JP2000316420A - Transgenic miniature pig - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a transgenic miniature pig as a human disease model, having a human or a miniature swine transduced gene, especially having an transduced human disease causative gene or a miniature swine homologous gene thereof, and useful as a human disease model animal and a donor for organ transplantation. SOLUTION: This transgenic miniature pig is the one having a transduced human or a miniature swine gene. The human disease causative gene is preferably the one based on a single gene mutation and/or the dominant form one. The homologous gene of the human disease causative gene in the miniature pig is preferably a cDNA of a mini swine huntingtin gene having a base sequence of the formula, or a cDNA of the mini swine huntingtin gene of the sequence in which the addition or deletion of at least one or more CAG codons or CAA codons is present in the 179th to 403rd bases in the sequence of the formula.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a transgenic minipig useful as a human disease model animal and as a donor for organ transplantation, and more particularly to a human disease model transgenic transfected with a human disease-causing gene or a homologous gene thereof in a minipig. About mini pigs.

[0002]

2. Description of the Related Art To date, transgenic animals have been reported in many animal species. A transgenic animal is a non-human animal into which at least one foreign gene has been incorporated, and has the gene in some or all of its cells. Initially, research was mainly conducted on the production of transgenic mice,
Since then, rabbits, sheep, pigs, cattle, goats, etc. have been produced. Technology for animal embryo manipulation and gene transfer has already been established for mice, and research has been made on livestock such as cows, pigs, and goats (Japanese Patent Application Laid-Open No.
298981, JP-A-10-150882, etc.).

[0003] In transgenic mice known so far, the "difference in complexity" from humans becomes a barrier,
Extrapolation to humans by analysis using transgenics as a disease model was difficult. Therefore, it has been necessary to create a disease model using a higher-order animal than a rodent such as a mouse. Among them, pigs are often physiologically and anatomically similar to humans, and their usefulness as experimental animals has been pointed out. However, since adult pigs weigh over 100 kg, their handling is very inconvenient, and it has not been possible to manage adult pigs in most research facilities. Many research facilities weigh 15-
Although piglets of about 20 kg are used, this is an immature individual like a baby in humans, and it is difficult to estimate the case of adult humans from experimental data obtained from such piglets. was there.

[0004] From such a point of view, mini-pigs are attracting attention as experimental animals because of easy handling and breeding management in experiments. Miniature pigs are thought to be similar to humans in anatomy and physiological characteristics like pigs, and mature miniature pigs have clearly different biological responses even if they are the same size as piglets, It is considered promising as a human disease model animal and as a donor for xenotransplantation. Miniature swine are suitable for pathological analysis because they have a complexity similar to humans compared to rodents. For example, when a high-fat diet is given to a miniature pig, a hyperlipidemia model can be created, and it has been proved to be useful as a model for angina pectoris.

On the other hand, Huntington's disease is a so-called triplet repeat disease (Osuka et al., Clinical Laboratory, vol. 43, no.
2, 1999), a progressive neurodegenerative disease with an autosomal dominant inheritance pattern, with specific involuntary movements, mental disorders and intellectual disorders. Pathologically, it is characterized by the selective degeneration of the middle spiny neuron in the caudate nucleus and putamen of the cerebrum. Experimental research using model animals is essential for the diagnosis and development of treatment methods for Huntington's disease, but it is thought that mouse and rat, which are common experimental animals, are deeply involved in the development of Huntington's disease Even if an attempt is made to induce elongation of the CAG / CAA repetitive sequence, so-called “dynamic mutation” does not occur due to the absence of polymorphism, which is a prerequisite thereof, and it cannot be considered as an appropriate model animal. In fact, it is known that a gene causing Huntington's disease is introduced into mice (Mangiari
ni et al., Cell, vol. 87, 493-506, 1996) were difficult to analyze due to their small size, and treatment was difficult even if symptoms were observed.

[0006] The present inventors have previously described the chromosomal DNA of minipigs.
Isolated the gene encoding huntingtin, a protein related to Huntington's disease, and analyzed the nucleotide sequence of its cDNA. As a result, it was found that the miniature swine huntingtin gene has a CAG / CAA repeat polymorphism. ,
We have filed applications for various genetically engineered materials that enable experimental manipulation of the gene (Japanese Patent Application No. 09-314020).
issue).

[0007]

However, the creation of a disease model in minipigs is performed by dieting, drug administration or surgical treatment as described above, and examples of genetic modeling have been reported so far. I didn't. Therefore, the present inventors have found that an adult weight of 25
Production of transgenic animals using the microinjection method was studied in minipigs weighing ３０30 kg and having anatomical physiological similarity to humans.

[0008] Our studies have shown that immature female minipigs do not induce tolerance during estrus and uterine maturation, unlike porcines, although hormone treatment induces superovulation. Therefore, natural mating cannot be expected, and artificial fertilization must be performed. However, even if semen is injected from the outside through the vagina, the muscular layer of the uterus is undeveloped, there is no peristalsis, and the injected semen leaks out as it is,
Fertilization was not achieved, and this particularly made it difficult to produce transgenic miniature pigs.

[0009] Therefore, by solving these problems and establishing gene transfer technology in miniature swine, it can be used as a human disease model animal for research on etiology elucidation, treatment and prevention, drug development, and for organ donation. It has been desired to produce transgenic minipigs that are also useful as donors (cerebral nerve cells, heart, lung, liver, pancreas, kidney, cornea, skin, etc.).

[0010]

As a result of various studies, the present inventors succeeded in collecting fertilized eggs by performing laparotomy on female minipigs in which follicular growth and ovulation were induced, and performing artificial insemination. However, they have found that a transgenic miniature pig can be produced by transplanting a fertilized egg into which a gene has been introduced by a microinjection method into a recipient female.

Accordingly, the present invention relates to transgenic minipigs into which human or minipig genes have been introduced, which are useful as human disease models and donors for organ donation.
The foreign gene to be introduced is not particularly limited as long as it can be introduced into a miniature swine fertilized egg by the method of the present invention, and is not particularly limited. Is preferably a homologous gene thereof. When used as a donor for organ donation, the range of organs that can be considered as a donor for xenogeneic organ transplantation into humans can be all organs other than the brain as transplant candidates, and specifically, Examples include nerve cells, heart, lung, liver, pancreas (especially Langerhans islet cells), kidney, cornea, skin, and the like, which can be used for treating Parkinson's disease and the like. In this case, the introduced gene is, for example, a gene having a function of reducing rejection in xenotransplantation, or a gene having a function of expecting an increase in the survival rate.

The present invention further relates to a human disease model transgenic mini-pig into which a human disease-causing gene or a homologous gene thereof in mini-pigs has been introduced. The human disease-causing gene may be introduced into minipigs and may be any as long as the resulting transgenic minipigs can be subjected to research as a human disease model animal, and are not particularly limited, but may be based on mutations of a single gene, It is preferably a human disease-causing gene in a dominant form.
Further, as a human disease-causing gene, a causative gene of triplet repeat disease can be suitably used. Triplet repeat disease is known to have a disease in which a three-base repeat sequence such as CAG and CCG has been abnormally elongated and found to occur in healthy individuals (Osuka et al., Clinical Laboratory, vol. 43, no.2). , 1999).

