JP7659828B2 - Method for producing transgenic pigs, somatic cell cloning material, transgenic pigs, established somatic cells, and tissues or organs - Google Patents

Description

The present invention relates to a method for producing transgenic pigs, somatic cell cloning materials used in the method, transgenic pigs, somatic cells established from the transgenic pigs, and tissues or organs extracted from the transgenic pigs.

There is a need to develop transgenic pigs that can express specific functional proteins in tissue- or organ-selective, and even time-selective, tissues or organs.

Since breeding or mating of pigs is complicated compared to rodents such as mice, the inventors initially attempted to produce transgenic pigs by introducing a vector containing both a control gene cassette for conditional gene expression and a functional gene cassette containing a gene encoding a functional protein into pigs.
However, the inventors have found that when a vector containing both a control gene cassette for conditional gene expression and a functional gene cassette containing a gene encoding a functional protein is introduced into a pig, it is difficult to produce a transgenic pig into which a group of genes for conditionally expressing the functional protein in a specific tissue or organ has been introduced (Comparative Example described below).

The present invention has been made in consideration of the problems of the prior art as described above, and the object of the present invention is to provide a method for producing transgenic pigs into which a group of genes for expressing specific functional proteins in a tissue or organ is selectively introduced, as well as the somatic cell cloning material, transgenic pigs, somatic cells established from the transgenic pigs, and tissues or organs extracted from the transgenic pigs.

As a result of intensive research into the above-mentioned problems, the present inventors have found that by dividing gene transfer using a functional gene cassette containing a gene encoding a functional protein and gene transfer using a vector containing a control gene cassette for conditional gene expression into two steps, it is possible to produce a transgenic pig into which a group of genes for conditionally expressing a functional protein in a tissue or organ have been introduced. In other words, it has been found that by constructing a genetically modified cloned pig into which a functional gene cassette containing a gene encoding a functional protein has been introduced, and then further gene-transforming the constructed genetically modified cloned pig with a vector containing a control gene cassette, it is possible to produce a transgenic pig into which a group of genes for conditionally expressing a functional protein in a tissue or organ have been introduced.
The present invention has been completed based on the above findings.
That is, the present invention is as follows.

<1> Sperm of a genetically modified cloned pig obtained by somatic cell cloning of a genetically modified pig cell obtained by introducing a gene cassette containing a gene encoding a functional protein and a construct capable of expressing the functional protein by a recombinase into a safe harbor region;
A method for producing a transgenic pig, comprising the step of introducing a gene into an egg by insemination with a liquid containing the vector (1),
The vector (1),
The production method includes a promoter that is expressed in a specific tissue or organ, and a gene encoding the recombinant enzyme that is located downstream of and under the control of the promoter that is expressed in the specific tissue or organ.
<2> The method according to <1>, wherein the safe harbor region is a ROSA region.
<3> The method according to <1>, wherein the vector (1) is a plasmid vector or an artificial chromosome vector.
<4> A somatic cell established from a transgenic pig produced by the method according to any one of <1> to <3> claims 1 to 3.

<5> A somatic cell cloning material comprising a genetically modified porcine cell containing a gene encoding a functional protein and a gene cassette containing a construct capable of expressing the functional protein by a recombinase in a safe harbor region,
A somatic cell cloning material for providing a material for gene transfer by intracytoplasmic sperm injection in the presence of the following vector (1).
(1) A vector comprising a promoter expressed in a specific tissue or organ, and a gene encoding the recombinase located downstream of the promoter expressed in a specific tissue or organ and under its control. <6> A vector comprising a promoter expressed in a specific tissue or organ, and a gene encoding the recombinase located downstream of the promoter expressed in a specific tissue or organ and under its control,
A transgenic pig comprising a gene encoding a functional protein, the transgenic pig comprising a gene cassette in a safe harbor region, the gene cassette comprising a construct that enables expression of the functional protein by the recombinant enzyme.
<7> A somatic cell established from the transgenic pig described in <6> above.
<8> A tissue or organ isolated from the transgenic pig described in <6> above.

The present invention provides a method for producing a transgenic pig into which a group of genes for conditionally expressing a specific functional protein in a tissue or organ selectively (preferably in a tissue or organ specific manner) has been introduced, as well as a somatic cell cloning material, a transgenic pig, somatic cells established from the transgenic pig, and a tissue or organ extracted from the transgenic pig.

FIG. 1 is a schematic diagram showing a gene cassette containing the loxP-Venus gene constructed in Reference Example 1. FIG. 2 is a schematic diagram showing the CreERT2 expression vector constructed in Reference Example 2. FIG. 3 is a schematic diagram showing a gene cassette containing the loxP-DTA gene constructed in Reference Example 3. FIG. 4 is an electrophoretic diagram showing the results of confirming the knock-in of the above gene cassette into the ROSA region. FIG. 5 shows the results of confirming induction of cell death by tamoxifen in genetically modified porcine cells in which a gene cassette containing the loxP-DTA gene was introduced into the porcine ROSA region. FIG. 6 shows electrophoretograms of the sex determination results. FIG. 7 shows an electrophoretic diagram showing the results of gene introduction evaluation. FIG. 8 is an electrophoretogram showing the results of confirming CreERT2 expression in fetal metanephroi. FIG. 9 shows the results of transgene copy number determination in individuals (diploid genome). FIG. 10 is a schematic diagram showing an all-in-one vector 1 containing both the mSix2 promoter and the CreERT2 and loxP-DTA genes as a comparative example. FIG. 11 is a schematic diagram showing an all-in-one vector 2 containing both the mSix2 promoter and the ERT2CreERT2 and loxP-DTA genes as a comparative example.

The following describes in detail the embodiments of the present invention, but the present invention is not limited to the following embodiments and can be modified as appropriate within the scope of the present invention.

<Production method for transgenic pigs>
The first aspect of the present invention relates to a method for producing a pig that is capable of expressing a functional protein by a recombinant enzyme, comprising: introducing a gene cassette containing a construct into a safe harbor region to produce a genetically modified pig cell; and a spermatozoa of a genetically modified cloned pig obtained by somatic cloning of the genetically modified pig cell.
The present invention relates to a method for producing a transgenic pig, the method including the step of introducing a gene into an egg by microinjection with a liquid containing the vector (1).
(1) A vector comprising a promoter that is expressed in a specific tissue or organ, and a gene encoding the recombinase that is located downstream of and under the control of the promoter that is expressed in a specific tissue or organ.

The transgenic pig produced by the production method according to the first aspect has undergone gene transfer (first gene transfer) of the gene cassette into the safe harbor region and second gene transfer by the vector (1) via the in vitro fertilization, and can also be called a double transgenic pig (double genetically modified pig).
In the present invention, the transgenic pig is preferably a transgenic pig that can conditionally express a specific functional protein in a tissue or organ selectively.

The transgenic pig produced by the production method according to the first aspect comprises a gene encoding a functional protein, and a gene cassette including a construct that enables the functional protein to be expressed by a recombinase.
In addition, the transgenic pig produced by the production method according to the first aspect comprises a promoter that is expressed in a specific tissue or organ, and a gene encoding the recombinant enzyme that is located downstream of the promoter and under its control.
Thereby, by coexisting the recombinant enzyme with the construct (preferably within the cell, more preferably within the nucleus), the functional protein can be conditionally expressed (preferably, dependent on the expression of the recombinant enzyme).
In other words, in the production method according to the first aspect, a liquid containing a vector (1) containing a gene encoding the recombinant enzyme is microinjected into an egg, thereby producing a transgenic pig that is capable of conditionally (preferably, dependent on the expression of the recombinant enzyme) expressing the functional protein.
As a result, the transgenic pig produced by the production method according to the first aspect can express the functional protein in a tissue or organ selectively (preferably, tissue or organ specifically) in the tissue or organ.

In pigs, a large amount (high copy number) of control gene cassettes is required for conditional gene expression, whereas a small amount (low copy number) of functional gene cassettes containing genes encoding functional proteins is sufficient. When sufficient gene transfer (transfection) is achieved to achieve conditional expression by the control gene cassette, the copy number of the functional gene cassette is too large, and the quantitative balance between the control gene cassette and the functional gene cassette is not adequate for pigs. The inventors speculate that this is a problem of the prior art that makes it difficult to produce the transgenic pigs. The inventors also speculate that the occurrence of non-specific expression (leakage) of the functional gene cassette is also a reason for the problem of the prior art that makes it difficult to produce the transgenic pigs.

More specifically, for example, when the integrated copy number of an all-in-one vector (see Comparative Example described below) containing both the control gene cassette and the functional gene cassette is low, the expression level of the gene that expresses a recombinase in a tissue-specific manner becomes insufficient, and as a result, the expression of the gene encoding the functional protein becomes insufficient.
On the other hand, if the integrated copy number of an all-in-one vector containing both the control gene cassette and the functional gene cassette is high, the expression of the gene encoding the functional protein will be excessive due to leakage, which will affect the efficiency of producing transgenic pigs.

