JP4174519B2 - Pathological model animal using hyaluronic acid synthase gene expression inducible transgenic animal - Google Patents

Description

The present invention, hyaluronic which to investigate the mechanism of onset and progression of hyaluronic acid synthesis diseases caused by abnormal use in order to you develop the treatment, the DNA fragments of hyaluronic acid synthase gene transfer plasmid was introduced The present invention relates to a human pathological model animal which is composed of an animal species of an acid synthase gene expression-inducible transgenic animal and has an organ-specific disease onset and is spontaneous . Further, the DNA fragment is introduced hyaluronic acid synthase gene expression inducible transgenic animals, and human pathology model is spontaneous an organ specific disease onset consists mated animal species it about the animal.

  Hyaluronic acid (HA) is a major constituent molecule of the extracellular matrix that is abundantly contained in articular cartilage and skin, and consists of a high molecular weight glycosaminoglycan sugar chain. Hyaluronic acid is composed of repetitive sugar units formed by beta-1,4 bonds of disaccharide units (GlcUAbeta1-3GlcNAc) in which glucuronic acid (GlcUA) is beta-1,3 linked to N-acetylglucosamine (GlcNAc).

  Such hyaluronic acid is biosynthesized by hyaluronic acid synthase (HAS) in animal cells. As hyaluronic acid synthase, there are three types of family molecules, hyaluronic acid synthase 1, 2 or 3 (HAS1, HAS2 or HAS3, respectively) showing homology on the primary structure. Each enzyme has hyaluronic acid synthesis activity. Hyaluronic acid synthase in mice is denoted as Mouse Has1, Has2, and Has3, respectively.

  Hyaluronic acid in animal cells works to promote cell growth and migration, and is considered to be important for the regulation of biological reactions such as tissue structure maintenance, functional regulation, inflammation, and wound healing. The synthetic abnormality is said to be deeply related to carcinogenesis, invasion and metastasis of cancer cells, and various diseases such as arteriosclerosis and fibrosis.

  In order to investigate the mechanism of the onset and progression of the above-mentioned various diseases caused by abnormal hyaluronic acid synthesis and to develop a therapeutic method, a model animal is indispensable.

  Non-Patent Document 1 discloses a transgenic mouse in which a human hyaluronic acid synthase 2 (HAS2) gene is introduced to express hyaluronic acid synthesis specifically in smooth muscle cells. An association between acid and arteriosclerosis has been suggested.

  Patent Document 1 discloses a non-human transgenic animal that includes a reporter gene linked to a skin metabolism-related promoter, and that the reporter gene encodes a luminescent or fluorescent product. This transgenic animal is for examining changes in expression of a reporter gene, and is capable of evaluating the influence of a compound on gene expression in the skin.

  These transgenic animals can be model animals for diseases such as arteriosclerosis and abnormal skin metabolism, but cannot be model animals for other cancer diseases. In addition, these transgenic animals prepared by conventional methods are designed to control the expression of the transgene by placing a tissue-specific promoter directly upstream of the transgene. It was necessary to individually produce transgenic animals according to the purpose of the organs and diseases to be used.

Chai, S. et al, Circ. Res. 96: 583-591 (2005) Japanese translation of PCT publication No. 2003-520946

The present invention was made in order to solve the above-mentioned problems, and is a human pathological model animal composed of a transgenic animal species that is hyaluronic acid synthase gene expression-inducible , which can be produced simply and economically , It is a first object to provide a disease state model animal using a DNA fragment of a hyaluronic acid synthase gene introduction plasmid that expresses hyaluronic acid synthase by the action of a site-specific recombinant enzyme.

Moreover, this DNA fragment is introduced, and hyaluronic acid synthase gene expression-inducible transgenic that can control the expression of hyaluronic acid synthase gene and the synthesis of hyaluronic acid for each individual using site-specific recombinase A second object is to provide a human pathological model animal comprising animal species .

Furthermore, it is used for research on various diseases related to hyaluronic acid synthesis abnormality, and consists of animal species of hyaluronic acid synthase gene expression inducible and organ-specific disease pathogenic model transgenic animals The third object is to provide a human pathological model animal .

The pathological model animal according to claim 1, which has been made to achieve the above object, is sandwiched between a transcription initiation regulatory base sequence and a pair of recognition base sequences recognized by a site-specific gene recombinase. The inactive spacer base sequence excised by the enzyme, the base sequence of the hyaluronic acid synthase Has2 gene, or the trans-expression induction control base sequence, the base sequence of the hyaluronic acid synthase Has2 gene is the 5 ′ end from the 3 'end, the DNA fragment have Ruhi hyaluronic acid synthase gene Has2 introduced plasmid linked in that order, are introduced into a non-human mammal having a breast cancer resistance gene,
Residues of the DNA fragment from which the spacer base sequence was excised by the enzyme by mating with the same kind of parent animal having the site-specific gene recombinase are recombined via one of the recognition base sequences and short-circuited. A hyaluronic acid synthase gene having Has2 genotype in which Has2 transcription is induced by recombination and induced by a substance that induces Has2 transcription at the trans-expression-inducing regulatory base sequence Consisting of animal species of expression-inducing transgenic animals,
The animal species is naturally pathogenic by the DNA fragment, the site-specific gene recombinase that expresses the gene of the hyaluronic acid synthase or the trans-expression-inducing control base sequence, and the breast cancer-causing gene. In addition, the animal model is a pathological model of hard cancer, characterized in that it has the onset of breast cancer in the mammary gland .