[0013] In the present invention, the human disease-causing gene or the homologous gene thereof in minipigs used as the transgene may be the full-length cDNA gene or the cDNA gene if the resulting transgenic minipig can be used as a human disease model transgenic minipig. Gene fragment, full length gene of genomic DNA, or genomic DN
Any of the gene fragments of A may be used and is not particularly limited.

Particularly preferred transgenes in the present invention include a gene causing human Huntington's disease (hereinafter sometimes referred to as HD gene) or a homologous gene thereof in minipigs. In particular, the minipig huntingtin gene (homologous gene to the human HD gene) filed by the present inventors earlier is an example of a gene that can be suitably used. For example, a miniature pig having the nucleotide sequence of SEQ ID NO: 1
Huntingtin gene cDNA, or a mini-swine huntingtin gene having a nucleotide sequence in which at least one or more CAG codons or CAA codons are added or deleted in the nucleotide sequence of bases 179 to 403 in the nucleotide sequence of SEQ ID NO: 1 cDNA is preferably used. The human HD gene has a CAG repeat sequence exhibiting polymorphism in its exon, and it is considered that the specific increase of the CAG repeat sequence is deeply involved in the development of Huntington's disease. In the case of humans, the CAG repetition number due to polymorphism in healthy individuals is about 9-30, but when this exceeds 36, it is considered that Huntington's disease develops, and furthermore, the onset age becomes younger with the increase in the repetition number. Have been. Therefore, in order to create a model animal for Huntington's disease, it is necessary to consider the length (number of repeats) of this repetitive sequence in the transgene. When creating a human disease model transgenic minipig in the present invention, CAG in the transgene
The repeat sequence is preferably 30 or more, more preferably 36 or more. Further, the disease gene to be introduced does not necessarily require the full length of the gene, and the cDNA of the miniature pig huntingtin gene having the nucleotide sequence of SEQ ID NO: 1 or the 17th nucleotide in the nucleotide sequence of SEQ ID NO: 1 is not required.
It may be a DNA fragment consisting of a partially continuous sequence of the cDNA of the mini swine huntingtin gene having a nucleotide sequence in which at least one or more CAG codons or CAA codons are added or deleted in the sequence of nucleotides 9 to 403. . In this case, the fragment is 179-40 in the nucleotide sequence of SEQ ID NO: 1.
Any partial nucleotide sequence of the cDNA of the miniature pig huntingtin gene having a nucleotide sequence in which at least one or more CAG codons or CAA codons are added or deleted in the sequence of the third base (please confirm) ,
The effect is expected if it is 10 bp or more, but it is more preferable if it contains a CAG repeat sequence.

Further, the present invention provides a transgenic minipig line established using the above-mentioned transgenic minipig into which a human or minipig gene has been introduced as a founder. The present invention further relates to organs, tissues, eggs, sperm, and fertilized eggs derived from the above-described transgenic minipigs or transgenic minipigs, or cell lines established from the above-described transgenic minipigs or transgenic minipigs. provide.

Further, the present invention provides a transgenic mini-pig as described above, or a cloned mini-pig produced from the transgenic mini-pig strain.
Furthermore, the present invention relates to a medicament for treating and / or preventing a disease, which uses the above-mentioned transgenic mini-pig or the above-mentioned transgenic mini-pig strain, the above-mentioned organ, tissue and / or the above-mentioned cell line. A screening method is provided. Furthermore, the present invention relates to a method for producing the above-mentioned transgenic miniature pig.

[0017]

DESCRIPTION OF THE PREFERRED EMBODIMENTS Construction of Transgene The transgene is not particularly limited as long as it can be introduced into fertilized mini swine eggs by the method of the present invention, and is not particularly limited. It is preferably a disease gene. The disease gene may be any gene that can be introduced into a miniature swine and the obtained transgenic miniature swine can be subjected to research as a human disease model animal, and is not particularly limited, but is based on a mutation of a single gene, and A / cause of a dominant form of a human hereditary disease or a homologous gene thereof in minipigs is preferred, and preferably, for example, a human HD gene or a homologous gene thereof in minipigs. In particular, the mini-pig huntingtin gene (homologous gene to the human HD gene) filed by the present inventors is an example of a gene that can be suitably used (Japanese Patent Application No. 9-31).
No. 4020). For example, in the cDNA of the miniature pig huntingtin gene having the nucleotide sequence of SEQ ID NO: 1, or in the sequence of nucleotides 179 to 403 in the nucleotide sequence of SEQ ID NO: 1,
A cDNA of a mini-swine huntingtin gene having a nucleotide sequence in which at least one or more CAG codons or CAA codons are added or deleted, and a DNA fragment consisting of a partially continuous sequence thereof are preferably used. In this case, the fragment is 179-403 in the nucleotide sequence of SEQ ID NO: 1.
Any partial nucleotide sequence of the cDNA of the miniature pig huntingtin gene having a nucleotide sequence in which at least one or more CAG codons or CAA codons are added or deleted in the sequence of the base number, and the effect is 10 bp or more Is expected, but it is more preferable to use a CAG repeat sequence.

As described above, the human huntingtin gene is considered to be closely related to the development of Huntington's disease due to the specific increase in the CAG repeat sequence showing the polymorphism.
The number of CAG repeat sequences in the transgene is preferably 30 or more, more preferably 36 or more. Therefore, when aiming to develop Huntington's disease in miniature pigs,
The transgene is constructed as follows.

First, cDNA of the human huntingtin gene
Using a DNA fragment derived from the PCR-amplified human HD genome as a probe, cDNA of the mini-porcine huntingtin gene, which is a homologous gene of the human hunting-tin gene, is isolated and identified from, for example, a minipig testis-derived cDNA library.
Next, using the obtained mini-porcine huntingtin gene as a material, a CAG repeat sequence fragment separately prepared by PCR was inserted into a repeat site in exon 1 using a random insertion method known to those skilled in the art, A mini-porcine huntingtin gene fragment containing multiple glutamic acids is obtained. This fragment was incorporated into an introduction vector,
To construct. As the vector, any of those commonly used in the art can be suitably used and are not particularly limited.
As the promoter used in the vector for introduction, those known in the art (for example, CMV, EF
1α or the like (Invitrogen) can be used, and is not particularly limited. For example, rat NSE promoter specifically expressed in nerve cells (Sakimura et al., PNAS. 82.7453-74)
57, 1985; Sakimura et al., Gene 60, 103-113, 1987) and a promoter which is expressed systemically, and can be appropriately used depending on the type and purpose of the gene. Depending on the type of transgene, it may be fatal. In such a case, a tissue-specific promoter is preferable to a tissue-specific promoter instead of a systemically expressed promoter to prevent death at an early stage of development. on the other hand,
When there is no particular reason, it may be reasonable to select a promoter that is expressed systemically.

Disease Genes, Promoters and Poly A
The gene for introduction having a signal sequence is digested with a unique site of a restriction enzyme, for example, SphI or ClaI, and the obtained gene fragment is purified by, for example, electroelution, and added to an injection buffer so as to have a concentration of 3.7 μg / ml. Dissolve and store at -20 ° C.

Collection of fertilized eggs Immature female miniature pigs (140-150 days old, weight 14-1
8 kg) to induce ovulation, follicle growth hormone such as 、 ampoule PG600 (Intervet)
) Or 500 units of eCG (pregnant horse serum gonadotropin; PMS, Nippon Zenyaku Kogyo) intramuscularly, and after a lapse of time determined by the physiology and experience of miniature pigs, preferably 70 to 80 hours Later, an ovulation-inducing hormone, for example, 100 or 500 units of hCG (human chorionic gonadotropin; Peverogen, Sankyo) is injected intramuscularly and subjected to superovulation.