In the transgenic pig produced by the production method of the first aspect and the transgenic pig produced by the transgenic pig of the third aspect described below, the copy number of the gene encoding the functional protein is not particularly limited, but may be, for example, one or more copies per pig individual (diploid genome), preferably two or more copies, and more preferably four or more copies.
There is no particular upper limit on the number of copies of the gene encoding the functional protein, but examples of this include 200 copies or less, 100 copies or less, or 550 copies or less per pig individual (diploid genome).

In the transgenic pig produced by the production method of the first aspect and the transgenic pig of the third aspect described below, the copy number of the gene encoding the recombinant enzyme is not particularly limited, but may be, for example, 1 or more copies per pig individual (diploid genome), preferably 2 or more copies, more preferably 4 or more copies, even more preferably 7 or more copies, particularly preferably 15 or more copies, and particularly preferably 30 or more copies.
There is no particular upper limit to the number of copies of the recombinase, but examples of the upper limit include 500 copies or less, 300 copies or less, and 100 copies or less per pig individual (diploid genome).

In the transgenic pig produced by the production method according to the first aspect and the transgenic pig according to the third aspect described below, the ratio of the copy number of the gene encoding the functional protein to the copy number of the gene encoding the recombinant enzyme is, for example, 1/2 to 1/300, preferably 2/3 to 2/100, more preferably 3/5 to 3/50, and even more preferably 4/6 to 4/20.

In the first aspect, the construct is not particularly limited as long as it contains a gene encoding the functional protein and the functional protein can be expressed by a recombinant enzyme, and examples of the construct include nucleic acid constructs such as DNA constructs, cDNA constructs, and RNA constructs (preferably gene constructs).
In a first aspect, the construct preferably comprises a specific sequence that is selectively (preferably specifically) recognized by the recombinase, and more preferably comprises at least one pair of specific sequences that are selectively (preferably specifically) recognized by the recombinase.
As a result, the recombinase selectively recognizes at least one pair of specific sequences (preferably at least one pair of specific sequences), thereby enabling conditional (preferably dependent on expression of the recombinase)) recombination to occur.

Combinations of the above-mentioned recombinase and specific sequences (preferably at least one pair of specific sequences) selectively recognized thereby include the following combinations (a) to (d), of which the following combination (a) of Cre recombinase and at least one pair of Lox sequences is preferred.
(a) a combination of Cre recombinase and at least one pair of Lox sequences;
(b) A combination of the recombinase FLP derived from Saccharomyces cerevisiae and the following FRT sequence (Broach, JR, Guarascio, VR, & Jayaram, M. (1982). Recombination within the yeast plasmid 2mu circle is site-specific. Cell, 29(1), 227-34.)
FRT sequence: 5'-GAAGTTCCTATTCTCTAGAAAGTATAGGAACTTC-3' (SEQ ID NO: 7)
(c) A combination of recombinant enzyme R and RS sequences derived from soy sauce yeast (Zygosaccharomyces rouxii) (Araki, H., Jearnpipatkul, A., Tatsumi, H., Sakurai, T., Ushio, K., Muta, T., & Oshima, Y. (1985). Molecular and functional organization of yeast plasmid pSR1. Journal of molecular biology, 182(2), 191-203.)
(d) Combination of the recombinase Gin and gix sequences derived from bacteriophage Mu (Maeser, S., & Kahmann, R. (1991). The Gin recombinase of phage Mu can catalyse site-specific recombination in plant protoplasts. Molecular & general genetics: MGG, 230(1-2), 170-6.)

As the specific sequence, the above Lox sequence is preferable, and among the above Lox sequences, a representative example is the 34-base loxP sequence derived from bacteriophage _P1 , which allows the construction of a so-called Cre-loxP system.
An example of a pair of loxP sequences is shown below.
5'-ATAACTTCGTATAGCATACATTATACGAAGTTAT-3' (SEQ ID NO: 1)
As described above, it is seen that the 13 bases at both ends, which are the Cre binding sites, are symmetrical, while the central 8 bases are asymmetrical.
loxP has a directionality, and if a pair of Lox sequences are arranged in the same direction (same orientation), the sequence between them is excised as a circular DNA by Cre. The excised circular DNA rarely returns to its original state, and the excision can be said to be substantially irreversible.
On the other hand, if a pair of Lox sequences are arranged in opposite directions (opposite orientations), the sequence between them is inverted (the orientation is swapped).

In the first embodiment, the at least one pair of Lox sequences is preferably 1 to 4 pairs of Lox sequences, more preferably 1 to 3 pairs of Lox sequences, and even more preferably 1 or 2 pairs of Lox sequences.
Representative examples of the at least one pair of Lox sequences include not only the above-mentioned loxP sequence, but also lox511 sequences, lox2272 sequences, loxFAS sequences, loxRE sequences, and loxLE sequences, and two or more pairs of these may be used in combination.

Examples of the lox511 sequence, the lox2272 sequence, the loxFAS sequence, the loxRE sequence, and the loxLE sequence are shown below. In the sequences below, the lowercase letters indicate bases that differ from the loxP sequence.
lox511: 5'-ATAACTTCGTATAGtATACATTATACGAAGTTAT-3' (SEQ ID NO: 2)
lox2272: 5'-ATAACTTCGTATAGgATACtTTATACGAAGTTAT-3' (SEQ ID NO: 3)
loxFAS: 5'-ATAACTTCGTATAtacctttcTATACGAAGTTAT-3' (SEQ ID NO: 4)
lox RE: 5'-ATAACTTCGTATAGCATACATTATACGAAcggta-3' (SEQ ID NO: 5)
lox LE: 5'-taccgTTCGTATAGCATACATTATACGAAGTTAT-3' (SEQ ID NO: 6)

It is known that specific recombination occurs between loxP-loxP, between lox511-lox511, and between lox2272-lox2272, while recombination does not occur between loxP-lox511 or between loxP-lox2272.
By utilizing this specific recombination property, two or more pairs of Lox sequences (placed in the opposite direction between each pair of sequences) can be used to place a target gene (a gene encoding a functional protein) in the opposite direction (inactive type), and then cause "irreversible" recombination with Cre to activate it by exon flipping, making it a FLEx (Flip-excision) switch (Experimental Medicine, November 2014, Vol. 32, No. 18). This overcomes the reversibility of inversion when a pair of Lox sequences is placed in the opposite direction (inverse direction).

When using a FLEx switch, examples of combinations of two pairs of Lox sequences include a combination of one pair of loxP and one pair of lox2272, or a combination of one pair of loxP and one pair of lox511.

In addition to the FLEx switch, an embodiment using a pair of Lox sequences will be described below.
In the above gene cassette, a gene of interest (a gene encoding a functional protein) can be arranged in an active orientation, and a STOP sequence flanked by a pair of Lox sequences arranged in the same direction (same orientation) can be arranged upstream of the gene of interest.
Since a pair of Lox sequences are arranged in the same direction (same orientation), the STOP sequence is essentially irreversibly excised by Cre, allowing the expression of a target gene (a gene encoding a functional protein).
The STOP sequence is not particularly limited as long as it is a sequence for terminating transcription, but examples thereof include the SV40 poly A signal sequence, and a sequence in which three SV40 poly A signal cassettes are linked is preferred.

From the viewpoint of preventing non-specific recombination (leakage), a Pause sequence may be included in addition to the STOP sequence as a sequence sandwiched between a pair of Lox sequences arranged in the same direction (same orientation) (Floxed), and it is preferable that a Pause sequence is included downstream of the STOP sequence.
The pause sequence is not particularly limited, and examples thereof include the human α2 globin gene RNA polymerase II transcriptional pause signal sequence.

In the production method according to the first aspect, the recombinant enzyme is preferably a fusion protein with a nuclear transport protein, i.e., the gene encoding the recombinant enzyme is preferably fused directly or indirectly with a gene encoding the nuclear transport protein.
The nuclear transport protein is not particularly limited as long as it can be transported into the nucleus, but examples thereof include proteins having a nuclear transport signal, and specifically, the estrogen receptor ER is preferred.
It is more preferable that the recombinase is Cre recombinase, and the gene encoding the recombinase is a gene encoding a fusion protein containing the Cre recombinase and the estrogen receptor ER.
The Cre recombinase is known as an enzyme (derived from bacteriophage _P1 ) that recognizes a specific base sequence (for example, a pair of two Lox sequences) and causes DNA homologous recombination.
The estrogen receptor ER is known to have a nuclear localization signal.
When the fusion protein is a fusion protein containing Cre recombinase and the estrogen receptor ER, a liquid containing the vector (1) is microinjected into an egg, whereby the Cre recombinase portion of the fusion protein recognizes at least one pair of Lox sequences, thereby producing a transgenic pig that undergoes conditional (preferably Cre expression-dependent) recombination.