The pathological model animal according to claim 2 is the animal model according to claim 1, wherein the pair of recognition base sequences is a loxP base sequence or a Frt base sequence.

In the pathological model animal, the hyaluronic acid synthase is preferably vertebrate hyaluronic acid synthase 2 .
The disease animal model according to claim 3 is the animal model according to claim 2, wherein the site-specific gene recombinase is Cre recombinase, and the inactive spacer base sequence is the pair of loxP bases. Since the base sequence of the neomycin resistance gene Neo sandwiched between the sequences, the Cre recombinase cuts out the base sequence of the Neo by the homologous recombination and becomes the Has2 genotype in which the transcription of the Has2 was induced. It is characterized by that.

Arsenate hyaluronic acid synthase gene expression inducible transgenic animals, DNA fragments of human hyaluronic acid synthase gene transfer plasmids that have been introduced into nonhuman mammals.

The disease model animal according to claim 4 is the animal model according to claim 1 , wherein the non-human mammal is a mouse, a rat, a hamster, a rabbit, or a pig.

Pathological model animal according to claim 5, in which according to claim 1, wherein the animal species, wherein the hyaluronic acid synthase gene expression inducible transgenic animals, and the breast cancer resistance with it the same kind of It is characterized by the animal species that the animals are mated with.

Pathological model animal according to claim 6, in which according to claim 1, wherein the animal species, wherein the hyaluronic acid synthase gene expression inducible transgenic animals, and the breast cancer resistance with it the same kind of It is characterized by being an animal species in which an animal is crossed and an animal having the site-specific recombinase is crossed.

Disease status model animals, those pathological model animal scirrhous carcinoma that has a breast cancer resistance in breast is preferable.

  The DNA fragment of the hyaluronic acid synthase gene transfer plasmid of the present invention uses an inducible gene expression system based on a site-specific gene recombinase such as Cre-loxP and a pair of base sequences recognized by it. Alternatively, it is useful for easily and economically producing a transgenic animal that can control hyaluronic acid synthase gene expression and hyaluronic acid synthesis at the individual level by a trans-expression induction control base sequence.

  The hyaluronic acid synthase gene expression inducible transgenic animal of the present invention uses this inducible gene expression system, optionally depending on the desired pathological model creation time and the purpose of the pathological model of the organ-specific disease. It is possible to control hyaluronic acid synthase gene expression and hyaluronic acid synthesis.

  In contrast to the conventional invention, the analysis of the change in the expression of the hyaluronic acid synthase gene is the object of evaluation, whereas the present invention is a transgenic animal that expresses the hyaluronic acid synthase itself, It is excellent in that the function can be evaluated.

  By crossing this inducible transgenic animal with an organ-specific transgenic animal, it is possible to control the expression of the hyaluronic acid synthase gene and inducible hyaluronic acid synthesis. In addition, it is not necessary to individually create an organ-specific transgenic animal that matches the target disease state.

  Furthermore, inducible promoters whose expression is controlled in trans by exogenous substances by introducing site-specific gene recombination enzymes such as Cre into cells using common gene transfer techniques such as viral vectors. It is also possible to control hyaluronic acid synthase gene expression and inducible hyaluronic acid synthesis.

  As a result, the animals in which the expression of the hyaluronic acid synthase gene is induced are highly useful as test animals for analyzing the function of hyaluronic acid in vivo, and treatment methods and therapeutic drugs for various diseases associated with abnormal hyaluronic acid synthesis are also available. It is useful as a model animal for development.

BEST MODE FOR CARRYING OUT THE INVENTION

  EXAMPLES Hereinafter, although the preferable Example of this invention is described in detail, the scope of the present invention is not limited to these Examples.

  The DNA fragment of the hyaluronic acid synthase gene introduction plasmid of the present invention has a base sequence of a drug resistance gene sandwiched between a transcription start control base sequence and a hyaluronic acid synthase gene base sequence by a pair of loxP base sequences. Furthermore, a transcription termination control base sequence is arranged on the downstream 3 ′ end side.

  The DNA fragment of this hyaluronic acid synthase gene transfer plasmid is an enzyme that site-specifically recombines the DNA region sandwiched between the paired loxP base sequences in order to control the expression of the inducible hyaluronic acid synthase gene. The recombination system by can be used.

  In the presence of a site-specific recombinase, the DNA fragment of the hyaluronic acid synthase gene transfer plasmid is excised from the base sequence of the drug resistance gene together with one of the loxP base sequences and recombined as a circular DNA. The remaining DNA fragments have the property of recombining with each other via the other loxP base sequence.

  Referring to FIG. 1 schematically showing an outline of a DNA fragment of a hyaluronic acid synthase gene introduction plasmid which is one embodiment of the present invention, and FIG. 2 showing a partial base sequence of DNA after gene recombination This will be described more specifically.

  The hyaluronic acid synthase gene transfer plasmid is Neo (neomycine resistant gene) + poly A (SV40 early mRNA polyadenylation) sandwiched between the loxP base sequence of the pair at the 3 'end downstream of the CAG promoter which is the transcription initiation regulatory base sequence. signal), the nucleotide sequence of the neomycin resistance gene, which is a drug resistance gene, and the cDNA of the mouse hyaluronan synthase 2 (Mouse Has2) translation region and b-globin poly A (b- A construct linking globin mRNA polyadenylation signal) was constructed on the pCALNL5 vector (obtained from RIKEN Bioresource Center DNA Bank, URL: http://www.brc.riken.jp/lab/dna/ja/) .

