hybridizes to a sequence that is not affected by Cre recombination, so it hybridizes equally to both the full-length and recombined loxP-Reaper transgene.

  To estimate the level of Cre activity expressed by the various pLenti-ERNI-Cre transgenic lines, the ratio of full-length (non-recombinant) and recombinant transgene band intensities is measured. If Cre is not active, little or no recombinant Reaper is observed and only the full length is observed. If Cre is moderately active, both SacI fragments are observed, indicating that recombination has occurred in some cells but not in other cells. If Cre was very active, only the loxP-Reaper transgene was observed because the recombinant loxP-Reaper transgene was recombined in every cell.

  Data summarizing the activity of the Cre strain is shown in FIG. The Cre activity of the 11 strains tested was very variable between strains and in some cases varied similarly within the strain. Only 1 out of 11 strains catalyzed 100% recombination of the loxP-Reaper transgene (Cre4 strain). In this strain, all embryos tested (18 of 18) showed 100% recombination. In other strains, recombination levels varied from about 5% up to about 80%. There was considerable variability at the level of recombination from embryo to embryo for some of these strains (Cre1, Cre2, Cre11 and Cre20). For example, Cre11 and Cre20 catalyzed only about 10% recombination in some embryos, but up to 60% recombination in other embryos. For most embryos, brain and skeletal muscle tissue were analyzed and the level of recombination was similar in both tissues in all cases. Since the ERNI promoter is believed to be active in neural tissue, it was expected that the brain would show increased levels of recombination, but brain and muscle recombination was always about the same. It is unclear whether the observed changes in Cre recombinase activity are due to changes in the level of Cre expression or due to changes in the length of time that Cre activity is expressed. For example, in the Cre4 strain, Cre is constantly expressed at low levels, leading to recombination that is accumulated up to day 15 of 100% development, while other strains are in early stages before recombination occurs in any cell. There is a possibility that Cre expression does not occur. Alternatively, Cre4 can express very high levels of Cre protein only during early development and catalyzes 100% recombination.

  The only strains that did not show recombination were those with rearranged or deleted transgenes.

Example 35: Successful recombination of three chicken strains carrying different rearper inserts
To demonstrate that the Cre recombinase enzyme in pLenti-ERNI-Cre transgenic chickens can catalyze the recombination of different loxP substrates, the Cre4 strain was divided into three different loxP-Reaper strains (6-03, 6-51 9-51). The Cre4 strain was selected because it previously showed 100% recombination. Embryos that inherited one of the loxP-Reaper transgenes and a copy of the Cre4 transgene were selected.

  Southern to measure the ability of Cre to catalyze the recombination of double transgenic embryos carrying one copy of the Cre4 transgene and one copy of the loxP-Reaper transgene to measure the level of recombination of the three loxP-Reaper transgenes Analyzed by blot. The loxP-Reaper transgene contains a 1.4 kb sequence (also called a STOP cassette) and is flanked in the same direction. Recombination between the two loxP sites results in excision of the 1.4 kb intervening sequence from the chromosome, leaving behind a single loxP site. The intervening sequence is then lost because it is no longer linked to the chromosome. After excision, the loxP-Reaper transgene is reduced in size by 1.4 kb. A Southern blot assay has been developed, in which the reduction in loxP-Reaper transgene size is used to measure Cre recombinase activity. When hybridized to a probe consisting of the Reaper gene and the lentiviral vector backbone (blasticidin gene and SV40 sequence), digestion with the restriction enzyme SacI results in a full length (non-recombinant) loxP-Reaper fragment of about 2.8 kb. Produce. Upon Cre-mediated recombination and excision of the 1.4 kb STOP sequence, the size of the Reaper SacI fragment is reduced to about 1.4 kb when hybridized to the same probe. The probe hybridizes to a sequence that is not affected by Cre recombination, so it hybridizes equally to both the full-length and recombined loxP-Reaper transgene.