In the production method according to the first aspect, it is more preferable that the estrogen receptor ER is ERT2 and the fusion protein is CreERT2 or ERT2CreERT2.
Tamoxifen compounds (tamoxifen, 4-hydroxytamoxifen (4-OHT), etc.) can bind to ER present in the cytoplasm and translocate into the nucleus.
ERT2 is known to be a protein that has been modified to bind ER only to tamoxifen compounds and lacks a nuclear localization signal (with estrogen).
Both CreERT2 and ERT2CreERT2 are fusion proteins of the above-mentioned ERT2 and Cre, and can translocate into the nucleus in the presence of a tamoxifen compound and cause recombination by Cre.
(Feil R, Brocard J, Mascrez B, LeMeur M, Metzger D and Chambon P (1996): Ligand-activated site-specific recombination in mice. Proc Natl Acad Sci USA, 93: 10887-10890. 1996.
Feil R, Wagner J, Metzger D and Chambon P (1997): Regulation of Cre recombinase activity by mutated estrogen receptor ligand-binding domains. Biochemical and Biophysical Research Communications, 237: 752-727. 1997.)
In a first embodiment, CreERT2 or ERT2CreERT2 can be used to induce conditional (eg, time-specific) recombination by tamoxifen compounds.
In the first and second aspects, said estrogen receptor ER is preferably ERT2, and said fusion protein is preferably CreERT2 or ERT2CreERT2.

(Genetically modified porcine cells containing a gene encoding the functional protein and a gene cassette containing a construct that enables the functional protein to be expressed by a recombinase in a safe harbor region)
The present invention is a somatic cell cloning material comprising a genetically modified pig cell containing a gene encoding the functional protein and containing, in a safe harbor region, a gene cassette containing a construct that enables the functional protein to be expressed by a recombinase, and the somatic cell cloning material is also an invention relating to a somatic cell cloning material for providing a material for gene introduction by intracytoplasmic sperm injection in the presence of the vector (1). A second aspect of the present invention is the somatic cell cloning material.

In the first and second aspects, the genetically modified porcine cells are preferably established (e.g., established strains or cloned) genetically modified porcine cells, from the viewpoints of stable culture and high cell homogeneity.
Furthermore, by establishing this system, it is possible to omit the various steps required prior to establishment, such as introducing a gene cassette into a safe harbor region, and to produce transgenic pigs in which a group of genes for conditionally expressing the functional protein in a specific tissue or organ has been introduced in a single step (e.g., essentially one step) of intracytoplasmic sperm injection, which makes it excellent for industrial mass production of transgenic pigs.

The above-mentioned genetically modified porcine cells are not particularly limited as long as they are porcine cells having a safe harbor region (preferably in the genome), but examples include fibroblasts, preadipocytes, embryo-derived pluripotent cells, somatic stem cells, induced pluripotent stem cells, etc., with fibroblasts being preferred and fetal fibroblasts being more preferred.

The safe harbor region into which the gene cassette is introduced in the first and second aspects refers to a region on the genome into which a transgene can be safely inserted, and examples of such regions include non-coding regions on the genome into which a transgene can be safely inserted.
As the safe harbor region, the porcine ROSA region or the H11 region is preferable, and the porcine ROSA region is more preferable.
The porcine ROSA region is not particularly limited as long as it is a ROSA region, but the ROSA region located between exons 1 and 2 of the 13th porcine chromosome is preferred.

In the first and second aspects, the functional protein can be expressed by the action of the recombinant enzyme in cells throughout the pig (systemic expression possibility) or in cells of a specific tissue or organ (localized expression possibility), but it is preferable that the functional protein be expressed at least in the "specific tissue or organ" defined by the vector (1).
Compared to rodents such as mice, for example, breeding or mating of pigs is complicated.
From the viewpoint that various gene-introduced pig variations capable of conditionally expressing the functional protein in each specific tissue or organ can be produced in a single step (e.g., substantially one process) of the above-mentioned microinsemination by preparing various vectors (1) containing promoters expressed in various tissues or organs and performing the above-mentioned vectors (1) in the presence of each of the various vectors (1), it is preferable that the functional protein can be expressed (systemically expressible) in cells throughout the entire body of the pig individual.
When the gene cassette containing the construct is introduced into a safe harbor region in the genome, it can be expressed in cells throughout the body of the pig (systemic expressibility).

In the gene cassettes of the first and second aspects, the functional protein is not particularly limited as long as it is a protein that exerts any function, and examples thereof include proteins that exert any function, such as a light-emitting (e.g., fluorescence, UV light) function, a labeling function (radioactive label, any tag), a gene expression regulating function, a gene recognition function, and a function that is lethal to a cell (e.g., acting on the cytoplasmic membrane to destroy it), and more specifically, examples thereof include fluorescent proteins (e.g., modified GFPs such as Green Fluorescent Protein (GFP) and Venus), metabolic enzymes (e.g., thymidine kinase, drug metabolizing enzymes (CYP)), cell proliferation-related factors (e.g., apoptosis regulator BAX, fibroblast growth factor FGF, etc.), growth-related gene proteins (e.g., growth hormone receptor, insulin-like growth factor receptor, etc.), cytotoxins, suicide gene proteins (e.g., iCaspase9, etc.), and the like.
Fluorescent proteins are generally known to be cytotoxic.
The cytotoxin is not particularly limited as long as it has a lethal effect on cells (e.g., acts on and destroys the cytoplasmic membrane), and is preferably a toxic substance with a high molecular weight, more preferably a toxic protein. Specific examples of the cytotoxin include diphtheria toxin, θ toxin, streptolysin S, α, δ toxin, leukocidin, etc., and is preferably diphtheria toxin, and more preferably diphtheria toxin A.
Diphtheria toxin is a bacterial toxin produced by the diphtheria bacterium, and consists of two domains, the A chain and the B chain. The A chain acts as the toxin itself, while the B chain binds to a receptor on the cell surface and takes the A chain into the cell.

In the gene cassettes of the first and second aspects, the gene encoding the cytotoxin includes the genes encoding the above-mentioned cytotoxins, preferably a gene encoding diphtheria toxin, more preferably a gene encoding diphtheria toxin A (hereinafter also simply referred to as "DTA").

In the gene cassettes of the first and second aspects, in order to allow the gene cassette to accept transcription by the promoter of the safe harbor region or a promoter located upstream of the safe harbor region, the gene cassette may or may not further include a splicing acceptor sequence (splice acceptor sequence) containing AG or the like, but it is preferable that the gene cassette further includes the splice acceptor sequence, and it is more preferable that the gene cassette further includes the splice acceptor sequence upstream of a "construct containing a gene encoding a functional protein and enabling the functional protein to be expressed by a recombinase," and it is even more preferable that the gene cassette further includes the splice acceptor sequence upstream of a "specific sequence and a gene encoding a functional protein."

In the gene cassette containing the above construct in the first and second aspects, in order to control the expression of the above functional protein, an arbitrary promoter may or may not be further included upstream of the gene encoding the functional protein (preferably a specific sequence and a gene encoding the functional protein). The arbitrary promoter may be a promoter that functions in any cell (preferably various types of cells), and preferred are CAG (cytomagalovirus immediate early enhancer-chiken β-actin hybrid), CMV, RSV promoter, etc., and more preferred is the CAG promoter.

In the gene cassettes containing the above constructs in the first and second aspects, a drug selection sequence for selecting genetically modified porcine cells (gene knock-in porcine cells) may or may not be further included. If such a sequence is further included, it is preferably included downstream of a gene encoding a functional protein.
The above-mentioned drug selection sequence is not particularly limited, and examples thereof include a gene cassette composed of SV40 promoter, puromycin N-acetyltransferase, and pA (polyA) of herpes simplex virus thymidine kinase (HSV-TK).

In the first and second aspects, a genome editing method can be used as a method for introducing the gene cassette into the safe harbor region (first gene modification method), and it is preferable to introduce the gene cassette and genome editing tool into a cell (preferably into the nucleus) by any transgene introduction technique such as lipofection, electroporation, or calcium phosphate, and then introduce them into the safe harbor region by genome editing.

Introduction by genome editing methods includes introduction using a genome editing tool containing an artificial nuclease for genome editing such as CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas nuclease, and may also be introduction using a genome editing tool containing an artificial restriction enzyme (artificial nuclease) using a Transcription Activator-Like Effector Nuclease (TALEN) and a zinc finger nuclease (ZFN).

The CRISPR/Cas nuclease comprises a guide RNA and a Cas nuclease (preferably Cas9).
At least a partial sequence within the safe harbor region that is targeted includes an oligonucleotide contained within the safe harbor region.

A gene cassette containing the above construct can be introduced by transfecting a guide RNA or a DNA encoding a guide RNA specific to at least a partial sequence in the above safe harbor region, and a composition containing a nucleic acid encoding a Cas nuclease or a Cas nuclease into a eukaryotic cell or organism containing the above safe harbor region.
Nucleic acids encoding Cas nuclease or Cas nuclease and guide RNA or DNA encoding guide RNA can be introduced into cells by various methods known in the art, such as, but not limited to, microinjection, electroporation, DEAE-dextran treatment, lipofection, nanoparticle-mediated transfection, protein transduction domain-mediated transduction, virus-mediated gene delivery, and PEG-mediated transfection into protoplasts. Nucleic acids encoding Cas nuclease or Cas nuclease and guide RNA can also be introduced into organisms by various methods known in the art for administering genes or proteins, such as injection. Nucleic acids encoding Cas nuclease or Cas proteins can be introduced into cells in the form of a complex with guide RNA or separately. Cas nuclease fused to a protein transduction domain such as Tat can also be efficiently delivered into cells.
Preferably, the eukaryotic cell or organism is co-transfected or sequentially transfected with the Cas9 nuclease and the guide RNA.
Sequential transfection can be performed by first transfection with a nucleic acid encoding a Cas nuclease, followed by a second transfection with a naked guide RNA, preferably, but not limited to, the second transfection 3, 6, 12, 18, 24 hours later.