  For the production of a transgenic animal, a DNA fragment obtained by digesting this hyaluronic acid synthase gene transfer plasmid with two types of restriction enzymes (Sal I and Sfi I) is used.

  The DNA fragment of the hyaluronic acid synthase gene transfer plasmid is that the polynucleotide encoding hyaluronic acid synthase is linked to a promoter that controls transcription initiation in cells of non-human mammals. Alternatively, it is arranged at a position where the initiation of transcription can be controlled in the cell of a non-human mammal into which this DNA fragment has been introduced.

  The transcription initiation control base sequence may be any sequence that expresses the transcription initiation function, and may be a viral promoter, for example, SV40-derived early promoter, cytomegalovirus LTR, Rous sarcoma virus LTR, MoMuLV-derived LTR, adenovirus-derived early promoter. A promoter of a constituent protein gene, for example, β-actin gene promoter, PGK gene promoter, transferrin gene promoter, CAG promoter is preferred.

  The spacer base sequence only needs to have a length sufficient to prevent the expression of the hyaluronic acid synthase gene from the transcription initiation control base sequence, and drug resistance genes such as neomycin, tetracycline, ampicillin, kanamycin, hygromycin A gene that confers resistance to a phosphinothricin drug; a gene that complements an auxotrophic mutation.

  The gene of hyaluronan synthase may be derived from vertebrates, and specifically, (U52524, U86408 D84424, U54804, U86409 literature: Accession No. D82964, Itano N., Kimata K .. J. Biol. Chem. 271: 9875-9878 (1996), Accession No.U59269, Shyjan AM, et al. J. Biol. Chem. 271: 23395-23399 (1996), Accession No.U52524, Spicer AP, et al. J. Biol. Chem. 271: 23400-23406 (1996), Accession No.U54804, Watanabe K., Yamaguchi YJ Biol Chem.271: 22945-22948 (1996), Accession No.U86408, U86409, Spicer AP, et al. J Biol. Chem. 272: 8957-8961 (1997), Liu N., et al. Cancer Res. 61: 5207-5214 (2001), mouse and human hyaluronic acid synthase 1 2 or 3 (for example, Mouse Has1, Has2, Has3, Human HAS1, HAS2, HAS3).

  The DNA fragment of the hyaluronic acid synthase gene introduction plasmid preferably has a transcription termination base sequence, that is, a terminator region, downstream of the polynucleotide encoding the hyaluronic acid synthase gene.

  The vector used for the preparation of the DNA fragment of the hyaluronic acid synthase gene transfer plasmid is not particularly limited as long as it can be administered to a non-human mammal, but it is not limited to retrovirus, adenovirus, adeno-associated virus, herpes virus, vaccinia virus. , Virus vectors such as poxvirus, poliovirus, Sindbis virus, Sendai virus may be used.

  The DNA fragment of the hyaluronic acid synthase gene-introduced plasmid may be one obtained by excising the transgene portion with Sal I and Sfi I restriction enzymes from a vector into which the hyaluronic acid synthase gene has been introduced.

  In the transgenic animal of the present invention inducible for expression of the hyaluronic acid synthase gene, this DNA fragment is introduced into a non-human mammal, preferably a mouse.

  This transgenic animal can induce the expression of the hyaluronic acid synthase gene using the properties of the DNA fragment of the hyaluronic acid synthase gene transfer plasmid. To introduce this DNA fragment, a method of microinjection into a fertilized egg; a viral vector method; a transfection method for introducing the vector into embryonic stem cells (ES cells); an infection method using a retroviral vector; electroporation This is done by general gene transfer techniques such as law.

  Said transgenic animals do not have a site-specific gene recombinase such as Cre recombinase. Therefore, the CAG promoter and the base sequence of the hyaluronic acid synthase 2 gene remain separated by the neomycin resistance gene, and transcription of the hyaluronic acid synthase gene is not induced.

  The hyaluronic acid synthase gene expression-inducible model animal of the present invention is an animal species obtained by mating this hyaluronic acid synthase gene expression-inducing transgenic animal and an animal of the same species that causes organ-specific diseases. It is.

  When a site-specific gene recombination enzyme acts on this hyaluronic acid synthase gene expression-inducible model animal, the induction of the hyaluronic acid synthase gene and the development of an organ-specific disease are expressed. Specifically, a hyaluronic acid synthase gene expression-inducible model animal is a hybrid animal that is hyaluronic acid synthase gene expression-inducible transgenic animal, and an animal that is the same species that has an organ-specific disease-causing animal. It can be obtained by mating animals with site-specific recombinase such as Cre recombinase. The offspring has a Cre recombinase and a hyaluronic acid synthase gene-introduced plasmid DNA fragment and an organ-specific disease-causing gene, each derived from a parent animal. Therefore, as shown in FIG. 1, this Cre recombinase acts on the DNA fragment of the hyaluronic acid synthase gene-containing vector, and the DNA recombination reaction causes gene recombination at two loxP sites in the transgene. As a result, one loxP base sequence and Neo + poly A base sequence are excised as circular DNA, and the remaining DNA fragments are recombined and short-circuited via the other loxP base sequence, causing homologous recombination. Finally, transcription of mouse hyaluronan synthase 2 (Has2) is induced from the CAG promoter, which is a transcription initiation regulatory base sequence.