  In order to evaluate the amount of Cre-lox recombination in each of the LoxP-Reaper strains, the ratio of band intensity to the full-length (non-recombinant) recombinant transgene is measured. If Cre4 is able to excise the STOP cassette in all three Reaper strains, then only the recombinant Reaper band is observed, which means that the LoxP-Reaper transgene is recombined in all cells. This is because the. The results are shown in FIG. 29, which shows that all three Reaper strains undergo 100% excision of the STOP cassette in the presence of the Cre4 transgene.

(Example 37: Cre-mediated excision of EGFP and neo from a docking site integrated into a chicken genome)
Cre recombination can be performed in cultured PGCs in vitro and likewise in transgenic birds. To perform Cre-lox recombination in cultured PGCs, cells are transiently transfected with a Cre expression vector.

  DOC2 cells were used to transfect with the Cre expression vector. This PGC strain carries a docking site construct integrated into chromosome 21 with a CpG island bound to Prkz and several ESTs. All of the cells in the starting DOC2 culture were green fluorescent because the cells carry the CX-EGFP gene in the docking site construct. Two Cre expression vectors were used: pBS185 with the Cre gene under the transcriptional control of the human CMV promoter, or the ERNI-Cre construct where the ERNI promoter causes Cre expression.

  The Cre expression construct was transiently transfected into DOC2 cells. Several days later, the culture was monitored for loss of green fluorescence, and it was between loxP sites on the docking site vector where the cells picked up the Cre construct, expressed Cre, and Cre contains CX-EGFP-CX-neo. Was considered an indicator that caused the sequence to be cut out. After Cre transfection, the culture consisted of green and non-green cells. To purify the two populations, cultures were sorted by flow cytometry based on green fluorescence. Millions of cells from each population (green and non-green) were collected.

  Southern blot analysis was used to demonstrate that the EGFP gene was excised into a non-green population of cells (see: FIG. 30). Genomic DNA from two populations of cells (green and non-green) was prepared and digested with HindIII restriction enzyme. The DNA was fractionated on an agarose gel, transferred to a nylon membrane, and hybridized to a radiolabeled sequence from the puromycin resistance gene present at the docking site. The puro gene is in the region of the docking site construct that is not excised by Cre-lox recombination, so the puro probe detects fragments of genomic DNA from both DOC2 excised and non-excised cells. The predicted size HindIII fragment is as follows: EGFP + (uncut), 5521 bp; EGFP- (cut), 1262 bp. The expected fragment was observed and in non-green cells Cre-lox recombination resulted in the deletion of the CX-EGFP-CX-neo sequence present between the two loxP sites in the docking site construct integrated into DOC2 cells. Showed that.

Example 38: Preparation of IgL pKO5B targeting vector
In order to target the chicken IgL locus, a targeting vector was prepared that lacked the endogenous J and C regions of the locus during targeted recombination (FIG. 31). The vector was the same as previously described IgL pKO5 except that the selectable marker was changed. The 5 ′ homology region of the vector consisted of a 2327 bp fragment near the IgLV region, and the 3 ′ homology region consisted of a 6346 bp fragment from the downstream of the C region. The homology arm was cloned from isogenic DNA obtained from the cell line used during targeting transfection. The targeting construct included one or more methods to prevent expression (eg, stop codon, nonsense sequence, attP site or combinations thereof). The vector also included a selectable marker gene and a site-specific recombination site.
HS4 ERNI-neo: The 804 bp neomycin resistance gene was placed under transcriptional control of the 800 bp ERNI promoter for expression in PGCs. ERNI expression in chickens is restricted to very early embryos, so the selectable marker should not be expressed in mature chickens. The 250 bp core HS4 insulator element from the chicken β-globin locus was tandemly replicated and the replicated insulator was placed on either side of the ERNI-neo selectable marker. A single loxP site (for Cre-mediated recombination) was cloned upstream of HS4-ERNI-neo.

attP-puro: The 600 bp puro gene was linked to a 43 bp attP site (for phiC31 mediated recombination) without a promoter. Then attP-puro was cloned downstream of HS4-ERNI-neo. Together, the loxP-HS4-ERNI-neo-attP-puro selectable marker cassette is 4089 bp.