The expression of the guide RNA may be performed using a guide RNA expression unit. The guide RNA expression unit is preferably a transcription unit of the CRISPR-Cas9 system including a target sequence (at least a partial sequence in the above-mentioned safe harbor region) and a guide RNA, and preferably has a promoter region for expressing the guide RNA (a promoter of RNA polymerase III (e.g., a promoter selected from U6 promoter and H1 promoter)), a target sequence (at least a partial sequence in the above-mentioned safe harbor region) and a guide RNA, and more preferably, the promoter, a sequence complementary to the target sequence (at least a partial sequence in the above-mentioned safe harbor region) and the guide RNA are seamlessly linked.
In order to prevent off-target, the CRISPR/Cas nuclease can also use a Cas9 mutant that cuts only one strand of double-stranded DNA as a nickase. An example of a single-stranded cleavage type Cas9 mutant is Cas9 (D10A). For example, when a single-stranded cleavage type Cas9 mutant is used in combination with a guide RNA having a target sequence complementary to one strand of a target DNA and a guide RNA having a target sequence complementary to the other strand in close proximity, one strand is cleaved with a specificity of 20 bases, and the other strand is further cleaved with a specificity of 20 bases, so that the DNA is cleaved with a specificity of 40 bases in total, and the specificity of the target can be greatly improved.

Genetically modified porcine cells (gene knock-in porcine cells) obtained by introducing the gene cassettes in the first and second aspects into the above-mentioned safe harbor region can be selected and established by any method, such as limiting dilution of less than one cell per well, genetic analysis, or drug resistance.

The following documents (A) to (C) describe the establishment of genetically modified pig cells by genome editing and the production of genetically modified cloned pigs by somatic cloning of the established genetically modified pig cells.
(i) Masahito Watanabe, Kazuhiro Umeyama, Kazuaki Nakano, Hitomi Matsunari, Toru Fukuda, Kei Matsumoto, Susumu Tajiri, Shuichiro Yamanaka, Koki Hasegawa, Kazutoshi Okamoto, Ayuko Uchikura, Shuko Takayanagi, Masaki Nagaya, Takashi Yokoo, Hiromitsu Nakauchi and Hiroshi Nagashima (2022) Generation of heterozygous PKD1 mutant pigs exhibiting earlyonset renal cyst formation. Laboratory Investigation, 102:560-569; https://doi.org/10.1038/s41374-021-00717-z
(b) Hitomi Matsunari, Michiyo Honda, Masahito Watanabe, Satsuki Fukushima, Kouta Suzuki, Shigeru Miyagawa, Kazuaki Nakano, Kazuhiro Umeyama, Ayuko Uchikura, Kazutoshi Okamoto,Masaki Nagaya, Teruhiko Toyo-oka, Yoshiki Sawa, Hiroshi Nagashima (2020) Pigs with δ-sarcoglycan deficiency exhibit traits of genetic cardiomyopathy. Laboratory Investigation, 100:887-899; https://doi.org/10.1038/s41374-020-0406-7
(c) Masahito Watanabe, Kazuaki Nakano, Hitomi Matsunari, Taisuke Matsuda, Miki Maehara, Takahiro Kanai, Mirina Kobayashi, Yukina Matsumura, Rieko Sakai, Momoko Kuramoto, Gota Hayashida, Yoshinori Asano, Shuko Takayanagi, Yoshikazu Arai, Kazuhiro Umeyama, Masaki Nagaya, Yutaka Hanazono, Hiroshi Nagashima (2013) Generation of Interleukin-2 Receptor Gamma Gene Knockout Pigs from Somatic Cells Genetically Modified by Zinc Finger Nuclease-Encoding mRNA. PLOS ONE, Volume 8, Issue 10, e76478
The gene cassette can be introduced into the safe harbor region and genetically modified porcine cells can be established according to the methods described in the above references (i) to (iii).

(Genetically modified cloned pigs)
In the first aspect, there are no particular limitations on the somatic cell cloning (somatic cell cloning technology) used to obtain a genetically modified cloned pig (gene knock-in cloned pig) from the above-obtained (established) genetically modified pig cells (gene knock-in cells).
Somatic cell cloning technology is a technique in which pig cells from which a clone is to be produced (preferably genetically modified pig cells) are cultured to create donor cells, and the nucleus of this donor cell is transferred to a recipient enucleated egg (oocyte) by a fusion method such as cell fusion. After culturing, the donor cell is transferred to a foster parent and fertilized to create a clone.

Specifically, in the first nuclear transfer, the above-obtained (established) genetically modified porcine cells (gene knock-in porcine cells) can be introduced into a mature enucleated unfertilized egg. For this introduction, Sendai virus, cell fusion using electrical stimulation, or intracytoplasmic injection can be used. Then, an activation treatment can be performed to form a nucleus. Examples of the activation treatment include strontium, ethanol, electrical stimulation, etc. This nucleus can be introduced into an enucleated fertilized egg as a donor for the second nuclear transfer.
The above operations may or may not be performed freehand (eg, freehand under a microscope by a skilled technician), but may also be performed using a micromanipulator.
The resulting engineered eggs derived from the genetically modified pig cells can be cultured, and when they develop into transplantable embryos, they can be transplanted into the uterus of a foster parent to produce genetically modified cloned pigs (gene knock-in cloned pigs) derived from the genetically modified pig cells (gene knock-in pig cells).

As described above, the above documents (A) to (C) also describe the production of genetically modified cloned pigs by somatic cloning of genetically modified established pig cells, and the production of genetically modified cloned pigs by somatic cloning of genetically modified established pig cells can be carried out in accordance with the methods described in the above documents (A) to (C).

As the above-mentioned recipient enucleated eggs (oocytes), recipient enucleated eggs of any mammal such as pig, cow, horse, sheep, goat, primate (e.g., human, ape (chimpanzee, monkey, etc.)), rodent (e.g., mouse, rat) are preferred, recipient enucleated eggs of mammals whose physical characteristics are closer to humans than rodents, such as physique, are more preferred, recipient enucleated eggs of pig, sheep, goat, or primate (e.g., human, ape), and recipient enucleated eggs of pig are particularly preferred.
The foster parent into which the above-mentioned transplantable embryo is transplanted is preferably any mammal such as a pig, cow, horse, sheep, goat, primate (e.g., human, ape (chimpanzee, monkey, etc.)), or rodent (e.g., mouse, rat), of which a mammal whose physical characteristics are closer to those of humans than rodents is more preferable, with pigs, sheep, goats, and primates (e.g., human, ape) being even more preferable, and pigs being particularly preferable.

(Sperm from genetically modified cloned pigs)
Sperm (sperm of gene knock-in cloned pig) to be used for gene transfer by intracytoplasmic sperm injection, as described below, can be obtained or collected from the genetically modified cloned pig (gene knock-in cloned pig) obtained above by any method.

(Vector (1))
In the above vector (1) used for gene transfer by ICSI described later in the first aspect, examples of the specific organ or tissue include internal organs (e.g., pancreas, kidney, ureter, bladder, liver, heart, stomach, intestine, etc.), reproductive organs (e.g., ovaries, testes, etc.), fertilized eggs, embryos, fetuses, bone marrow (e.g., hematopoietic organs), brain, eyes, nose, mouth, skin, nerves, or tissues derived therefrom, or artificial tissues (cell sheets such as cartilage cell sheets, organoids, etc.), with internal organs, reproductive organs, fertilized eggs, embryos, and fetuses being preferred, internal organs and reproductive organs being more preferred, and pancreas (e.g., pancreatic islets), kidney (e.g., metanephros, particularly urinary organs including metanephros, ureters, and bladder) being even more preferred.

In the vector (1), the promoter expressed in the specific tissue or organ is not particularly limited as long as it is a promoter expressed in the specific tissue or organ, and examples thereof include a promoter that controls the transcription of Six2 (hereinafter also simply referred to as "Six2 promoter") or a promoter that controls the transcription of Sall1 (hereinafter also simply referred to as "Sall1 promoter"), Six2 promoter, FGF20 promoter, etc., and the Six2 promoter or Sall1 promoter is preferred.
Both Six2 and Sall1 are known to be expressed, for example, in the kidney, and in particular in renal progenitor cells, and in particular in the metanephric mesenchyme region, and more particularly in progenitor cells in the metanephric mesenchyme region.
Six2 is known to be expressed particularly in the undifferentiated metanephric mesenchyme region surrounding the tip of the ureteric bud.