  Such model animals can freely control the hyaluronic acid synthase gene expression-inducible expression time caused by site-specific gene recombination enzymes, and can also be used in various organ-specific disease-causing tissues. Gene expression-inducible expression can be controlled.

  Transgenic animals having site-specific recombinant enzymes such as Cre recombinase and Flp recombinase are available as various animal species and are described in Zadelaar SM, et al. Transgenic Res. 15: 31-36 (2006). SM-CreER (T2) (ki) / rosa26 mice, and commercially available (C57BL / 6-TgN (AlbCre) 21Mgn, Tg (Ins2-cre) 25Mgn, B6.Cg-Tg (Mx1-cre ) 1Cgn / J, B6.Cg-Tg (Nes-cre) 1Kln / J, B6.Cg-Tg (Tek-cre) 12Flv / J, B6.Cg-Tg (cre / Esr1) 5Amc / J, FVB-Tg (GFAP-cre) 25 Mes / J, STOCK Tg (MMTV-cre) 1 Mam / J, etc. (The Jackson Laboratory, Bar Harbor, Maine, USA).

  Organ-specific disease-causing animals can be obtained as various animal species having cancer-causing, arteriosclerosis-causing, organ fibrosis-causing cirrhosis, or inflammatory disease-causing disease including arthritis. Wakita, TJ Biol. Chem. 273: 9001-9006 (1998) and Wipke, BT, Allen, PMJ Immunol. 167: 1601-1608 (2001), and commercially available FVB / N-Tg (MMTVneu) 202Mul / J or B6.129P2-Apoetm1Unc / J (The Jackson Laboratory, Bar Harbor, Maine, USA) may be used. Of these, breast cancer-causing mice are preferred.

  The site-specific gene recombinase used in the vector is preferably a heterologous recombinase recognition sequence that is not recognized by a recombinase endogenous to a non-human mammal cell to be genetically introduced. Therefore, a combination of Cre recombinase which is a site-specific gene recombinase derived from bacteriophage P1 of E. coli and loxP sequence which is a recognition base sequence, or a site-specific gene recombination enzyme Flp recombinase derived from yeast and a recognition base sequence. Combinations with certain frt sequences are even more preferred.

  The DNA fragment of the hyaluronic acid synthase gene-introduced plasmid may be linked to the trans-expression induction control base sequence and the base sequence of the hyaluronic acid synthase gene.

  This DNA fragment has an inducible promoter which is a trans expression induction control base sequence whose expression is controlled in trans by an exogenous substance. When the inducible promoter is, for example, the promoter of the metallothionein-1 gene, transcription of hyaluronan synthase at a desired time by administering heavy metals, steroids, alkylating agents, chelating agents, and cytokines as exogenous substances. Is induced.

  The non-human mammal into which the DNA fragment of the hyaluronic acid synthase gene transfer plasmid is introduced is preferably a mouse, rat, hamster, rabbit, or pig. Among them, a mouse is more preferable, and either male or female may be used.

  Hereinafter, examples of producing a DNA fragment of the hyaluronic acid synthase gene-introduced plasmid of the present invention, a hyaluronic acid synthase gene expression-inducing transgenic animal, and a hyaluronic acid synthase gene expression-inducing model animal will be specifically described. To do.

(Example 1: Preparation of DNA fragment of hyaluronic acid synthase gene introduction plasmid)
Mouse Has2 cDNA [Accession No. U52524, Spicer, AP et al. J. Biol. Chem., 271: 23400-23406 (1996) by PCR using a mouse brain-derived cDNA library (Clontech, Mountain View, CA, USA) as a template. )]. According to a known method [Itano, N. et al, J. Biol. Chem., 274: 25085-25092 (1999)], BglII and SmaI were added to pFLAG-CMV2 vector (Sigma-Aldrich, St. Louis, MO, USA). A nucleotide sequence encoding a FLAG tag was added to the amino terminus by inserting it into the restriction enzyme cleavage site to produce a FLAG-mouse Has2 DNA fragment. This FLAG-mouse Has2 DNA fragment was inserted into the SacI and SmaI sites of the vector pCALNL5 (obtained from RIKEN Bioresource Center DNA Bank) using TDNA ligase (New England Biolabs, Inc. Beverly, MA, USA).

  A vector incorporating the FLAG-mouse Has2 cDNA fragment was named pCALNL5-Has2. A DNA fragment was excised from this CALNL5-Has2 vector using SalI and SfiI restriction enzymes (New England Biolabs, Inc. Beverly, MA, USA) to obtain a DNA fragment of a hyaluronic acid synthase gene transfer plasmid (FIGS. 1 and 2). .

  The pCALNL5 vector contains a CAG promoter (a combination of a chicken β-actin promoter sequence and a cytomegalovirus immediate early enhancer sequence), a first loxP base sequence on the 5 ′ end side, a neo resistance gene (Bgl II− from pSV2neo) Sma I fragment 1.0 kb), containing the second loxP strand at the 3 ′ end and the poly A signal from pCAGGS. This pCALNL5 vector has a known full nucleotide sequence, Kanegae, Y. et al., Gene, 181 (1-2), 207-12 (1996), Niwa, H. et al. Gene, 108 (2), 193-9 (1991) and Kanegae, Y. et al., Nucleic Acid Res., 23 (19), 3816-21 (1995).