5 'Homologous Arm: The 2327 bp fragment is

Is used to generate a 1994 bp NcoI-BamHI fragment from the PGC35 IgL SacI clone + 333 bp NotI-Ncol PCR product replicated from PGC35 genomic DNA. The PCR fragment contained an overlapping genomic NcoI site joining the two fragments.

The resulting 2327 bp fragment was released upstream of HS4 ERNI-puro using NotI and BamHI for cloning into the pKO vector backbone. The NotI site was not present in the genome and was added by PCR.
3'-Homologous Arm: A 6346 kb SpeI-BglII fragment was generated by ligating together the following three fragments: a SpeI-EcoRI fragment from the SacI genomic clone, and an EcoRI- from the EcoRI-MfeI genomic clone. ApaLI clone and 300 bp ApaLI-BglII PCR fragment replicated from EcoRI-MfeI clone

The BglII site was not present in the genome but was added by PCR. The 3′-homologous arm was cloned into the pKO vector backbone between HS4 ERNI-puro and HS4 b-actin EGFP.
HS4 b-actin EGFP: The 1.3 kb chicken b-actin promoter was used to cause expression of the 700 bp EGFP gene. One copy of the replicated HS4 insulator was added to the end to sequester the randomly inserted targeting vector from the position effect to allow EGFP expression.

  The final IgL pKO5B targeting vector had a size of 17,681 bp and was linearized with NotI before transfection into PGC.

Example 39 PGC Transfection and Generation of KO-07 IgL Knockout PGC Cell Line
38 aliquots of 100 μl of 5 × 10 6 cells of electroporation buffer (V buffer from Amaxa) were transfected with 10 μg of DNA each. All aliquots were electroporated with Amaxa nucleofector pulse A33 (Amaxa). Nine clones were obtained, 4 of which were GFP positive and were not followed further. Five GFP non-expressing clones were expanded for Southern analysis and designated KO-07, 08, 09, 10 and 11.

(Example 40: Southern blot analysis)
For the 5 ′ side of homologous recombination, genomic DNA from 5 clonal PGC strains transfected with IgL pKO5B was digested with SacI restriction enzyme and fractionated on a 0.7% agarose gel. The DNA was transferred to a nylon membrane and hybridized to a probe from the chicken IgL locus upstream from the region used as a homology arm (ie, exogenous probe). The probe is a 0.5 kb SacI-BstEII fragment that detects a wild type fragment of about 10 kb and a mutant fragment of about 4 kb (FIG. 32, left panel). For the 3 ′ side of targeting, genomic DNA was digested with BstEII and the blot was hybridized to the 3 ′ 1.7 kb NsiI-MfeI fragment (also outside the targeting vector) (FIG. 32, right panel). ).

Example 41 Production of Germline Chimeras
3000 PGCs were injected into the dorsal aorta for each stage 15-16 (Hamburger & Hamilton) embryo. Embryos were incubated on a surrogate shelf. The hatched chicks were grown to sexual maturity.

  Chimeric roosters were mated with wild-type Barred Rock hens by artificial insemination. Semen was collected from 9 roosters and fertilized. Six of the roosters transmitted the black feather phenotype to offspring and showed germline transmission of IgL knockout PGCs (Table 1). One of the roosters (IV75-41) transmitted at a rate exceeding 50%.

Example 42 Southern Blot Analysis of Knockout Embryos
Black feather embryos on day 14 of development were euthanized and genomic DNA was prepared from skeletal muscle. Southern blots were performed to show that knockout was transferred to 5 of the 7 embryos tested in the experiment (embryos 2, 3, 4, 6 and 7). Embryo 1 and Embryo 5 are wild type embryos, which inherited the wild type IgL allele from the heterozygous targeted KO-07 knockout PGC (FIG. 33).

