Disease model pigs showing a stable phenotype and method for producing the same

Abstract

To provide a disease model swine which is created by nuclear transplantation, a disease model swine indicating a stable phenotype, and a creation method of these items.SOLUTION: A method for creating a gene-modified disease model swine includes: a step (a) for transplanting a nucleus into an ooplasm by using a nucleus of a gene-modified cell; a step (b) for obtaining an offspring by generating an obtained clone embryo in a womb of a swine; and a step (c) for hybridizing the obtained offspring, or using it for sexual reproduction, and obtaining the gene-modified offspring as a disease model swine.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a disease model pig showing a stable phenotype and a method for producing the same. The present invention also relates to a chimeric pig, its gonad, and its germ cell for producing a disease model pig showing a stable phenotype.

Background Art

[0002] In large animals such as pigs, genetically modified animals can be produced from mammalian cells in which the function of a specific gene has been modified using genome editing technology, gene disruption technology, etc. by somatic cell cloning technology (Non-Patent Documents 1 to 4).

[0003] However, in many cases, the phenotype derived from the disease-causing gene is not necessarily stably obtained. This has greatly reduced the attractiveness of producing genetically modified animals by somatic cell cloning technology.

Prior Art Documents

Non-Patent Documents

[0004]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Summary of the Invention

Problems to be Solved by the Invention

[0005] The present invention provides a disease model pig showing a stable phenotype and a method for producing the same. The present invention also provides a chimeric pig, its gonad and its germ cell for producing a disease model pig showing a stable phenotype.

Means for Solving the Problems

[0006] The present inventors have found that there is a problem that the phenotype is unstable in genetically modified pigs produced by somatic cell cloning, and that this problem can be solved by eliminating epigenetic effects derived from somatic cells, and a genetically modified pig with a stable phenotype can be obtained. The present inventors have also found that a hyperammonemia model pig can be produced by disrupting the function of the ornithine transcarbamylase gene. The present inventors have further found that a model pig of dilated cardiomyopathy can be produced by disrupting the function of the γ-sarcoglycan gene. In addition, a method for reducing epigenetic effects in these model pigs has been devised. Furthermore, the present inventors have found that it is useful to produce chimeric embryos in order to produce a disease model pig showing a stable phenotype even if the gene to be modified is lethal. The present invention is based on these findings and inventions.

[0007] That is, the present invention provides the following inventions. (1) A method for producing a genetically modified disease model pig, comprising: (a) performing nuclear transplantation into an egg cytoplasm using the nucleus of a genetically modified cell; (b) allowing the obtained cloned embryo to develop in the uterus of a female pig to obtain offspring; (c) mating the obtained offspring or subjecting them to sexual reproduction to obtain further offspring that are genetically modified as the disease model pig; and a method comprising the steps. (2) The method according to (1) above, wherein the disease model pig is a model pig for a disease selected from the group consisting of diabetes, hyperammonemia, and dilated cardiomyopathy. (3) The method according to (2) above, wherein the disease model pig is a diabetes model animal, and the cells used in (a) above are cells genetically modified by introducing a dominant negative mutant of HNF-1α. (4) The method according to (2) above, wherein the disease model pig is a dilated cardiomyopathy model pig. (5) The method according to any one of (2) to (4) above, wherein the phenotype of the disease is a dominant trait. (6) A disease model pig obtained by the method according to any one of (1) to (5) above. (7) A method for producing a lethal genetic disease model pig, comprising: (i) performing nuclear transfer of the nucleus of genetically modified cells into the oocyte cytoplasm; (ii) making the cloned embryo obtained by nuclear transfer into a chimeric state with an embryo of a pig that does not develop the genetic disease, and allowing it to develop in the uterus of a female pig to obtain offspring; (iii) mating the obtained offspring or subjecting the germ cells of the obtained offspring to sexual reproduction to obtain female offspring having the genetic modification; (iv) mating the obtained female offspring with a male pig or subjecting it to sexual reproduction to obtain a lethal genetic disease model pig. The method comprising the above steps. (8) The method according to (7) above, wherein the lethal genetic disease is hyperammonemia. (9) The method according to (7) above, wherein the lethal genetic disease is dilated cardiomyopathy. (10) A lethal genetic disease model pig obtained by the method according to any one of (7) to (9) above. (11) A model pig for hyperammonemia in which the gene function of the ornithine transcarbamylase gene is disrupted. (12) A model pig for hyperammonemia in which a part of the second exon of the ornithine transcarbamylase gene is deleted, resulting in a nonsense mutation. A genetically modified pig having, in a somatic chimeric state, cells having an ornithine transcarbamylase gene with disrupted gene function and cells having a wild-type ornithine transcarbamylase gene. (14) A female pig heterozygous for an ornithine transcarbamylase gene with disrupted gene function, obtained by sexual reproduction from the pig described in (13) above. (15) A male pig, which is a model of hyperammonemia, obtained by sexual reproduction from the female pig described in (14) above. (16) A model pig for dilated cardiomyopathy in which the gene function of the γ-sarcoglycan gene is disrupted. (17) A model pig for dilated cardiomyopathy in which a part of the second exon of the γ-sarcoglycan gene is deleted, resulting in a nonsense mutation. (18) A genetically modified pig having, in a somatic chimeric state, cells having a γ-sarcoglycan gene with disrupted gene function and cells having a wild-type γ-sarcoglycan gene. (19) A female pig heterozygous for a γ-sarcoglycan gene with disrupted gene function, obtained by sexual reproduction from the pig described in (18) above. (20) A male pig, which is a model of dilated cardiomyopathy and homozygous for a γ-sarcoglycan gene with disrupted gene function, obtained by sexual reproduction from the female pig described in (19) above. (21) A gonad or germ cell obtained by the following method: (i) Performing nuclear transplantation into an egg cytoplasm using the nucleus of a genetically modified cell; (ii) Making a cloned embryo obtained by nuclear transplantation chimeric with an embryo of a pig that does not develop a genetic disease, and allowing it to develop in the uterus of a female pig to obtain offspring; (iii-a) Growing the obtained offspring to obtain gonads or germ cells. A method comprising: (22) The gonad or germ cell according to (21) above, wherein the genetically modified cell is a cell in which the gene function of the ornithine transcarbamylase gene is disrupted, or a cell in which the gene function of the γ-sarcoglycan gene is disrupted.