(Example 2: Transgenic animal inducible for expression of hyaluronic acid synthase gene)
The DNA fragment of this hyaluronic acid synthase gene-introduced plasmid was prepared according to a known method [Hogan, B et al, “Manipulating the mouse embryo. A Laboratory Manual” Cold Spring Harbor Laboratory (1986)]. A Has2 gene expression-inducible transgenic mouse was produced by injecting fertilized eggs derived from (manufactured by Co., Ltd.) into the fertilized eggs by microinjection and transplanting the fertilized eggs into the uterus of ICR mice to obtain offspring.

  Among the offspring, transgenic mouse strains in which the transgene is inserted into the chromosome are obtained using PCR primers (polymerase chain) as shown in the test method described below using DNA primers (Table 1) synthesized based on the Has2 gene sequence. reaction) method.

  In addition, a tissue extract was prepared from breast cancer of a transgenic mouse, and the expression of FLAG-Has2 fusion protein was detected using an anti-FLAG antibody by Western blot analysis shown below. In addition, RNA was extracted from breast cancer tissue, cDNA was synthesized using reverse transcriptase, and then the expression of Has2 gene was analyzed by real-time PCR shown below.

  When this transgenic mouse is mated with a C57BL / 6J mouse, the chromosomal DNA is extracted from the biopsy sample of the tail when the offspring mouse is 3 weeks old, and the gene incorporated by the PCR method as shown in the test method described later The presence of was screened.

(Example 3: Transgenic animal for disease model)
In order to spontaneously develop breast cancer in the Has2 gene expression-inducible transgenic mouse prepared in Example 2, FVB / N-Tg (MMTVneu) 202Mul / J (The Jackson Laboratory, Bar Harbor, Maine) , USA) and a breast cancer-onset Has2 gene expression-inducing transgenic mouse was obtained. Identification of Has2 / Neu double transgenic mice carrying both Has2 and Neu genes by PCR using DNA primers specific for each gene listed in Table 1 as shown in the test method described below. did.

  In parallel, MMTV-Neu transgenic mice and MMTV-Cre transgenic mice that specifically express Cre gene recombinase in the mammary gland (STOCK Tg (MMTV-cre) 1Mam / J, The Jackson Laboratory, Bar Harbor, Maine, USA) and Cre / Neu double transgenic mice expressing both the Neu and Cre genes in the mammary gland were prepared.

  The combinations of transgenes were analyzed by PCR using DNA primers specific for each gene listed in Table 1 as shown in the test method described later.

  Has2 / Neu mouse and Cre / Neu mouse mated and have 4 different combinations of genotypes (Neu, Cre / Neu, Has2 / Neu (Has2 + Neo), and Has2 / Neu / Cre (Has2ΔNeo)) Transgenic mice were obtained.

  Of these, females of the same age (6 months of age) with Has2 + Neo and Has2ΔNeo genotypes were then used to promote the development of breast cancer in FVB / N male mice (FVB / NJcl, CLEA Japan Inc., Tokyo, Japan).

  Various tests were performed on the transgenic mice obtained in Examples 2 and 3 as follows.

(Test 1: Genotype analysis by PCR method)
The tail DNA of each transgenic mouse was extracted according to a standard method, and PCR reaction was performed using Gene Amp PCR System 9700 (manufactured by Applied Biosystems) using DNA primers described in the following table. In the case of the Has2 and Neu genes, the PCR reaction was held at 94 ° C for 2 minutes, followed by a cycle consisting of denaturation at 94 ° C for 45 seconds, annealing at 59 ° C for 1 minute, and extension reaction at 72 ° C for 1 minute. Repeated 35 times, and finally, an extension reaction was performed at 72 ° C. for 7 minutes. In the case of the Cre gene, hold at 94 ° C for 3 minutes, and then repeat 35 cycles of 94 ° C denaturation for 30 seconds, 55 ° C annealing for 1 minute, and 72 ° C for 1 minute extension. The extension reaction was performed at 72 ° C. for 2 minutes. The amplified DNA fragment was subjected to agarose gel electrophoresis, stained with ethidium bromide, and a band was detected by infrared rays.

(Test 2: Confirmation of gene recombination reaction by expression of Cre recombinase)
Approximately 2 weeks after the onset of breast cancer, the cancer tissue was surgically excised, and genomic DNA was prepared according to a standard method. The success or failure of gene recombination was confirmed by PCR gene amplification using primers (GAG forward, Neo reverse, Has2-7 reverse) synthesized based on the CAG promoter, neomycin resistance gene and the base sequence of mouse Has2 gene. The amplified DNA fragment was subjected to agarose gel electrophoresis and stained with ethidium bromide to detect and calculate the size. The gene recombination reaction before and after expression of the Cre enzyme was analyzed by amplification and size change of the DNA fragment. In addition, after agarose gel electrophoresis, DNA was purified from the gel fragment excised from the gel using High pure PCR product purification kit (Roch Diagnostics GmbH), and pGEM-T Easy vector system I (Promega Corp) was purified. And incorporated into a plasmid vector. The base sequence of the vector was analyzed by ABI Prism 310 Genetic Analyzer (Applied Biosystems) to confirm gene recombination at the loxP sequence site by Cre recombinase.