The vector (1) in the first aspect includes a plasmid vector, a virus vector, an artificial chromosome vector, and the like, and is preferably a plasmid vector or an artificial chromosome vector.
The plasmid vector and the viral vector include any cloning vector, for example, pCR4Blunt-TOPO vector (manufactured by Thermo Fisher Scientific) and the like are commercially available as cloning vectors and can be used in the present invention.
Examples of artificial chromosome vectors include bacterial artificial chromosomes (BAC), yeast artificial chromosomes (YAC), P1 phage-derived artificial chromosomes (PAC), and porcine artificial chromosomes, with BAC, YAC, and PAC being preferred.
The vector (1) may be linear or circular, but is preferably linear from the viewpoint of use in the below-described ICSI and amplification. Linearization can be performed by any method.

(Step of introducing genes by intracytoplasmic sperm injection)
Further gene introduction (second gene modification) can be performed using the vector (1) via ICSI by microinsemination (also called intracytoplasmic sperm injection; ICSI) between a liquid containing sperm of the genetically modified cloned pig (sperm of the gene knock-in cloned pig) and the vector (1) and an egg (preferably an egg, more preferably a mature egg) (ICSI mediated gene transfer method).
The intracytoplasmic sperm injection can be performed by injecting a liquid containing the sperm and the vector (1) into the egg (preferably a mature egg) by any method (e.g., injection, glass tube, capillary tube) to allow fertilization.
The injection procedure may or may not be performed freehand (eg, freehand under a microscope by a skilled technician), but may also be performed using a micromanipulator.
The resulting fertilized eggs may or may not be activated by sonication.
The fertilized eggs can then be implanted into the uterus of a recipient female (foster mother) to generate the desired transgenic pig (double genetically modified pig) individual.

The liquid containing the sperm and the vector (1) is not particularly limited as long as it allows the sperm and the vector (1) to coexist, and examples thereof include any aqueous solution, any buffer solution, any culture solution, etc., having a pH of about 7.0 to 8.0, which may optionally contain inorganic salts, buffers, nutrients, serum albumin, etc.
The pig eggs to be subjected to the above-mentioned ICSI are preferably ova, more preferably mature ova.

<Transgenic pigs>
A third aspect of the present invention includes a promoter that is expressed in a specific tissue or organ, and a gene encoding the above-mentioned recombinase that is downstream of and under the control of the promoter that is expressed in the specific tissue or organ,
The transgenic pig comprises a gene encoding a functional protein and a gene cassette containing a construct that enables the functional protein to be expressed by the recombinant enzyme.
The "promoter expressed in a specific tissue or organ, and the gene encoding the recombinant enzyme present downstream of and under the control of the promoter expressed in a specific tissue or organ" may be present (integrated) at any position in the transgenic pig (preferably, cells of the transgenic pig), and may be present (integrated) in, for example, the cytoplasm, genome, mitochondrial genome, etc. of the transgenic pig (preferably, cells of the transgenic pig), and is preferably present (integrated) in the cytoplasm or genome. When present in the cytoplasm, it may be present in the form of a plasmid.
In the safe harbor region containing the gene cassette including the construct containing a gene encoding a functional protein, "a promoter expressed in a specific tissue or organ, and a gene encoding the recombinant enzyme that is present downstream of the promoter expressed in a specific tissue or organ and is under its control" may or may not be present (or incorporated); however, from the viewpoint of the balance of the expression levels of the control gene cassette and the functional gene cassette, it is preferable that they are not present (or not incorporated) in the safe harbor region.
The transgenic pig according to the third aspect can be produced by the production method according to the first aspect.

In the third aspect, specific examples and preferred examples of the "promoter expressed in a specific tissue or organ", "recombinant enzyme", "gene encoding a recombinant enzyme", "functional protein", "gene encoding a functional protein", "construct capable of expressing a functional protein", "gene cassette", and "transgenic pig" are the same as the specific examples and preferred examples described above in the first aspect.

The transgenic pig (double gene modified pig) produced by the production method according to the first aspect and the transgenic pig according to the third aspect may be a fetus, a young pig, or an adult, but is preferably a fetus from the viewpoint of low immunogenicity.

As described above, the transgenic pig produced by the production method of the first aspect and the transgenic pig produced by the production method of the third aspect can conditionally express a specific functional protein in a tissue or organ selectively (preferably in a tissue or organ-specific manner) in the tissue or organ.
The transgenic pig produced by the production method of the first aspect and the transgenic pig produced by the third aspect can conditionally kill at least a portion of the cells in the tissue or organ (preferably, the cells inside the tissue or organ excluding the cells on the surface and inner layer of the tissue or organ) when the functional protein is a cytotoxin.
This makes it possible to conditionally kill at least a portion of the cells inside only the desired tissue or organ (preferably the cells inside the tissue or organ excluding the cells on the surface and outer layer of the tissue or organ).

Furthermore, the transgenic pig produced by the production method of the first aspect and the transgenic pig produced by the production method of the third aspect can also express a specific functional protein in a time-specific conditional manner, as described above.
The transgenic pig produced by the production method of the first aspect and the transgenic pig produced by the third aspect can also conditionally kill at least a portion of the cells in a tissue or organ (preferably, cells inside the tissue or organ excluding cells on the surface and inner layer of the tissue or organ) in a time-specific manner when the functional protein is a cytotoxin.

The transgenic pig (double gene modified pig) produced by the production method according to the first aspect and the transgenic pig according to the third aspect may or may not be a material for somatic cell cloning.
Specifically, somatic cells may be obtained or harvested by any method from the transgenic pig (double gene-modified pig) produced by the production method according to the first aspect or the transgenic pig according to the third aspect, and the somatic cells may be used as donor cells. The donor cells may then be nuclear-transferred into a recipient enucleated egg (oocyte) by a fusion method such as cell fusion, and the donor cells may then be cultured and then transplanted into a foster parent and fertilized to produce a clone.
The present invention also relates to a transgenic pig (double gene modified pig) produced by the production method of the first aspect, and to somatic cells established (e.g., established or cloned) from the transgenic pig of the third aspect.
Since the somatic cells are established, the culture is stable and the cells are highly uniform, which results in excellent industrial mass production of the transgenic pig (double gene modified pig) produced by the production method according to the first aspect and the clone of the transgenic pig according to the third aspect.
The transgenic pig (double gene-modified pig) produced by the production method according to the first aspect and the transgenic pig according to the third aspect may contain the above-mentioned clone.
By using the transgenic pig (double gene-modified pig) produced by the production method according to the first aspect or the transgenic pig according to the third aspect as material for such somatic cell cloning, tissues or organs can be industrially produced from the transgenic pig (double gene-modified pig) produced by the production method according to the first aspect or the transgenic pig according to the third aspect.

<Tissue or organ>
A fourth aspect of the present invention is a transgenic pig (double gene-modified pig) produced by the production method according to the first aspect, or a tissue or organ extracted by any method from the transgenic pig according to the third aspect.
Examples of the organs or tissues include internal organs (e.g., pancreas, kidney, ureter, bladder, liver, heart, stomach, intestines, etc.), reproductive organs (e.g., ovaries, testes, etc.), fertilized eggs, embryos, fetuses, bone marrow (e.g., hematopoietic organs), brain, eyes, nose, mouth, skin, nerves, or tissues derived therefrom, or artificial tissues (cell sheets such as cartilage cell sheets, organoids, etc.), with internal organs, reproductive organs, fertilized eggs, embryos, and fetuses being preferred, internal organs and reproductive organs being more preferred, and pancreas (e.g., pancreatic islets), kidneys (e.g., metanephros, particularly the urinary system including the metanephros, ureters, and bladder) being even more preferred.
The tissue or organ according to the fourth aspect is capable of conditionally expressing a specific functional protein within the tissue or organ.
When the functional protein of the tissue or organ according to the fourth aspect is a cytotoxin, it is possible to conditionally kill at least a portion of the cells in the tissue or organ (preferably, cells inside the tissue or organ excluding cells on the surface and in the outer layer of the tissue or organ).

In the tissue or organ according to the fourth aspect, when the functional protein is a cytotoxin, at least a portion of the cells (preferably cells inside the tissue or organ excluding cells on the surface and outer layer of the tissue or organ) that have been conditionally killed can be replaced with a foreign substance.
Here, the foreign substance means a substance derived from other than the tissue or organ, and includes a substance derived from a human or animal, a drug (e.g., a pharmaceutical product), etc. Examples of the substance include a cell, a growth factor, a hormone, a cytokine, etc., among which a cell is preferable. The cell may or may not be an established cell (e.g., a genetically modified cell, an ES cell, an iPS cell).

The following examples of the present invention will be presented to explain the present invention in more detail, but the present invention is not limited to these examples, and various applications are possible within the scope of the technical concept of the present invention.