The abdomen is opened 100 to 112 hours, preferably 103 to 107 hours after the first hormone administration, and artificial insemination is performed by injecting semen directly into the uterine cavity.
The abdomen was then re-opened 120-128 hours, preferably 122-125 hours after the first hormone administration,
For example, 2 mg / ml BSA (bovine albumin, Sigma
Perfused with PBS (phosphate-buffered saline, Nissui) to which fertilized eggs were collected.

The fertilized egg obtained by gene transfer is opaque because it contains a large amount of fat granules in the cytoplasm, as in cows and pigs. Therefore, in order to visualize the pronucleus, it is preferable to perform centrifugation at, for example, 13000 × G for 10 minutes. For the introduction of a foreign gene into a fertilized egg, any method known in the art, such as microinjection, electroporation, transfection, and lipofection, may be employed, but usually, the microinjection method is often used. ing. The gene transfer to the fertilized egg by the microinjection method is preferably performed under an inverted microscope equipped with a differential interference device (Nikon) equipped with two micromanipulators (Narishige). One manipulator is fitted with a glass pipette for egg retention. A glass injection pipette is attached to the other manipulator, and the pipette is filled with PBS in which the transgene has been dissolved, and a positive pressure is applied to constantly spout the gene solution. The egg is fixed with a retaining pipette, the tip of the injection pipette is penetrated into the pronucleus, and the gene is injected. The presence or absence of injection can be confirmed by swelling of the pronucleus.

The cell membrane and nuclear membrane of minipigs are much more resilient than mice, and it is relatively difficult to penetrate a pipette. Therefore, it is preferable to use a piezo manipulator attached to the injection pipette. This facilitates penetration of the pipette. The fertilized egg into which the gene has been injected is cultured in a Whitten medium at 39 ° C. in a CO ₂ incubator until the fertilized egg is transferred.

The fertilized egg into which the fertilized egg transfer gene has been injected is transplanted into the oviduct of the recipient. In fertilized egg transplantation, it is important to synchronize the stage of development of the fertilized egg to be transplanted and the estrous cycle of the transplanted female. Therefore, it is necessary for the fertilized female to synchronize the estrous cycle in accordance with the schedule of the fertilized egg collection. For synchronization of estrus, it is preferable to use Regmate (Hoechst), which is a progesterone which is orally active. In this case, the estrous cycle of a female miniature pig that repeats the normal estrous cycle is 10
From the day (allowable start date is set to day 0) to 21 days of the estrous cycle, legmate is mixed with the food and administered at 5 to 10 mg / day. The last day of administration is determined according to the egg collection schedule so that transplantation can be performed on day 7 with the last day of administration as day 0.

The actual transplant operation is as follows. The recipient female is laparotomized under inhalation anesthesia to expose the fallopian tubes and tubules. First, a plastic tube is inserted into the fallopian tube through the opening of the fallopian tube. Using this cylinder as a guide, a glass pipette into which the fertilized egg has been sucked is inserted into the fallopian tube, and after the guide is pulled out, the fertilized egg is blown out together with a small amount of embryo culture solution. Thereafter, the abdomen is closed using an absorbent yarn.

Confirmation of the transgene A sample for confirming the transgene is taken on the day of delivery of the recipient female or on the day following the delivery day. The sampling site was good for the tail of the offspring.
cm is enough. For analysis, PCR and Southern blotting are used together. Using the PCR method, it is possible to confirm the transgene within a few days after delivery,
The transgene can be further confirmed by the analysis result of the Southern blotting method.

The transgenic mini-pig according to the present invention obtained as described above can be used as a founder using the transgenic mini-pig as a founder or an egg, sperm, or fertilized egg derived from the founder individual by means known in the art. Can be used to obtain homozygotes or heterozygotes. From the obtained homozygotes or heterozygotes, it is possible to establish a transgenic minipig strain that is more useful for research such as pathological analysis and drug screening. it can. In the establishment of the line, means such as cloning known in the art (Wilmut
Et al., Nature, vol. 385, 1997). It is also possible to establish a cell line from the founder, a transgenic mini-pig or a strain thereof, by a method also known in the art.

Using the transgenic minipigs of the present invention and their strains, it is possible to analyze the translation products of transgenes, particularly disease genes, and to confirm the onset of the disease. In addition, it becomes possible to carry out drug screening for treatment and prevention of diseases using transgenic minipigs or their strains, these organs, tissues, or cell lines, etc., which is very useful as a human disease model. is there.

[0030]

EXAMPLES Hereinafter, the present invention will be further described with reference to examples, but the present invention is not limited to these examples.
The minipig huntingtin gene was introduced by the following method.

Construction of the Transgene Using the cDNA of the human huntingtin gene and the DNA fragment derived from the PCR-amplified human huntington disease gene genomic clone as probes, the cDNA of the miniporcine huntingtin gene (12.7
kb) was isolated and identified. Exon 1 as a result of structural analysis
In the same manner as in humans, a CAG tribase repeat sequence showing copy number polymorphism was confirmed.

Next, using the obtained mini-porcine huntingtin gene as a material, a CAG repeat sequence fragment separately prepared by PCR was inserted into a repeat site in exon 1 using a random insertion method (SEQ ID NO: 1).
Nucleotides 197-223) and glutamic acid 7
5 pieces ((CAG) 5CAA (CAG) 66CAA (CA
G) Miniature pig huntingtin gene fragment phtt containing 2)
/ E ₂₄ Q ₇₅ (SEQ ID NO: 1) was obtained. The phtt / E ₂₄ Q _75, since the Huntington's disease is a neuron specific diseases where cell failure occurs, the rat NSE promoter used in the hope neuron-specific expression, poly A signal sequence from SV40 And a vector for introduction containing the same was constructed. Transgene restriction enzyme SphI, and digested with ClaI, resulting gene fragment (Fig. 1, hereinafter referred to phtt / E ₂₄ Q ₇₅ fragment) was purified by electroelution method, 3.7μg / ml
And dissolved at -20 ° C in an injection buffer.

Collection of Fertilized Eggs Fertilized mini pigs were obtained by adding 1/2 ampoule of PG60 to immature female mini pigs (140 to 150 days old, weight: 14 to 18 kg).
0 (Intervet) or 500 units of eCG (pregnant horse serum gonadotropin; PMS, Nippon Zenyaku Kogyo) is injected intramuscularly and 75 hours later, 100 or 500 units of hCG (human chorionic gonadotropin) ; Peverogen,
Sankyo) was intramuscularly injected and superovulated.

PG600 or eCG administration 103 to 10
Five hours later, the abdomen was opened, and artificial insemination was performed by injecting semen directly into the uterine cavity. 122-124 hours after administration of PG600 or eCG, the abdomen was re-opened, and 2 mg
Perfused with PBS (phosphate buffered saline, Nissui) supplemented with BSA (bovine albumin, Sigma) at a concentration of / ml, and fertilized eggs were collected.