(Test 3: Measurement of hyaluronic acid content)
Approximately 2 weeks after the onset of breast cancer, the cancer tissue was surgically removed and disrupted in 200 ml of cell lysate (50 mM Tris-HCl, 100 mM EDTA, 100 mM NaCl, 500 mg / ml protease K, pH 8.0). The reaction was adjusted at 50 ° C for a whole day and night. The amount of hyaluronic acid in the tissue was measured with reference to Itano, N. et al. J. Biol. Chem. (2004) 279, pp18679-18687. Briefly, a 96-well plate was first coated with hyaluronic acid crosslinked with BSA. Next, b-HABP (a hyaluronic acid probe in which the N-terminal domain of bovine cartilage-derived aggrecan was biotinylated) and a sample containing hyaluronic acid were mixed and added to wells coated with BSA-crosslinked hyaluronic acid. After incubating at 37 ° C. for 1 hour, the plate was washed 3 times with TBS (50 mM Tris-Hcl, 0.15 M NaCl, pH. 7.5) containing 0.2% Tween 20. Streptavidin cross-linked with horseradish peroxidase was added to each well and incubated at 37 ° C. for 1 hour. Each well was washed 3 times with TBS containing 0.2% Tween 20, and then DAB (diaminobenzidine) chromogenic substrate was added and incubated at 37 ° C. for 1 hour. Absorbance at 490 nm was measured with a microplate reader, and the hyaluronic acid content in the tissue was calculated using a calibration curve obtained from a standard sample of hyaluronic acid with a known concentration.

(Test 4: Analysis of Has2 gene expression by real-time RT-PCR)
Total RNA was isolated from the removed breast cancer using RNeasy total RNA Isolation kit (QIAGEN). Using 200 ng equivalent of total RNA as a template, a reverse transcription reaction was performed by reacting at 42 ° C. for 1 hour with a reverse transcription reagent (manufactured by Takara Biochemicals) using a random primer. Real-time quantitative PCR was performed according to Itano, N. et al. J. Biol. Chem. (2004) 279, pp18679-18687. The relative expression level of the Has2 gene was calculated as the ratio of the relative amount of GAPDH mRNA measured as a control using TaqMan rodent GAPDH detection reagents (Applied Biosystems, Inc.).

(Test 5: Western blot analysis)
The cancer tissue was crushed in RIPA buffer (10 mM sodium phosphate, 150 mM NaCl, 0.1% SDS, 1% NP-40, 1% sodium deoxycholate and protease inhibitors, pH 7.2) to prepare a lysate. The lysate (10 μg protein equivalent) was separated by SDS polyacrylamide gel electrophoresis and electrically transferred to a PVDF nylon membrane. The mixture was reacted with an anti-FLAG antibody for 1 hour at room temperature. After washing 3 times with TBS containing 0.2% Tween 20, an anti-mouse antibody cross-linked with horseradish peroxidase was used as a secondary antibody and reacted at room temperature for 30 minutes. After washing three times with TBS containing 0.2% Tween 20, the reaction was performed with an ECL Western blot detection reagent, and the luminescence generated from the FLAG-fused Has2 protein band was exposed to an X-ray film and detected.
The results of these tests are as follows.

(Test result A: Results for Has2 gene expression inducible transgenic mice)
Among the offspring obtained by introducing the inducible expression vector of the Has2 gene into fertilized eggs, the gene was introduced into 9 strains as a result of PCR analysis. Of these, one strain with the highest expression of the introduced Has2 gene was selected as an inducible transgenic mouse. The established Has2 gene expression-inducing transgenic mice appeared to be normal in appearance and maintained fertility.

(Test result B: Results of mast breast cancer model mice using Has2 gene expression inducible transgenic mice)
The Has2 gene expression inducible transgenic mouse of the present invention and a breast cancer model mouse MMTV-Neu are crossed, and among the resulting offspring, a Has2 / Neu double transgenic mouse having both Has2 and neu genes is obtained by PCR. Sorted. Cre / Neu double transgenic that expresses both neu and cre genes in the mammary gland by crossing between MMTV-Neu transgenic mouse and MMTV-Cre transgenic mouse designed to express Cre recombinase specifically in mammary gland Mice were made. Has2 / Neu double transgenic mice and Cre / Neu double transgenic mice are crossed and 4 different combinations of transgenic mice (Neu, Cre / Neu, Has2 / Neu (Has2 ^{+ Neo} ) and Has2 / Cre / Neu (Has2 ^ΔNeo )).

(Test result C: Result of Cre-loxP gene recombination in inducible transgenic mice)
Among transgenic mice having the Has2 ^{+ Neo} and Has2 ^ΔNeo genotypes, female mice of the same age (6 months old) were mated with FVB / N male mice to promote the development of breast cancer. The developed breast cancer tissue was surgically excised to prepare genomic DNA. Then, using genomic DNA as a template, gene amplification by PCR and determination of the base sequence of the amplified gene product were performed, and the success or failure of the gene recombination reaction by Cre enzyme was determined.