<Reference Example 1>
(Construction of a gene cassette containing loxP-Venus gene)
A knock-in vector was constructed that is composed of a splicing acceptor sequence followed by a loxP-STOP-Pause-loxP sequence, downstream of which is Venus-pA and PuroR (puromycin resistance gene cassette) for drug selection. The ROSA region sequences (approximately 700 base pairs each) are linked to both ends (5' and 3') of the vector as homology arms for knock-in.
FIG. 1 is a schematic diagram of the gene cassette containing the loxP-Venus gene constructed above.
In FIG. 1 , SA represents a splicing acceptor sequence for allowing the gene cassette to accept transcription by the ROSA promoter, STOP represents a sequence in which three SV40 poly A signal cassettes are linked, Pause represents a human α2 globin gene RNA polymerase II transcriptional pause signal sequence, Venus represents a Venus sequence, pA represents the 3′-UTR (untranslated region) of the rabbit β-globin gene, and PuroR represents the SV40 promoter, puromycin N-acetyltransferase, and herpes simplex virus thymidine kinase. The gene cassette composed of pA (polyA) of HSV-TK is shown.

<Reference Example 2>
(Construction of tissue- or organ-specific CreERT2 expression vector)
Approximately 7 kb of bases upstream from the region surrounding the start codon of the mouse Six2 gene were used as the mSix2 promoter, and a gene cassette in which CreERT2 and pA were linked to the 3' end of the mSix2 promoter was introduced into the pCR4Blunt-TOPO vector (Thermo Fisher Scientific) to construct a tissue- or organ-specific CreERT2 expression vector.
FIG. 2 is a schematic diagram showing the above-constructed CreERT2 expression vector.
In FIG. 2, pA represents the 3′-UTR of the rabbit β-globin gene.
The obtained tissue- or organ-specific CreERT2 expression vector was linearized and then used in the ICSI mediated gene transfer method described below.

<Reference Example 3>
(Construction of a gene cassette containing loxP-DTA gene)
A knock-in vector was constructed that is composed of a splicing acceptor sequence followed by a loxP-STOP-Pause-loxP sequence, downstream of which is DTA-pA and PuroR (puromycin resistance gene cassette) for drug selection. The ROSA region sequences (approximately 700 base pairs each) are linked to both ends (5' and 3') of the vector as homology arms for knock-in.
FIG. 3(a) is a schematic diagram of the gene cassette containing the loxP-DTA gene constructed above.
In FIG. 3(a), SA indicates a splicing acceptor sequence for promoting transcription by the ROSA promoter, STOP indicates a sequence in which three SV40 poly A signal cassettes are linked, Pause indicates a human α2 globin gene RNA polymerase II transcriptional pause signal sequence, DTA indicates a diphtheria toxin A fragment sequence, pA indicates the 3′-UTR (untranslated region) of the rabbit β-globin gene, and PuroR indicates the SV40 promoter, puromycin N-acetyltransferase, and herpes simplex virus thymidine kinase. The gene cassette composed of pA (polyA) of HSV-TK is shown.
FIG. 3(b) is a schematic diagram of the knock-in of the gene cassette into the ROSA region.

(Establishment of cells into which a gene cassette containing a loxP-DTA gene was introduced by CRISPR/Cas9 in the ROSA region)
An RNP complex consisting of a gene cassette containing the above-mentioned loxP-DTA gene, a gRNA complex, and recombinant Cas9 was introduced into 5.0×10 ⁵ cells of porcine fetal fibroblast cells by electroporation.
Here, the gRNA complex consists of tracrRNA (Trans-activating crRNA) and a porcine ROSA region-gRNA (guide RNA) represented by the following sequence. The following sequence possessed by the porcine ROSA region-gRNA (guide RNA) is located at 65,757,140 to 65,757,159 on the 13th porcine chromosome and is a sequence present between exons 1 and 2 of ROSA. However, the position numbers of the above genes are position numbers in the UCSC genome browser as of May 24, 2022, and may change due to database updates.

Pig ROSA region-gRNA
5'-GTAGTGCTCTAGATTAGCAC-3' (SEQ ID NO: 8)

After selection based on puromycin resistance, limiting dilution was performed to less than one cell per well, and the clones derived from the grown single-cell cells were subjected to genetic analysis to establish genetically modified porcine cells in which the gene cassette containing the above-constructed loxP-DTA gene was introduced into the porcine ROSA region.
FIG. 4 is an electrophoretic diagram showing the results of confirming the knock-in of the above gene cassette into the ROSA region.
As is clear from the results shown in FIG. 4, it was confirmed that the above gene cassette was knocked into the ROSA region.

(Confirmation of cell death induction by ERT2CreERT2 and tamoxifen in genetically modified porcine cells in which a gene cassette containing the above loxP-DTA gene was introduced into the porcine ROSA region)
The ERT2CreERT2 expression vector constructed separately was introduced by electroporation into genetically modified porcine cells in which a gene cassette containing the above-mentioned loxP-DTA gene had been introduced into the porcine ROSA region.
Thereafter, the cells were cultured in a medium containing 10 nM tamoxifen (4-hydroxytamoxifen (4-OHT)). The results are shown in FIG.
As is clear from the results shown in FIG. 5, cell death was confirmed in the genetically modified porcine cells into which a gene cassette containing the loxP-DTA gene had been introduced into the porcine ROSA region.

(Construction of genetically modified cloned pigs from the above genetically modified pig cells)
Genetically modified porcine cells in which a gene cassette containing the loxP-DTA gene was introduced into the porcine ROSA region were seeded at 5 x 10 ⁵ cells in Dulbecco's modified Eagle's (DMEM) culture medium supplemented with 10% fetal bovine serum (FBS) on a 35 mm diameter dish and passaged once. Half of the culture medium was removed 14 to 18 hours after passage, and after 2 hours of culture, the culture medium was collected after gentle pipetting and centrifuged at 1000 rpm for 3 minutes to collect the cells. These were used as nuclear donor cells for nuclear transplantation.

For nuclear transfer, unfertilized eggs in the metaphase of the second meiotic division, obtained by in vitro maturation of eggs collected from the ovaries of wild-type female pigs, were used as recipient cytoplasm after enucleation. The nuclear transfer procedure was carried out in an egg manipulation solution. Electrical fusion was used to fuse the nuclear donor cells with the recipient cytoplasm. The eggs that had completed the fusion were cultured in a culture solution for 30 minutes to an hour, and then subjected to a direct current electrical stimulus. At least some of these eggs were activated normally and began to develop.

Of the in vitro cultured nuclear transfer embryos, those that cleaved normally on days 1-2, or developed into blastocysts on days 5-6, were transferred into the oviducts or uteruses of recipient pigs whose estrus cycles had been synchronized.
At least some of the transplanted embryos gave birth to liveborn offspring, and genetically modified cloned pigs were constructed. Sperm were collected from the resulting genetically modified cloned pigs. The sperm can be used in the subsequent ICSI mediated gene transfer method.

<Reference Example 4>
(Construction of transgenic pigs using tissue- or organ-specific CreERT2 expression vectors via ICSI)
Sperm used in ICSI were ultrasonicated to separate the head and tail. The ultrasonic treatment was performed at an intensity that caused separation of the head and tail in about 70% of sperm. After co-culturing the tissue- or organ-specific CreERT2 expression vector constructed in the above-mentioned <Reference Example 2> and the porcine sperm head in a culture medium, one sperm head was sucked into an injection pipette, and the injection pipette was injected into an egg using one or more piezo pulses. After confirming that the injection pipette had reached the egg cytoplasm, the sperm head was quickly injected into the egg. Sperm injection was performed in an egg manipulation solution.

Pre-implantation culture: After ICSI, the sperm-injected eggs were transferred to a PZM5 culture medium drop prepared on a plastic culture dish (IWAKI). After 20 and 44 hours of culture, normal one-cell stage embryos and normal cleavage embryos were observed. After 116 and 140 hours, blastocyst stage embryos were observed.

Embryo transfer: Embryos cultured in microdroplets of PZM medium for the above period were transferred into the oviduct or uterus of embryo recipient pigs. Normal one-cell embryos and normal cleavage embryos were transferred into the oviduct within one day after ovulation, and blastocysts were transferred into the uterus on the fourth day after ovulation.

(Sexing test)
A total of 42 fetuses were obtained from four embryo recipient pigs (#236, #365, K364, #238). After obtaining tails from the fetuses, genomic DNA was extracted using a DNA extraction kit (DNeasy Blood and Tissue Kit, manufactured by QIAGEN), and sex determination was performed by PCR using primers targeting amelogenins (AMEL X and AMEL Y).
The electrophoretograms of the sex determination results for the 42 animals obtained are shown in FIG.
As is clear from Figure 6, #236-1, 5, 6, 8, #365-1, 2, 4, 9, 10, 11, 12, 13, K364-3, 4, 5, 6, 8, and #238-1, 3, 5, 6 were male, and #236-2, 3, 4, 7, 9, #365-3, 5, 6, 7, 8, K364-1, 2, 7, and #238-2, 4, 7 to 12 were male.

(Gene transfer determination test)
Using the genomic DNA of the 42 fetuses (described above), PCR was carried out using a primer targeted to CreERT2 from the mSix2 promoter on the transgene.
The PCR products were electrophoresed using 1% agarose gel (Tris-acetate-EDTA) to determine gene transfer. The results are shown in FIG.