Claims (25)

A transgenic chicken derived from a cloned transfected chicken primordial germ cell (PGC), wherein the genome of the PGC and the genome of the transgenic chicken are J and C gene segments of an endogenous immunoglobulin light chain gene A targeting construct that disrupts and an exogenous promoter operably linked to the transgene, the targeting construct being located between the two homologous regions for the endogenous immunoglobulin light chain gene and the homologous region A transgenic chicken comprising a selectable marker. The transgenic chicken of claim 1, wherein the targeting construct inserts a stop codon, an att-p site, a nonsense sequence, or a combination thereof into the endogenous immunoglobulin light chain gene. The transgenic chicken of claim 1, wherein the targeting construct is comprised of a positive selectable marker. The transgenic chicken of claim 1, wherein the targeting construct is comprised of a negative selectable marker. The transgenic chicken of claim 1, wherein the targeting construct is comprised of at least one insulator. The transgenic chicken according to claim 5, wherein the insulator is a chicken β-globin 5'HS4 insulator. 2. The transgenic chicken of claim 1, wherein the targeting construct is comprised of an early response to neural induction (ERNI) promoter. 2. The transgenic chicken of claim 1, wherein the endogenous immunoglobulin light chain gene comprises a deletion of at least 10 kb. A method for producing a transgenic chicken comprising:
The targeting construct is integrated into the cultured chicken PGC such that the targeting construct is integrated into the genome of the cultured chicken primordial germ cell (PGC) and destroys the J and C regions of the endogenous immunoglobulin light chain gene, thereby Generating a transformed PGC, the targeting construct comprising a nucleotide sequence encoding the J and C segments of the immunoglobulin light chain and an exogenous promoter used to drive expression of the transgene And a targeting sequence comprising two homologous regions for the endogenous immunoglobulin light chain gene and a selectable marker located between the homologous regions A process comprising:
Selecting a cloned transformed PGC;
Inserting the cloned transformed PGC into a recipient chicken embryo; and
Hatching a transgenic chicken from the embryo, the chicken comprising the targeting construct that disrupts the J and C regions of an endogenous immunoglobulin light chain gene and the promoter operably linked to the transgene Comprising germline cells derived from said cloned transformed PGC comprising: and
Mating a chimeric chicken to produce a transgenic chicken, wherein the genome of the transgenic chicken is derived from the germline cell and disrupts the J and C regions of the endogenous immunoglobulin light chain gene The targeting construct and the promoter operably linked to the transgene.
Including the method. 10. The method of claim 9, wherein the targeting construct inserts a stop codon, nonsense sequence, att-p site, or a combination thereof into the endogenous immunoglobulin light chain gene. The method of claim 9, wherein the targeting construct is comprised of a positive selectable marker. The method of claim 9, wherein the targeting construct is comprised of a negative selectable marker. The method of claim 9, wherein the targeting construct is comprised of at least one insulator. 14. The method of claim 13, wherein the insulator is a chicken [beta] -globin 5'HS4 insulator. 10. The method of claim 9, wherein the targeting construct is composed of an early response to neural induction (ERNI) promoter. At least one clone-derived chicken primordial germ cell (PGC) comprising in its genome a targeting construct that disrupts the J and C gene segments of the endogenous immunoglobulin light chain gene and an exogenous promoter operably linked to the transgene. × 10 ⁵ Wherein the targeting construct is composed of two homologous regions for the endogenous immunoglobulin light chain gene and a selectable marker located between the homologous regions. 17. The culture of claim 16, wherein the PGC is derived from whole blood of stage 12-17 chicken embryos. 17. The culture of claim 16, wherein the targeting construct inserts a stop codon, an att-p site, a nonsense sequence, or combinations thereof into the endogenous immunoglobulin light chain gene. 17. The culture of claim 16, wherein the targeting construct is composed of a positive selectable marker. 17. A culture according to claim 16, wherein the targeting construct is composed of a negative selection marker. 17. A culture according to claim 16, wherein the targeting construct is composed of at least one insulator. The culture according to claim 21, wherein the insulator is a chicken β-globin 5'HS4 insulator. 17. The culture of claim 16, wherein the targeting construct is comprised of an early response to neural induction (ERNI) promoter. 10. A method for producing a protein according to claim 9, comprising the step of expressing the protein encoded by the nucleotide sequence of the transgene in the transgenic chicken according to claim 9. 25. The method of claim 24, further comprising isolating the protein expressed by the transgenic chicken.