Specifically, a genomic DNA prepared from a tumor in the Cre non-expression group (Has2 ^{+ Neo} ) was used as a template, and a PCR reaction (# 1 reaction) with a CAG promoter and a neomycin resistance gene primer was performed. Fragment amplification was observed. On the other hand, when DNA ^prepared from the Cre expression group (Has2 ^ΔNeo ) was used as a template, amplification of a DNA fragment of the same size was not observed. In addition, as a result of PCR (reaction of # 2) using the CAG promoter and Has2 gene primer, gene amplification of a DNA fragment having a size of 1880 bp was observed in the Cre non-expression group, but 670 bp in the Cre expression group. Gene amplification was observed. This result indicates that a neomycin resistance gene sandwiched between two loxP sequences was excised by genetic recombination. As a result of isolating the amplified DNA fragment of 670 bp and determining the base sequence, it was confirmed that gene recombination occurred with two loxP sequences by Cre recombination enzyme, and neomycin resistance gene was excised ( (See FIG. 3 showing a photograph in which the success or failure of the gene recombination reaction by the Cre enzyme was determined by the PCR method). None of the gene amplifications were observed when using a sample of breast cancer developed in Cre: Neu and Neu mice. M represents a migration image of the DNA size marker, and the numerical value on the side surface represents the size of the marker.

  As a result of measuring the hyaluronic acid content by adjusting the tissue extract from the breast cancer tissue, excessive production of hyaluronic acid was not observed in the absence of Cre. On the other hand, in the breast cancer of the Cre expression group, the hyaluronic acid content increased more than 5 times compared to the Cre non-expression group (see Table 2).

Hyaluronic acid sugar chain length was examined by gel filtration chromatography. In comparison with the control group (Has2 + Neo, Cre: Neu and Neu: black circle), the acceleration of tumor formation rate was observed in Has2ΔNeo (white circle). It was revealed that hyaluronic acid from Has2 ^ΔNeo breast cancer was increased in the amount of high molecular hyaluronic acid and low molecular hyaluronic acid compared to Has ^{2 + Neo} breast cancer (see FIG. 4).

  As a result of real-time quantitative PCR, the expression of Has2 was about 16 times higher in the breast cancer of the Cre expression group than in the non-expression group (Table 2). From the above results, it was revealed that the inducible transgenic mouse of the hyaluronic acid synthase gene of the present invention increases the expression of the introduced Has2 hyaluronic acid synthase gene and the synthesis of hyaluronic acid by the expression of Cre.

(Evaluation test: Evaluate the utility value as a disease model animal in the above model experiment system for developing breast cancer)
The utility value of the inducible transgenic mouse of the hyaluronic acid synthase gene of the present invention as a disease model animal was evaluated in the above-described model experimental system for developing breast cancer.

Transgenic mice with Has2 ^{+ Neo} and Has2 ^ΔNeo genotypes have neu genes for carcinogenesis experiments, and breast cancer occurred frequently in female mice due to pregnancy due to the Neu oncogene.
The incidence of this tumor was significantly increased in the Cre expression group, that is, the group in which hyaluronic acid production was artificially increased.

  As shown in FIG. 5, as a result of measuring the growth rate of the tumor after the onset of the tumor, compared to the control group (Has2 + Neo, Cre: Neu and Neu: black circle), in the Cre expression group Has2ΔNeo (white circle) An increase in the rate of tumor formation was observed.

  FIG. 6 is a photograph of a histopathological analysis result of breast cancer under Cre expression and non-expression. As shown, the histopathological analysis of breast cancer with and without Cre expression revealed host fibroblast invasion and significant stromal formation in the breast cancer tissue in Has2ΔNeo mice compared to Has2 + Neo mice. It was. The lower part shows a strong magnified image.

  FIG. 7 is a photograph of intrastromal stromal reaction and blood vessels observed under a fluorescent microscope by immunofluorescence staining. The upper left and lower photographs are images observed by staining blood vessels in the tumor with an anti-CD31 antibody, and the upper right and lower photographs are images stained with an anti-type I collagen antibody and an anti-fibronectin antibody, respectively. As shown, Has2ΔNeo tumors have more microvessels than Has2 + Neo tumors and are composed of extracellular substrates such as fibronectin and type I collagen produced by host fibroblasts in Has2ΔNeo tumors. It became clear that it was.

  FIG. 8 shows the size distribution of tumor blood vessels. The tumor blood vessel size distribution was measured under a fluorescent microscope after immunostaining with anti-CD31 antibody. As a result, in the Cre expression group of breast cancers, significant formation promotion of tumor blood vessels, which seems to be a result of stromal reaction, was observed. In particular, it was revealed that the number of microvessels in Has2ΔNeo tumor is about twice that of Has2 + Neo tumor.

  The biological reaction as described above is observed in hard cancers of extremely high malignancy among human breast cancers. Therefore, in this example, hyaluronic acid-specific hyaluronic acid is introduced into a hyaluronic acid synthase gene-inducible transgenic mouse. It was proved that it can be a model animal of malignant breast cancer by imparting production ability.

  The inducible transgenic animal into which the hyaluronic acid synthase gene of the present invention has been introduced can be obtained by using hyaluronic acid under the control of a site-specific gene recombination enzyme such as Cre by using an inducible gene expression system such as Cre-loxP. Production can be artificially controlled.

  As described above, when a site-specific gene recombination enzyme such as Cre is expressed specifically for the development of an organ-specific disease such as breast cancer, a significant stromal reaction is induced in cells such as tumors. The stromal reaction proceeds with the proliferation of fibroblasts and the accumulation of extracellular matrix such as fibronectin and type I collagen that they produce. Fibrosis of various organs is caused by such a remarkable interstitial reaction. For example, in cirrhosis, a persistent inflammation caused by hepatitis virus or the like causes a stromal reaction in the liver, resulting in liver sclerosis and dysfunction. Therefore, the hyaluronic acid synthase gene expression inducible transgenic mouse of the present invention is a cancer in which abnormal synthesis of hyaluronic acid is deeply linked to disease progression, invasion / metastasis of such cancer, as well as arteriosclerosis and cirrhosis. It can be applied as a model animal for fibrosis of various organs.