FIG. 7 is an electrophoretic diagram showing the results of gene transfer assessment.
In FIG. 7, M is a DNA molecular weight marker, N is a negative control using wild-type pig genomic DNA, and P is a positive control using a CreERT2 expression gene vector.
As is clear from Figure 7, out of the 42 fetuses, a total of 9 tissue- or organ-specific CreERT2 expression transgenic pig fetuses were obtained, and the production efficiency of transgenic pigs was 21% (#236-2, 5, 6, #365-3, 4, 12, K364-8, #238-5, 7). However, #365-12 and K364-8 were regressed fetuses.

(Test to confirm CreERT2 expression in fetal metanephroi)
Among the above-mentioned tissue- or organ-specific CreERT2 expression gene-transduced pig fetuses, one metanephroi was taken from each of #236-2, 5, 6, #365-3, 4, and 12, and homogenized using a BioMasher (manufactured by Nippi) in the tissue solution attached to the RNeasy Plus Mini Kit (manufactured by QIAGEN), and total RNA was collected according to the product protocol. Reverse transcription reaction was performed using SuperScriptIII Reverse Transcriptase (manufactured by Thermo Fisher Scientific) to obtain cDNA. An amplification reaction was carried out using a Veriti thermal cycler (Thermo Fisher Scientific) with CreERT2 primers and PigSix2 primers, and a test for confirming CreERT2 expression in the fetal metanephroi was carried out by RT-PCR (nested PCR).
The PCR product was subjected to electrophoresis using 1% agarose gel (Tris-acetate-EDTA) to carry out a test for confirming CreERT2 expression in fetal metanephroi. The results are shown in FIG.

CreERT2 primer sequence ^1st : 5'-CATGTCCATGCTGCCCACCTTCG-3' (SEQ ID NO: 9) / 5'-GTATATCCTGGCAGCGATCGC-3' (SEQ ID NO: 10)
^2nd : 5'-TCGCAAGAACCTGATGGACA-3' (SEQ ID NO: 11) / 5'-CGCCGCATAACCAGTGAAAC-3' (SEQ ID NO: 12)

PigSix2 primer sequence ^1st : 5'-AGTCGCAGCGTGCTGCGCGAG-3' (SEQ ID NO: 13) / 5'-CTAGGAGCCCAGGTCCACGA-3' (SEQ ID NO: 14)
^2nd : 5'-ACCACGCAGGTCAGCAACTGG-3' (SEQ ID NO: 15) / 5'-TCCGAGCTGCCTAGCACCGAC-3' (SEQ ID NO: 16)

FIG. 8 is an electrophoretogram showing the results of confirming CreERT2 expression in fetal metanephroi.
In FIG. 8, +RT indicates data obtained with reverse transcriptase for sample amplification.
As is clear from FIG. 8, CreERT2 gene expression under the mouse Six2 promoter was confirmed in the metanephros of all of the above-mentioned tissue- or organ-specific CreERT2 expression gene-modified pig fetuses (#236-2, 5, 6, #365-3, 4, 12).

(Determining the copy number of the transgene)
Genomic DNA was extracted from the tail tissue of the above-mentioned tissue- or organ-specific CreERT2 expression gene-introduced pig fetuses, #236-2, 5, 6, #365-3, 4, 12, #238-5, and 7, and the copy number of the transgene (introduced gene) in the genome was determined by digital PCR. The results are shown in FIG.
Figure 9 shows the results of transgene copy number determination in the diploid genome, in which β-actin is an internal standard (2 copies).

As is apparent from the results shown in FIG. 9 , in the diploid genome, the copy number of the transgene in #236-2 was 5 copies, the copy number of the transgene in #236-5 was 2 copies, the copy number of the transgene in #236-6 was 1 copy, the copy number of the transgene in #365-3 was 16 copies, the copy number of the transgene in #365-4 was 6 copies, the copy number of the transgene in #365-12 was 19 copies, the copy number of the transgene in #238-5 was more than 200 copies, and the copy number of the transgene in #238-7 was 10 copies.
As described above, transgenic pig fetuses were obtained in which one, two or more, or even 200 or more copies of tissue- or organ-specific CreERT2 gene were introduced per individual (diploid).

Example 1
(Establishment of cells into which a gene cassette containing a loxP-Venus gene was introduced by CRISPR/Cas9 in the ROSA region)
An RNP complex consisting of a gene cassette containing the loxP-Venus gene constructed as described above in Reference Example 1, a gRNA complex, and recombinant Cas9 was introduced into 5.0 × ¹⁰⁵ pig fetal fibroblast cells by electroporation using a Neon Transfection System (Thermo Fisher Scientific).
Here, the gRNA complex consists of tracrRNA (Trans-activating crRNA) and the porcine ROSA region-gRNA (guide RNA) represented by the sequence of SEQ ID NO: 8. The following sequence of the porcine ROSA region-gRNA (guide RNA) is located at 65,757,140 to 65,757,159 on the 13th porcine chromosome, and is a sequence present between exons 1 and 2 of ROSA. However, the position numbers of the above genes are the position numbers in the UCSC genome browser as of May 24, 2022, and may change due to database updates.
After selection based on puromycin resistance, limiting dilution was performed to less than one cell per well, and the clones derived from the grown single-cell cells were subjected to genetic analysis to establish genetically modified porcine cells in which the gene cassette containing the loxP-Venus gene constructed above was introduced into the porcine ROSA region.

(Construction of genetically modified cloned pigs from the above genetically modified pig cells)
Genetically modified porcine cells obtained by introducing a gene cassette containing the loxP-Venus gene into the porcine ROSA region are seeded at 5 x 10 ⁵ cells in a 35 mm diameter dish in Dulbecco's Modified Eagle's (DMEM) culture medium supplemented with 10% fetal bovine serum (FBS) and passaged once. Half of the culture medium is removed 14 to 18 hours after passage, and after 2 hours of culture, the culture medium is collected after gentle pipetting and centrifuged at 1000 rpm for 3 minutes to collect the cells. These are used as donor nuclei for nuclear transplantation.

For nuclear transfer, unfertilized eggs in the metaphase of the second meiotic division, obtained by in vitro maturation of eggs collected from the ovaries of wild-type female pigs, were used as recipient cytoplasm after enucleation. The nuclear transfer procedure was carried out in an egg manipulation solution. Electrical fusion was used to fuse the nuclear donor cells with the recipient cytoplasm. The eggs that had completed the fusion were cultured in a culture solution for 30 minutes to an hour, and then subjected to a direct current electrical stimulus. At least some of these eggs were activated normally and began to develop.

Of the in vitro cultured nuclear transfer embryos, those that cleaved normally on days 1-2, or developed into blastocysts on days 5-6, were transferred into the oviducts or uteruses of recipient pigs whose estrus cycles had been synchronized.
At least some of the transplanted embryos gave birth to liveborn offspring, and genetically modified cloned pigs were constructed. Sperm were collected from the resulting genetically modified cloned pigs. The sperm can be used in the subsequent ICSI mediated gene transfer method.

(Construction of transgenic pigs using tissue- or organ-specific CreERT2 expression vectors via ICSI)
The tissue- or organ-specific CreERT2 expression vector constructed in the above-mentioned <Reference Example 2> and the heads of the pig sperm are co-cultured in a culture medium, and a normally developed transgenic pig fetus is obtained from the embryo recipient pig by the same ICSI procedure, egg culture, and embryo transfer into the uterus of the embryo recipient pig as in the above-mentioned <Reference Example 4>.
By administering tamoxifen to a female pig carrying the above-mentioned normally developed fetus, or by transplanting the metanephroi harvested from the above-mentioned normally developed fetus into the body of an immunodeficient mouse and then administering tamoxifen to the mouse, or by culturing the harvested metanephroi in vitro and adding tamoxifen to the culture medium, Venus fluorescence can be conditionally observed in specific tissues or organs.

Example 2
(Construction of transgenic pigs using tissue- or organ-specific CreERT2 expression vectors via ICSI)
The sperm of a genetically modified cloned pig into which the tissue- or organ-specific CreERT2 expression vector constructed in the above-mentioned <Reference Example 2> and the gene cassette containing the loxP-DTA gene obtained in the above-mentioned <Reference Example 3> have been introduced are co-cultured in a culture medium, and normally developed fetuses of the transgenic pig are obtained from the embryo recipient pig by the same ICSI procedure, oocyte culture, and embryo transfer into the uterus of the embryo recipient pig as in the above-mentioned <Reference Example 4>.

Comparative Example
(Construction of all-in-one vector 1 containing both the mSix2 promoter and the CreERT2 and loxP-DTA genes)
An all-in-one knock-in vector was constructed in which the mSix2 promoter was approximately 7 kb upstream from the region surrounding the start codon of the mouse Six2 gene, CreERT2 and pA were linked to the 3' end of the mSix2 promoter, the CAG promoter was linked downstream of the pA, and the 3' end of the CAG promoter was followed by a loxP-STOP-loxP sequence, followed downstream of which was DTA-pA.
FIG. 10 is a schematic diagram showing the all-in-one vector 1 containing both the mSix2 promoter and the CreERT2 and loxP-DTA genes constructed as above.