Gene by microinjection method
Introduced fertilized eggs are 13000x to visualize the pronucleus
G, centrifugation was performed for 10 minutes. Gene transfer into the fertilized eggs was performed under an inverted microscope (Nikon) equipped with a differential interference device equipped with two micromanipulators (Narishige). One manipulator was fitted with a glass pipette for egg retention. Attaching a glass injection pipette on the other manipulator, the dissolution of the liquid (genes dissolved transgene (phtt / E ₂₄ Q ₇₅ containing fragment) in the pipette was added 0.1 mM EDTA PBS
) And positive pressure was applied to constantly spout the gene solution. Oocytes were fixed with a retaining pipette, the tip of the injection pipette was penetrated into the pronucleus, and the gene was injected. The presence or absence of injection can be confirmed by swelling of the pronucleus. A piezo manipulator was also used for the injection pipette. The fertilized egg into which the gene has been injected
The cells were cultured in an itten medium in a CO ₂ incubator at 39 ° C.

The fertilized egg into which the fertilized egg transfer gene was injected was implanted into the oviduct of the recipient. For synchronization of estrus, Regmate (Hoechst), a progesterone having oral activity, was used. Estrous cycle 10 of female miniature pigs repeating normal estrous cycle
From the day (allowable start date is set to day 0) and beyond the 21st estrus cycle, legmate was administered to the diet at 10 mg / day. The last day of administration was determined according to the schedule for egg collection so that transplantation could be performed on the seventh day, with the last day of administration as day 0.
Oviposited females with synchronized estrous cycle were laparotomized under inhalation anesthesia to expose the fallopian tubes and tubules. First, a plastic tube was inserted into the fallopian tube through the opening of the fallopian tube. Using this cylinder as a guide, a glass pipette from which a fertilized egg was aspirated was inserted into the fallopian tube, and after the guide was pulled out, the fertilized egg was blown out together with a small amount of embryo culture solution. Thereafter, the abdomen was closed using an absorbent yarn.

[0037] was carried out Sampling for the confirmation of the delivery date or the next day to transgene confirmation egg female of the transgene. The sampling site was the tail of the offspring, and DNA was extracted according to a conventional method. That is, the tail pieces of the collected neonatal mini pigs were placed in a DNA extraction buffer (50 mM Tris-HCl pH
8.0 / 100mM NaCl / 20mM EDTA / 1% SDS)
Proteinase K (200 μg / ml) and proteinase E (1 mg / m
l) was added and dissolved by stirring at 55 ° C. overnight. After phenol treatment, RNaseA (50 μg / ml) was added, reacted at 37 ° C. for 30 minutes, and DNA was extracted by ethanol precipitation. Using the obtained DNA, the introduced gene was confirmed by a PCR method and a Southern hybridization method according to a conventional method. FIG. 2 shows the positions and combinations of the PCR primers and the positions of the probes used for Southern hybridization.
Primers are DNA synthesizer (ABI394RNA / DNA Synthesizer)
Was prepared. The sequence of each primer is shown below.

NSE4 (+): AGTAAAGGTGATGGCAGGAA (SEQ ID NO: 3) NSE5 (−): TCTGCTTGAGAGCTGTGCTTAG (SEQ ID NO: 4) HD250 + 1: CCGTCCCGAAATCATGG (Ex6 in the figure) (SEQ ID NO: 5) HD235-1: CAGCCAGCTGTAGAAGTAGC (Sequence No. 3) No. 6) HD235 + 1: CCTTGCATTACACTCAGCACC (Ex9) (SEQ ID NO: 7) HDEx12 (−): CAGAGCTGTCTGAAGGGGTC (Ex12) (SEQ ID NO: 8) HDEx14-3 (+): CTTCAACAGTCACATGTGTTG (Ex14) (SEQ ID NO: 9) HD236-1 : AGGAGCACAATCCTCGTCG (Ex15) (SEQ ID NO: 1)
0) HD256 + 1: CTTCCACGTGGGGACTG (Ex17) (SEQ ID NO: 1)
1) HD256-1: CGGAGGATTCATCCTTCAAC (Ex18) (SEQ ID NO: 12)

3A to 3D show the results of PCR using Ex9 to Ex12 as primers. In FIG. 3A, rows 1 to 6 show D extracted from each mini-pig used.
NA, column 7 is a positive control (transgenic fragment + normal genomic DNA), column 8 is a negative control (no template). As shown in FIG. 3A, approximately in the second row of minipigs 7-2l
A clear band of 700 bp was observed, confirming the introduction of the gene. Similarly, in FIG. 3B, rows 1 to 5 show DNA extracted from each mini-pig used, row 6 shows a positive control (transgenic fragment), and row 7 shows a positive control (transgenic fragment + normal genome). DNA), 8th
Rows are the positive control (normal genomic DNA), row 9 is the negative control (no template), row 10 is the λHindIII / φX174HaeIII marker. As shown in FIG. 3B, the first row of minipigs 25-1l
, A clear band of about 700 bp was observed, confirming the introduction of the gene.

In FIG. 3C, the first row is a DNA ladder marker of 50 bp, the second to fifth rows are DNA extracted from each mini-pig used, the sixth row is a positive control (transgenic fragment), and the seventh row. Indicates a positive control (transgenic fragment + normal genomic DNA), the eighth column indicates a positive control (normal genomic DNA), and the ninth column indicates a negative control (no template).
As shown in FIG. 3C, a clear band of approximately 700 bp was observed in the mini-pig 26-2l in the third row, confirming the introduction of the gene. In FIG. 3D, the first to third rows show DNA extracted from each mini-pig used, and the fourth row shows a positive control (transgenic fragment + normal genomic DNA). As shown in FIG. 3D, a clear band of about 700 bp was observed in the mini-pig 31-3L in the third row, confirming the introduction of the gene.

Next, the obtained 25-2l and 26-2 according to the present invention are obtained.
l, using DNA extracted from 26-3L of 31-3L and Comparative Examples, the probe fragment (MP HD Ex.9 fragments) corresponding to exon 9 of miniature swine-Hanchintin gene phtt / E ₂₄ Q ₇₅ Southern hybridization was performed. FIG. 4 shows the results. As shown in FIG. 4, in the DNA of the mini-pig in which the introduction of the gene was confirmed by PCR (columns 1 to 3), apart from the endogenous huntingtin gene, 3.0% of the transgene represented by a positive control having a different copy number was used. A kb band was confirmed.

[0042] As shown in FIGS. 3 and 4, in transgenic Buni pig according to the present invention, it was confirmed that the introduced gene (phtt / E ₂₄ Q ₇₅ containing fragment) is incorporated. By the present invention described in detail above, five transgenic minipigs have been obtained so far. Table 1 summarizes the production efficiency and the like of transgenic minipigs.

[0043]

[Table 1]

As shown in Table 1, transgenic minipigs could be produced with very high efficiency.

[0045]

As described above, according to the present invention, it has become possible to produce a transgenic miniature pig which has not been reported hitherto. The transgenic mini-pig of the present invention contributes to the creation of a human disease model animal and a donor for organ transplantation, whose necessity is expected to increase in the future. Furthermore, the transgenic mini-pig according to the present invention can establish its lineage and further cell lines using founder individuals or eggs, sperm, or fertilized eggs derived therefrom, whereby pathological analysis, drug screening can be performed. It is more useful in research such as. If cloned individuals are created to establish a line, more efficient research can be expected.