  When assuming a model animal of cirrhosis, by crossing a transgenic animal that expresses a site-specific gene recombinase such as Cre specifically with the liver, and a transgenic animal that induces hyaluronic acid synthase gene expression, It is possible to induce an increase in hyaluronic acid production in the liver. Next, a model animal of cirrhosis can be created by inducing an inflammatory state by liver injury.

  The feature of the present invention is that, in addition to the liver, by expressing a site-specific gene recombinase such as Cre specifically in the lung, kidney, artery, etc., organs such as lung fibrosis, renal fibrosis, arteriosclerosis A model animal that develops fibrosis can be easily and economically generated.

  An inducible transgenic animal of the hyaluronic acid synthase gene of the present invention capable of inducing a stromal reaction under the control of a site-specific gene recombinase such as Cre is an expression site of a site-specific gene recombinase such as Cre It is possible to control the expression of hyaluronic acid synthase and the production of hyaluronic acid spatially and spatially in all tissues and organs. Spatiotemporal expression control of Cre recombinase is possible by controlling the gene encoding it with a tissue-specific promoter or by expressing it locally using a viral vector.

  Furthermore, by constructing a site-specific gene recombinase expression system such as Cre, it is possible to induce hyaluronic acid production at any place and at any time as needed, and animals that also have new diseases Can be created.

  These model animals are extremely useful for supporting preclinical animal studies in the development of therapeutics and therapeutic agents for the treatment of the above diseases.

It is a figure which represents typically the structure of the DNA fragment of the hyaluronic acid synthase gene introduction plasmid used for the transgenic animal production to which this invention is applied, and gene recombination by Cre recombinase.

It is a figure which shows a part of gene sequence of the DNA fragment of the hyaluronic acid synthase gene introduction | transduction plasmid to which this invention is applied.

It is the photograph which judged the success or failure of the gene recombination reaction by Cre enzyme by PCR method about the transgenic animal for pathological models to which this invention is applied.

It is a graph which shows the result of having analyzed the hyaluronic acid sugar chain length by the gel filtration chromatography about the transgenic animal for a disease state model to which this invention is applied.

It is a graph which shows the result of having measured the growth rate of the tumor about the transgenic animal for pathological models to which this invention is applied.

It is a photograph which shows the histopathological-analysis result of the breast cancer under Cre expression and non-expression about the transgenic animal for pathological models to which this invention is applied.

It is the photograph which observed the intrastromal stromal reaction and the blood vessel under the fluorescence microscope by immunofluorescent dyeing | staining about the transgenic animal for pathological models to which this invention is applied.

It is a graph which shows the size distribution of a tumor blood vessel about the transgenic animal for pathological conditions to which this invention is applied.

Claims (6)

It is sandwiched between the transcription initiation regulatory base sequence, the recognition base sequence of the pair recognized by the site-specific gene recombinase, and the inactive spacer base sequence cut out by the enzyme and the base sequence of the gene of hyaluronic acid synthase Has2 or trans induced expression control nucleotide sequences, nucleotide sequence of the gene of hyaluronan synthase Has2 is 5 distally 'to 3' end, the DNA fragment has led in this order Ruhi hyaluronic acid synthase gene Has2 introduced plasmid , Introduced into non-human mammals with breast cancer-onset genes,
Residues of the DNA fragment from which the spacer base sequence was excised by the enzyme by mating with the same kind of parent animal having the site-specific gene recombinase are recombined via one of the recognition base sequences and short-circuited. A hyaluronic acid synthase gene having Has2 genotype in which Has2 transcription is induced by recombination and induced by a substance that induces Has2 transcription at the trans-expression-inducing regulatory base sequence Consisting of animal species of expression-inducing transgenic animals,
The animal species is naturally pathogenic by the DNA fragment, the site-specific gene recombinase that expresses the gene of the hyaluronic acid synthase or the trans-expression-inducing control base sequence, and the breast cancer-causing gene. A disease model animal of hard cancer, characterized in that said breast cancer is onset in the mammary gland. The pathological model animal according to claim 1, wherein the pair of recognition base sequences is a loxP base sequence or a Frt base sequence.   The site-specific gene recombinase is Cre recombinase, and the inactive spacer base sequence is the base sequence of the neomycin resistance gene Neo sandwiched between the pair of loxP base sequences, so that the Cre recombinase is The pathological model animal according to claim 2, wherein the Neo base sequence is excised by the homologous recombination to form the Has2 genotype in which the transcription of the Has2 is induced. The disease model animal according to claim 1 , wherein the non-human mammal is a mouse, a rat, a hamster, a rabbit, or a pig. The disease state according to claim 1, wherein the animal species is the transgenic animal that induces expression of the hyaluronic acid synthase gene expression and the animal species that is the same species and mates the animal that develops breast cancer. Model animal . The animal species is a cross of the hyaluronic acid synthase gene expression-inducing transgenic animal and the same species of the breast cancer-causing animal, and an animal having the site-specific recombinase is crossed. It is an animal species, The disease state model animal of Claim 1 characterized by the above-mentioned .

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