(Construction of all-in-one vector 2 containing both the mSix2 promoter and the CreERT2 and loxP-DTA genes)
An all-in-one knock-in vector 2 was constructed in which the mSix2 promoter was approximately 7 kb upstream of the region surrounding the start codon of the mouse Six2 gene, P2A (self-cleaving 2A peptide derived from porcine Teschovirus-1) was linked to the 3' end of the mSix2 promoter, ERT2CreERT2 and pA were further linked, a CAG promoter was linked downstream of the pA, and a DTA-pA sequence (reverse) sandwiched between FLEx switch-type loxPlox2272 and loxPlox2272 (reverse) was linked to the 3' end of the CAG promoter.
FIG. 11 is a schematic diagram showing the all-in-one vector 2 containing both the mSix2 promoter and the CreERT2 and loxP-DTA genes constructed above.
The obtained all-in-one vectors 1 and 2 containing both the mSix2 promoter and the CreERT2 and loxP-DTA genes were linearized and then used in the ICSI described below.

(Construction of transgenic pigs using all-in-one vectors 1 and 2 via ICSI)
A transgenic pig was constructed via ICSI in the same manner as in Example 1, except that the "tissue- or organ-specific CreERT2 expression vector" was changed to the all-in-one vector 1 or 2 containing both the mSix2 promoter and CreERT2 and loxP-DTA genes.
The results of constructing transgenic pigs using ICSI-mediated all-in-one vector 1 or 2 are summarized in Tables 1 and 2 below.

表１から明らかなように、ＩＣＳＩを介したオールインワン型ベクター１による遺伝子導入では、正常発達遺伝子導入胎仔が１頭得られたものの、遺伝子導入ブタ胎仔の作出効率は０．３５％未満であり作出効率は極めて低かった。ＣｒｅＥＲＴ２遺伝子のコピー数に対する、ｌｏｘＰ－ＤＴＡ遺伝子のコピー数のバランスが悪かった（過剰であった）ことが影響しているものと考えられる。

As is clear from Table 1, in the gene transfer using the all-in-one vector 1 via ICSI, one normally developed gene-transferred fetus was obtained, but the production efficiency of gene-transferred pig fetuses was less than 0.35%, which was extremely low. This is thought to be due to the poor balance (excessive) of the copy number of the loxP-DTA gene relative to the copy number of the CreERT2 gene.

表２から明らかなように、ＩＣＳＩを介したオールインワン型ベクター２による遺伝子導入では、正常発達遺伝子導入胎仔は得られなかった（遺伝子導入ブタ胎仔の作出効率０％）。また、遺伝子導入された胎仔は全て退行してしまった。

As is clear from Table 2, gene transfer using all-in-one vector 2 via ICSI did not result in normal development of gene-transferred fetuses (production efficiency of gene-transferred pig fetuses: 0%). Furthermore, all gene-transferred fetuses regressed.

As described above, it was difficult to construct transgenic pigs using an all-in-one vector containing both the mSix2 promoter and the CreERT2 and loxP-DTA genes.

Claims (17)

Sperm of a genetically modified cloned pig obtained by somatic cloning of a genetically modified pig cell obtained by introducing a gene cassette containing a gene encoding a functional protein and a construct capable of expressing the functional protein by a recombinase into a safe harbor region;
A method for producing a transgenic pig, comprising the step of introducing a gene into an egg by insemination with a liquid containing the vector (1),
The vector (1),
A gene encoding the recombinase is contained in the downstream of a promoter that is expressed in a specific tissue or organ and is under the control of the promoter that is expressed in the specific tissue or organ,
A production method in which the ratio of the copy number of the gene encoding the functional protein to the copy number of the gene encoding the recombinant enzyme is 1/2 to 1/300. Sperm of a genetically modified cloned pig obtained by somatic cloning of a genetically modified pig cell obtained by introducing a gene cassette containing a gene encoding a functional protein and a construct capable of expressing the functional protein by a recombinase into a safe harbor region;
A method for producing a transgenic pig, comprising the step of introducing a gene into an egg by insemination with a liquid containing the vector (1),
The vector (1),
A gene encoding the recombinase is contained in the downstream of a promoter that is expressed in a specific tissue or organ and is under the control of the promoter that is expressed in the specific tissue or organ,
The method for producing the present invention, wherein the functional protein is at least one selected from the group consisting of a metabolic enzyme, a cell proliferation-related factor, a growth-related gene protein, a cytotoxin, and a suicide gene protein. A gene cassette including a construct that includes a gene encoding a functional protein and enables the functional protein to be expressed by a recombinase;
The method according to claim 1 or 2, which has improved production efficiency compared to a method for producing a transgenic pig, which includes a gene transfer step of microinsemination into an egg a vector containing both a promoter expressed in a specific tissue or organ and a gene cassette containing a gene encoding the recombinant enzyme that is located downstream and under the control of the promoter expressed in a specific tissue or organ. The method according to claim 1 or 2, wherein the promoter is a Six2 promoter, a Sall1 promoter, or an FGF20 promoter. The method according to claim 1 or 2, wherein the gene cassette is introduced into the safe harbor region by genome editing. The method of claim 1 or 2, wherein the safe harbor region is a ROSA region. The method according to claim 1 or 2, wherein the vector (1) is a plasmid vector or an artificial chromosome vector. A somatic cell cloning source comprising a genetically modified porcine cell comprising a gene encoding a functional protein and a gene cassette in a safe harbor region comprising a construct capable of expressing the functional protein by a recombinase,
A somatic cell cloning material for providing a material for gene transfer by intracytoplasmic sperm injection in the presence of the following vector (1).
(1) A vector comprising a promoter expressed in a specific tissue or organ, and a gene encoding the recombinase that is present downstream of and under the control of the promoter expressed in a specific tissue or organ, wherein the ratio of the copy number of the gene encoding the functional protein to the copy number of the gene encoding the recombinase is 1/2 to 1/300. A somatic cell cloning source comprising a genetically modified porcine cell comprising a gene encoding a functional protein, the gene cassette comprising a construct capable of expressing the functional protein by a recombinase in a safe harbor region,
A somatic cell cloning material for providing a material for gene transfer by intracytoplasmic sperm injection in the presence of the following vector (1):
A somatic cell cloning source material, wherein the functional protein is at least one selected from the group consisting of metabolic enzymes, cell proliferation-related factors, growth-related gene proteins, cytotoxins, and suicide gene proteins.
(1) A vector comprising a promoter that is expressed in a specific tissue or organ, and a gene encoding the recombinase that is downstream of and under the control of the promoter that is expressed in a specific tissue or organ. A gene cassette including a construct that includes a gene encoding a functional protein and enables the functional protein to be expressed by a recombinase;
The somatic cell cloning material described in claim 8 or 9, which has improved production efficiency by somatic cell cloning compared to a somatic cell cloning material containing genetically modified pig cells containing both a promoter expressed in a specific tissue or organ and a gene cassette containing a gene encoding the recombinant enzyme that is located downstream of the promoter expressed in a specific tissue or organ and under its control. 10. The somatic cell cloning material according to claim 8 or 9 , wherein the promoter is a Six2 promoter, a Sall1 promoter or an FGF20 promoter. A gene encoding the recombinase is contained in the downstream of a promoter that is expressed in a specific tissue or organ and is under the control of the promoter that is expressed in the specific tissue or organ,
A transgenic pig comprising a gene encoding a functional protein, a gene cassette comprising a construct that enables the functional protein to be expressed by the recombinant enzyme, in a safe harbor region, and a ratio of the copy number of the gene encoding the functional protein to the copy number of the gene encoding the recombinant enzyme of 1/2 to 1/300. A gene encoding the recombinase is contained in the downstream of a promoter that is expressed in a specific tissue or organ and is under the control of the promoter that is expressed in the specific tissue or organ,
A gene cassette including a construct that includes a gene encoding a functional protein and enables expression of the functional protein by the recombinase is included in the safe harbor region;
The transgenic pig, wherein the functional protein is at least one selected from the group consisting of a metabolic enzyme, a cell proliferation-related factor, a growth-related gene protein, a cytotoxin, and a suicide gene protein. A gene cassette including a construct that includes a gene encoding a functional protein and enables the functional protein to be expressed by a recombinase;
The transgenic pig according to claim 12 or 13, which has an improved survival rate compared to a transgenic pig produced by a production method including a gene transfer step of injecting an egg with a vector containing both a promoter expressed in a specific tissue or organ and a gene cassette containing a gene encoding the recombinant enzyme that is located downstream and under the control of the promoter expressed in a specific tissue or organ . The transgenic pig according to claim 12 or 13 , wherein the promoter is a Six2 promoter, a Sall1 promoter or an FGF20 promoter. A somatic cell established from the transgenic pig according to claim 12 or 13 . A tissue or organ isolated from the transgenic pig according to claim 12 or 13 .

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