Transgenic minipigs are compared to rodent models that have been primarily used so far,
It has human-like complexity and is suitable for pathological analysis. In addition, we will establish multiple reproducible strains from transgenic individuals obtained by introducing disease genes into fertilized eggs and analyze them in molecular biology, biochemistry, morphology, and physiology. This can elucidate the mechanism of disease establishment. Furthermore, it is also useful as a source of transformed cells and a transgene product, and enables efficient analysis. Based on the knowledge obtained in the process of analyzing the mechanism of disease formation, it is possible to develop therapeutic methods and preventive methods, including drug discovery by organ transplantation, gene therapy, drug screening, and the like.

[0047]

[Sequence List] SEQUENCE LISTING <110> SLA RESEARCH, INC. <120> Transgenic Miniture Swine <130> P99-0172 <140> <141> <160> 12 <170> PatentIn Ver. 2.0 <210> 1 <211> 3259 <212> DNA <213> Miniture Swine <220> <221> CDS <222> (128) .. (3256) <400> ctg gaa aag ctg atg aaa gct ttc gag tct ctc 169 Met Ala Thr Leu Glu Lys Leu Met Lys Ala Phe Glu Ser Leu 1 5 10 aag tcc ttc cag cag cag cag cag caa cag cag cag cag cag cag cag 217 Lys Ser Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln 15 20 25 30 cag cag cag cag cag cag cag cag cag cag cag cag cag cag cag cag 265 Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln 35 40 45 cag cag cag cag cag cag cag cag cag cag cag cag cag cag cag cag 313 Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln 50 55 60 cag cag cag cag cag cag cag cag cag cag cag cag cag cag cag cag 361 Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln 65 70 75 cag cag cag cag cag cag cag cag cag cag cag caa cag cag ctg ccg 409 Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Leu Pro 80 85 90 cca ccg ccg cct cag cct cct cag ccg cca ccg cag acg cag ccg ccg 457 Pro Pro Pro Pro Gln Pro Pro Gln Pro Pro Pro Gln Thr Gln Pro 95 95 105 105 cct cag ccg ccg ccg ccc ccg ccc ccg ccg ccg ccg ccg ccg ccc ggc 505 Pro Gln Pro Pro Pro Pro Pro Pro Pro Pro Pro Pro Pro Pro Pro Gly 115 120 125 ccg gca gtg gcc gag gag ccg ctg cac cga cca aag aag gag ctc tca 553 Pro Ala Val Ala Glu Glu Pro Leu His Arg Pro Lys Lys Glu Leu Ser 130 135 140 gcc acc aag aaa gat cgt gtg aat cat tgt ctg aca ata tgt gaa aac 601 Ala Thr Lys Lys Asp Arg Val Asn His Cys Leu Thr Ile Cys Glu Asn 145 150 155 ata gtg gca cag tct ctc aga aat tca cca gaa ttt cag aaa ctg ctg 649 Ile Val Ala Gln Ser Leu Arg Asn Ser Pro Glu Phe Gln Lys Leu Leu 160 165 170 ggc atc gct atg gaa ctt ttc ctg gtg tg gat gc a gag tca 697 Gly Ile Ala Met Glu Leu Phe Leu Leu Cys Ser Asp Asp Ala Glu Ser 175 180 185 190 gat gtc agg atg gtg gct gac gaa tgc ctc aac aaa gtc atc aag gct 745 Asp Val Arg Met Val Ala Asp Glu Leu Asn Lys Val Ile Lys Ala 195 200 205 ttg atg gat tcc aac ctt ccg agg tta cag cta gaa ctt tat aag gaa 793 Leu Met Asp Ser Asn Leu Pro Arg Leu Gln Leu Glu Leu Tyr Lys Glu 210 215 220 att aaa aag aac ggt gct cct cgg agc ctc cgt gct gcc ctg tgg agg 841 Ile Lys Lys Asn Gly Ala Pro Arg Ser Leu Arg Ala Ala Leu Trp Arg 225 230 235 ttt gct gag ctg gct cac ctg gtc cgg cct cag aag tgc agg Pg gg agg Ala Glu Leu Ala His Leu Val Arg Pro Gln Lys Cys Arg Pro Tyr 240 245 250 ttg gtg aac ctc ttg ccc tgc ctg acg cgc aca agc aag agg ccc gag 937 Leu Val Asn Leu Leu Pro Cys Leu Thr Arg Thr Ser Lys Arg Pro Glu 255 260 265 270 gag tct gtt cag gag acc ttg gct gca gcc gtc ccg aaa atc atg gcg 985 Glu Ser Val Gln Glu Thr Leu Ala Ala Ala Val Pro Lys Ile Met Ala 275 280 285 tcc ttt ggc aat ttc gca aac gat ga g att aag gtt ctg ttg aag 1033 Ser Phe Gly Asn Phe Ala Asn Asp Asn Glu Ile Lys Val Leu Leu Lys 290 295 300 gcc ttc gta gcg aac ctg aag tcc ggc tcc ccc acg gtc cgg cgc acg 1081 Ala Lehe Val Lys Ser Gly Ser Pro Thr Val Arg Arg Thr 305 310 315 gcg gcg ggc gcg gcg gtg agc gtg tgc cag cac tcc cgg cgg acg cgc 1129 Ala Ala Gly Ala Ala Val Ser Val Cys Gln His Ser Arg Arg Thr Arg 320 325 330 tac ttc tac agc tgg ctg ctc agc gtg ctc tta ggt ctg ctc gtg cct 1177 Tyr Phe Tyr Ser Trp Leu Leu Ser Val Leu Leu Gly Leu Leu Val Pro 335 340 345 350 gtg gag gag gaa cac ggc acc ctg cg ctc acc 1225 Val Glu Glu Glu His Gly Thr Leu Leu Ile Leu Gly Val Leu Leu Thr 355 360 365 ctg agg tat ctg gtg ccc ctg ctg cag cag cag gtc aag gac acg agcc 1273 Leu Arg Tyr Leu Val Pro Leu Leu Gln Gln Gln Gln Val Lys Asp Thr Ser 370 375 380 ctg aaa ggc agc ttc ggg gtg acc cgg aag gaa atg gag gtc tct ccc 1321 Leu Lys Gly Ser Phe Gly Val Thr Arg Lys Glu Met Glu Val Ser Pro 385 390 395 395 tcg acg gaa cag ctt gtc cag gtt tac gag ttg acc ttg cat tac act 1369 Ser Thr Glu Gln Leu Val Gln Val Tyr Glu Leu Thr Leu His Tyr Thr 400 405 410 cag cac caa gac cac aac gtt gtg acg ggg gcc ttg gag ctc ctg cag 1417 Gln His Gln Asp His Asn Val Val Thr Gly Ala Leu Glu Leu Leu Gln 415 420 425 430 cag ctg ctc agg acg cct ccc ccg gag ctc ctg cag gcg ctg acc tcg 1465 Gln Leu Leu Arg Thr Pro Pro Pro Glu Leu Leu Gln Ala Leu Thr Ser 435 440 445 ccg ggc ggc atc gcg cag ctc acc gcc aac aac gac gag ccc agg tgc 1513 Pro Gly Gly Ile Ala Gln Leu Thr Ala Asn Asn Asp Glu Pro Arg Cys 450 455 460 cga agt cgc agc ggg agc atc ata gct gga ggg ggt tct 1561 Arg Ser Arg Ser Gly Ser Ile Val Glu Leu Ile Ala Gly Gly Gly Ser 465 470 475 tca tgc agc cct gtc ctt tca aga aaa caa aaa ggg aag gtg ctc ttg 1609 Ser Cys Ser Pro Val Leu Ser Arg Lys Gln Lys Gly Lys Val Leu Leu 480 485 490 gga gac gca gcg gcc ttg gag gat gag cct gcg tcg cgg tcg gag ggc 1657 Gly Asp Ala Ala Ala Leu Glu Asp Glu Pro Ala Ser Arg Ser Glu Gly 495 505 505 510 agc agc ccc gcc ctc aca gcc tcg gtc acg ggt gag ctg ggt ggt gag 1705 Ser Ser Pro Ala Leu Thr Ala Ser Val Thr Gly Glu Leu Gly Gly Glu 515 520 525 ctg gcc ccc tcc tcc ggt gtc tcc act cct ggc agc tcc gct 1753 Leu Ala Pro Ser Ser Gly Val Ser Thr Pro Gly Ser Ala Ser Ser Ala 530 535 540 gcc gac tct gtg ggt cac gac atc atc aca gaa cag ccc cgg tct cag 1801 Ala Asp Ser Val Gly His Asp Ile Ile Thr Glu Gln Pro Arg Ser Gln 545 550 555 cac acg ctg cag ccg gac tcg gtg gat ctg agc ggc tgt gac ttg agc 1849 His Thr Leu Gln Pro Asp Ser Val Asp Leu Ser Gly Cys Asp Leu Ser 560 565 570 agcat gcc acc ggg gac gag gag gac atc ttg agc cac agc tcc 1897 Ser Ala Ala Thr Asp Gly Asp Glu Glu Asp Ile Leu Ser His Ser Ser 575 580 585 590 590 agc cag atg agt gcc gtc ccg tcg gac cct gcc atg gac ttg Serac Gln Met Ser Ala Val Pro Ser Asp Pro Ala Met Asp Leu Asn Asp 595 600 605 ggg acc cag gcc tcc tca ccc gtc agc gac agc tcc cag acc acc acc 1993 Gly Thr Gln Ala Ser Ser Pro Val Ser Asp Ser Ser Gln Thr Thr Thr 610 615 620 gag ggc cct gac tcg gct gtg acc cct tca gac agc tct gaa att gtg 2041 Glu Gly Pro Asp Ser Ala Val Thr Pro Ser Asp Ser Ser Glu Ile Val 625 630 635 635 ttg gat ggt gca gac ggc cag tac tct ggg ctg cgg ttg gga cag ccc 2089 Leu Asp Gly Ala Asp Gly Gln Tyr Ser Gly Leu Arg Leu Gly Gln Pro 640 650 650 cag gac gcg gac gcg gac gag gac gcc gca ggg ctc ctt ccc cac gga 2137 Gln Asp Ap Asp Glu Asp Ala Ala Gly Leu Leu Pro His Gly 655 660 665 670 ggc ccg gat gct ttc aga agc tcc ccc acc gcc ctt caa cag tca cat 2185 Gly Pro Asp Ala Phe Arg Ser Ser Pro Thr Ala Leu Gln Gln Ser His 675 680 685 gtg ttg aaa agc ctg ggc cac agc agg cag ccc tcc gac agc agt gta 2233 Val Leu Lys Ser Leu Gly His Ser Arg Gln Pro Ser Asp Ser Ser Val 690 695 700 gat aaa ttt gtc tcg aga gac gaa gct gct gag gca gag cca gaa 2281 Asp Lys Phe Val Ser Arg Asp Glu Ala Ala Glu Ala Gly Glu Pro Glu 705 710 715 aac aag cct tgc cgc atc aaa ggt gac ata ggc cag tcc acc gac gag 2329 Asn Lys Pro Cys Arg Ile Lys Gly A sp Ile Gly Gln Ser Thr Asp Glu 720 725 730 gat tct gct cct ctc gtc cac tgt gtc cgc ctt tta tct gct tca ttt 2377 Asp Ser Ala Pro Leu Val His Cys Val Arg Leu Leu Ser Ala Ser Phe 735 740 745 750 750 ttg ctg acg ggg aaa aga aac gac gag cag tac gtg tcg gac atc ctg 2425 Leu Leu Thr Gly Lys Arg Asn Asp Glu Gln Tyr Val Ser Asp Ile Leu 755 760 765 agc tac atc cac cac ggc gac ccg cag gtc cgaggc gcc 2473 Ser Tyr Ile His His Gly Asp Pro Gln Val Arg Gly Ala Thr Ala Ile 770 775 780 ctc tgc gga acc ctg gtc tcc tcc atc ctc agc agg tcc cgc ttc cac 2521 Leu Cys Gly Thr Leu Val Ser Ser Ile Leu Ser Arg Ser Arg Phe His 785 790 795 gtg ggg gac tgg atg ggc gcc gtc cgg gct ctg aca gga aat gca ttt 2569 Val Gly Asp Trp Met Gly Ala Val Arg Ala Leu Thr Gly Asn Ala Phe 800 805 810 tct ctg gtg gac tgc atg ctg caa aag acg ttg aag gat gaa 2617 Ser Leu Val Asp Cys Ile Pro Leu Leu Gln Lys Thr Leu Lys Asp Glu 815 820 825 830 tcc tcc gtt acg tgc aag ttg gcg tgc aca gcc gtg agg ctc tg Ser gtg 2 l Thr Cys Lys Leu Ala Cys Thr Ala Val Arg Leu Cys Val 835 840 845 gcg gct ctc tgc ggc agc agc tac agc cag tgg ggg ctg cag ctc atc 2713 Ala Ala Ala Leu Cys Gly Ser Ser Tyr Ser Gln Trp Gly Leu Gln 850 855 860 acc gac ctg ctg acc ctg agg agc agc tcc tac tgg ctg gtg cgg acc 2761 Thr Asp Leu Leu Thr Leu Arg Ser Ser Ser Tyr Trp Leu Val Arg Thr 865 870 875 gag ctc ctg gag acc gtg gca gag att gac agg ttg gtg agc ttt 2809 Glu Leu Leu Glu Thr Val Ala Glu Ile Asp Phe Arg Leu Val Ser Phe 880 885 890 ttg gag gca aaa gca gaa aac cta cac aga ggg gcc cat cac tac aca 2857 Leu Glu Ala Lys Ala Glu Asn His Arg Gly Ala His His Tyr Thr 895 900 905 910 ggg ctg tta aga ctg caa gaa cga gtg ctc aat aat gtt gtc atc tac 2905 Gly Leu Leu Arg Leu Gln Glu Arg Val Leu Asn Asn Val Val Ile Tyr 915 920 925 ctc ggg gac gag gac ccc agg gtg cga cat gtt gct gca gct tcg 2953 Leu Leu Gly Asp Glu Asp Pro Arg Val Arg His Val Ala Ala Ala Ser 930 935 940 tta gtg agg ctc gtc cca aag ctg ttt tat aaa tgt gac caa cag 3001 Leu Val Arg Leu Val Pro Lys Leu Phe Tyr Lys Cys Asp Gln Gly Gln 945 950 955 gct gac cca gtg gtg gcc gtg gcg aga gat caa agc agt gtt tac ctg 3049 Ala Asp Pro Val Val Ala Val Ala Arg Asp G Ser Ser Val Tyr Leu 960 965 970 aag ctc ctc atg cac gag acg cag cct ccg tcg cac ttc tcc gtc agc 3097 Lys Leu Leu Met His Glu Thr Gln Pro Pro Ser His Phe Ser Val Ser 975 980 985 990 acc gtc acc aga aca tac aga ggc tat aac cta cta ctg agc gtc acc 3145 Thr Val Thr Arg Thr Tyr Arg Gly Tyr Asn Leu Leu Leu Ser Val Thr 995 1000 1005 gac gtc act ctg gaa aat aac ctt tcg cga gtt act gca gca gtt tct 3193 Asp Val Thr Leu Glu Asn Asn Leu Ser Arg Val Thr Ala Ala Val Ser 1010 1015 1020 cat gaa ctc atc acg tca acc acc cga gca ctc aca ttt gga tgc tgt 3241 His Glu Leu Ile Thr Ser Thr Thr Arg Ala Leu Thr Phe Gly Cys Cys 1025 1030 1035 gaa gct ttg atc cac tag 3259 Glu Ala Leu Ile His 1040 <210> 2 <211> 1043 <212> PRT <213> Miniture Swine <400> 2 Met Ala Thr Leu Glu Lys Leu Met Lys Ala Phe Glu Ser Leu Lys Ser 1 5 10 15 Phe Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln 20 25 30 Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln 35 40 45 Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln 50 55 60 Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln 65 70 75 80 Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Leu Pro Pro Pro 85 90 95 Pro Pro Gln Pro Pro Gln Pro Pro Pro Gln Thr Gln Pro Pro Pro Gln 100 105 110 Pro Pro Pro Pro Pro Pro Pro Pro Pro Pro Pro Pro Gly Pro Ala 115 120 125 Val Ala Glu Glu Pro Leu His Arg Pro Lys Lys Glu Leu Ser Ala Thr 130 135 140 Lys Lys Asp Arg Val Asn His Cys Leu Thr Ile Cys Glu Asn Ile Val 145 150 155 160 Ala Gln Ser Leu Arg Asn Ser Pro Glu Phe Gln Lys Leu Leu Gly Ile 165 170 175 Ala Met Glu Leu Phe Leu Leu Cys Ser Asp Asp Ala Glu Ser Asp Val 180 185 190 Arg Met Val Ala Asp Glu Cys Leu Asn Lys Val Ile Lys Ala Leu Met 195 200 205 Asp Ser Asn Leu Pro Arg Leu Gln Leu Glu Leu Tyr Lys Glu Ile Lys 210 215 220 Lys Asn Gl y Ala Pro Arg Ser Leu Arg Ala Ala Leu Trp Arg Phe Ala 225 230 235 240 Glu Leu Ala His Leu Val Arg Pro Gln Lys Cys Arg Pro Tyr Leu Val 245 250 255 Asn Leu Leu Pro Cys Leu Thr Arg Thr Ser Lys Arg Pro Glu Glu Ser 260 265 270 Val Gln Glu Thr Leu Ala Ala Ala Val Pro Lys Ile Met Ala Ser Phe 275 280 285 Gly Asn Phe Ala Asn Asp Asn Glu Ile Lys Val Leu Leu Lys Ala Phe 290 295 300 Val Ala Asn Leu Lys Ser Gly Ser Pro Thr Val Arg Arg Thr Ala Ala 305 310 315 320 Gly Ala Ala Val Ser Val Cys Gln His Ser Arg Arg Thr Arg Tyr Phe 325 330 335 Tyr Ser Trp Leu Leu Ser Val Leu Leu Gly Leu Leu Val Pro Val Glu 340 345 350 Glu Glu His Gly Thr Leu Leu Ile Leu Gly Val Leu Leu Thr Leu Arg 355 360 365 Tyr Leu Val Pro Leu Leu Gln Gln Gln Val Lys Asp Thr Ser Leu Lys 370 375 380 Gly Ser Phe Gly Val Thr Arg Lys Glu Met Glu Val Ser Pro Ser Thr 385 390 395 400 400 Glu Gln Leu Val Gln Val Tyr Glu Leu Thr Leu His Tyr Thr Gln His 405 410 415 Gln Asp His Asn Val Val Thr Gly Ala Leu Glu Leu Leu Gln Gln Leu 420 425 430 Leu Arg T hr Pro Pro Pro Glu Leu Leu Gln Ala Leu Thr Ser Pro Gly 435 440 445 Gly Ile Ala Gln Leu Thr Ala Asn Asn Asp Glu Pro Arg Cys Arg Ser 450 455 460 Arg Ser Gly Ser Ile Val Glu Leu Ile Ala Gly Gly Gly Ser Ser Cys 465 470 475 480 480 Ser Pro Val Leu Ser Arg Lys Gln Lys Gly Lys Val Leu Leu Gly Asp 485 490 495 Ala Ala Ala Leu Glu Asp Glu Pro Ala Ser Arg Ser Glu Gly Ser Ser 500 505 510 Pro Ala Leu Thr Ala Ser Val Thr Gly Glu Leu Gly Gly Glu Leu Ala 515 520 525 Pro Ser Ser Gly Val Ser Thr Pro Gly Ser Ala Ser Ser Ala Ala Asp 530 535 540 Ser Val Gly His Asp Ile Ile Ile Thr Glu Gln Pro Arg Ser Gln His Thr 545 550 555 560 Leu Gln Pro Asp Ser Val Asp Leu Ser Gly Cys Asp Leu Ser Ser Ala 565 570 570 575 Ala Thr Asp Gly Asp Glu Glu Asp Ile Leu Ser His Ser Ser Ser Gln 580 585 590 Met Ser Ala Val Pro Ser Asp Pro Ala Met Asp Leu Asn Asp Gly Thr 595 600 605 Gln Ala Ser Ser Pro Val Ser Asp Ser Ser Gln Thr Thr Thr Glu Gly 610 615 620 Pro Asp Ser Ala Val Thr Pro Ser Asp Ser Ser Glu Ile Val Leu Asp 625 630 635 640 Gly Ala A sp Gly Gln Tyr Ser Gly Leu Arg Leu Gly Gln Pro Gln Asp 645 650 655 Ala Asp Ala Asp Glu Asp Ala Ala Gly Leu Leu Pro His Gly Gly Pro 660 665 670 Asp Ala Phe Arg Ser Ser Pro Thr Ala Leu Gln Gln Ser His Val Leu 675 680 685 Lys Ser Leu Gly His Ser Arg Gln Pro Ser Asp Ser Ser Val Asp Lys 690 695 700 Phe Val Ser Arg Asp Glu Ala Ala Glu Ala Gly Glu Pro Glu Asn Lys 705 710 715 715 720 Pro Cys Arg Ile Lys Gly Asp Ile Gly Gln Ser Thr Asp Glu Asp Ser 725 730 735 Ala Pro Leu Val His Cys Val Arg Leu Leu Ser Ala Ser Phe Leu Leu 740 745 750 Thr Gly Lys Arg Asn Asp Glu Gln Tyr Val Ser Asp Ile Leu Ser Tyr 755 760 765 Ile His His Gly Asp Pro Gln Val Arg Gly Ala Thr Ala Ile Leu Cys 770 775 780 Gly Thr Leu Val Ser Ser Ile Leu Ser Arg Ser Arg Phe His Val Gly 785 790 795 800 Asp Trp Met Gly Ala Val Arg Ala Leu Thr Gly Asn Ala Phe Ser Leu 805 810 815 Val Asp Cys Ile Pro Leu Leu Gln Lys Thr Leu Lys Asp Glu Ser Ser 820 825 830 Val Thr Cys Lys Leu Ala Cys Thr Ala Val Arg Leu Cys Val Ala Ala 835 840 845 Leu Cys GlySer Ser Tyr Ser Gln Trp Gly Leu Gln Leu Ile Thr Asp 850 855 860 Leu Leu Thr Leu Arg Ser Ser Ser Tyr Trp Leu Val Arg Thr Glu Leu 865 870 875 880 Leu Glu Thr Val Ala Glu Ile Asp Phe Arg Leu Val Ser Phe Leu Glu 885 890 895 Ala Lys Ala Glu Asn Leu His Arg Gly Ala His His Tyr Thr Gly Leu 900 905 910 Leu Arg Leu Gln Glu Arg Val Leu Asn Asn Val Val Ile Tyr Leu Leu 915 920 925 Gly Asp Glu Asp Pro Arg Val Arg His Val Ala Ala Ala Ser Leu Val 930 935 940 940 Arg Leu Val Pro Lys Leu Phe Tyr Lys Cys Asp Gln Gly Gln Ala Asp 945 950 955 960 Pro Val Val Ala Val Ala Arg Asp Gln Ser Ser Val Tyr Leu Lys Leu 965 970 975 Leu Met His Glu Thr Gln Pro Pro Ser His Phe Ser Val Ser Thr Val 980 985 990 Thr Arg Thr Tyr Arg Gly Tyr Asn Leu Leu Leu Ser Val Thr Asp Val 995 1000 1005 Thr Leu Glu Asn Asn Leu Ser Arg Val Thr Ala Ala Val Ser His Glu 1010 1015 1020 Leu Ile Thr Ser Thr Thr Arg Ala Leu Thr Phe Gly Cys Cys Glu Ala 025 1030 1035 1040 Leu Ile His <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220 > <223> De scription of Artificial Sequence: PCR primer <400> 3 agtaaaggtg atggcaggaa 20 <210> 4 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: PCR primer <400> 4 tctgcttgag agctgtgctt ag 22 <210> 5 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: PCR primer <400> 5 ccgtcccgaa atcatgg 17 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: PCR primer <400> 6 cagccagctg tagaagtagc 20 <210> 7 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: PCR primer <400> 7 ccttgcatta cactcagcac c 21 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: PCR primer <400> 8 cagagctgtc tgaaggggtc 20 <210> 9 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: PCR primer <400> 9 cttcaacagt cacatgtgtt g 21 <210> 10 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: PCR primer <400> 10 aggagcacaa tcctcgtcg 19 <210> 11 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: PCR primer <400> 11 cttccacgtg gggactg 17 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: PCR primer <400> 12 cggaggattc atccttcaac 20

[0048]

[Sequence List Free Text]

SEQ ID NO: 3: PCR primer used to confirm transgene SEQ ID NO: 4: PCR primer used to confirm transgene SEQ ID NO: 5: PCR primer used to confirm transgene SEQ ID NO: 6: Used to confirm transgene PCR primer SEQ ID NO: 7: PCR primer used to confirm transgene SEQ ID NO: 8: PCR primer used to confirm transgene SEQ ID NO: 9: PCR primer used to confirm transgene SEQ ID NO: 10: Confirmation of transgene PCR primer used SEQ ID NO: 11: PCR primer used for confirmation of transgene SEQ ID NO: 12: PCR primer used for confirmation of transgene

[Brief description of the drawings]

Minipigs & Hanchintin gene were used for the production of transgenic mini-pig of the present invention; FIG phtt / E ₂₄ Q ₇₅
1 shows the outline of the structure.

Figure 2 shows the position and the combination of the PCR primers used for the confirmation of transgene phtt / E ₂₄ Q _75, and the position of Southern hybridization probes.

Figure 3 shows the results of PCR analysis was performed using phtt / E ₂₄ Q ₇₅ was extracted from the miniature swine introduced DNA.

4 shows a Southern hybridization results were performed using phtt / E ₂₄ Q ₇₅ was extracted from the miniature swine introduced DNA.

 ──────────────────────────────────────────────────続 き Continued on the front page (72) Inventor Tokuko Matsuyama 1672-5, Izumi-cho, Izumi-ku, Yokohama-shi, Kanagawa Prefecture (72) Inventor Kuichiro 999-19-B, Oiso-cho, Oiso-cho, Naka-gun, Kanagawa Prefecture (72) Inventor Ikeda Joei 5-31-1, Kameguro, Meguro-ku, Tokyo F-term (reference) 4B024 AA01 AA11 CA01 CA03 CA04 CA06 DA02 GA12 GA18 HA01 4B063 QA05 QA19 QA20 QQ02 QQ08 QQ42 QQ61 QR32 QR60 QR72 QR77 QR80 QS40 4B065 AA90AB AA90A CA60

Claims (17)

[Claims] 1. A transgenic miniature pig into which a human or miniature pig gene has been introduced. 2. The transgenic mini-pig according to claim 1, which is used as a human disease model. 3. The transgenic mini-pig according to claim 1, which is used as a donor for organ donation. 4. A human disease model transgenic mini-pig into which a human disease-causing gene or its homologous gene in mini-pigs has been introduced. 5. The human disease-causing gene is based on a single gene mutation and / or is in a dominant form.
The human disease model transgenic mini-pig according to claim 4. 6. The human disease model transgenic minipig according to claim 4, wherein the human disease causative gene is a causative gene of triplet repeat disease. 7. A human disease-causing gene or a homologous gene thereof in a miniature pig, wherein the full-length cDNA, cD
The human disease model transgenic minipig according to any one of claims 4 to 6, which is a gene fragment of NA, a full length gene of genomic DNA, or a gene fragment of genomic DNA. 8. The human disease model transgenic miniature pig according to claim 4, wherein the human disease causative gene is a causative gene of human Huntington's disease. 9. A homologous gene of a human disease-causing gene in a miniature pig, wherein the cDNA of the miniature pig huntingtin gene having the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 1
The cDNA of the miniature swine huntingtin gene having a nucleotide sequence in which at least one or more CAG codon or CAA codon is added or deleted in the sequence of the 179th to 403rd nucleotides in the nucleotide sequence of claim 4. Human disease model transgenic mini pig. 10. The human disease model transgenic mini-pig according to claim 8, wherein the CAG repeat sequence is 30 or more. 11. The human disease model transgenic mini-pig according to claim 9 or 10, wherein the gene is a DNA fragment comprising a partial continuous sequence of the cDNA according to claim 9. 12. A transgenic minipig line established as a founder using the transgenic minipig according to any one of claims 1 to 11, into which a human or minipig gene has been introduced. 13. An organ derived from the transgenic minipig according to any one of claims 1 to 11, or the transgenic minipig according to claim 12,
Tissue, eggs, sperm, and fertilized eggs. 14. A cell line established from the transgenic mini-pig according to any one of claims 1 to 11, or the transgenic mini-pig according to claim 12. 15. A transgenic mini-pig according to any one of claims 1 to 11, or a cloned mini-pig produced from the transgenic mini-pig strain according to claim 12. 16. The transgenic mini-pig according to any one of claims 1 to 11, or the strain of the transgenic mini-pig according to claim 12,
A method for screening a drug for treating and / or preventing a disease, using the organ, tissue, and / or cell line according to claim 14. 17. The method for producing a transgenic miniature pig according to any one of claims 1 to 11.