Brief Description of the Drawings

[0008]

Figure 1

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[0009] As used herein, "genetically modified" means that the gene has been modified by genetic engineering techniques and its function has been at least partially or completely lost, or its function has been over-enhanced. Genetic modifications include those by gene introduction and those by modification of genomic genes. In large animals such as pigs, genetic modification can be achieved by subjecting the nuclei of genetically modified cells to somatic cell cloning to obtain genetically modified offspring as genetically modified animals.

[0010] As used herein, "disease model pig" means a pig that exhibits symptoms of a specific disease due to genetic modification. The diseases are not particularly limited, and examples include diabetes, hyperammonemia, and dilated cardiomyopathy. In certain embodiments, the disease can be hereditary.

[0011] In this specification, "somatic cell cloning" refers to a technique for obtaining an individual having the same genome as a somatic cell by introducing the nucleus of a somatic cell into a fertilized egg whose nucleus has been inactivated or removed in advance. In large animals including pigs, it is known to use somatic cell cloning when producing genetically modified animals such as transgenic animals and knockout animals. Somatic cell cloning generally involves transplanting the nucleus taken out from a somatic cell differentiated into skin, tissue, or an organ into an egg cell (oocyte cytoplasm) derived from another individual from which the nucleus has been removed to produce a cloned embryo, and then transplanting the cloned embryo into the uterus of a surrogate mother, which is another individual with synchronized estrus.

[0012] In this specification, "mating" means mating a male and a female. In this specification, "subjecting to sexual reproduction" means contacting sperm and an egg in vivo or in vitro to effect fertilization and allowing development in the uterus to obtain offspring.

[0013] In this specification, "dominant trait" means that when a mutant gene and a wild-type gene are heterozygous, the trait based on the mutant gene is expressed as a phenotype. In this specification, "recessive trait" means that when a mutant gene and a wild-type gene are heterozygous, the trait based on the mutant gene is not expressed as a phenotype.

[0014] In this specification, "ornithine carbamoyltransferase" is used synonymously with "ornithine transcarbamylase" and "OTC". In this specification, "γ-sarcoglycan" is used synonymously with "SGCD".

[0015] The inventors have found that in a model pig of type 3 young-onset adult diabetes, the phenotypes of the first-generation offspring obtained by somatic cell cloning are unstable. The inventors have also found that when the first-generation offspring are used to obtain further offspring (second generation) through sexual reproduction, the diabetic phenotype is stable in the second-generation offspring. This suggests that the epigenetic state characteristic of embryos, fetuses or individuals created by somatic cell cloning affects the phenotype of the first generation, and this epigenetic state is eliminated in the second generation, suggesting that the phenotype based on genetic mutations clearly appears.

[0016] Therefore, according to the present invention, A method for producing a genetically modified disease model pig, comprising: (a) performing nuclear transplantation into an egg cytoplasm using the nucleus of a genetically modified cell; (b) allowing the obtained cloned embryo to develop in the uterus of a female pig to obtain offspring; (c) mating the obtained offspring or subjecting them to sexual reproduction to obtain the further genetically modified offspring as disease model pigs. A method is provided.

[0017] Hereinafter, the method of the present invention will be described step by step.

[0018] (a) Using the nucleus of a genetically modified cell to perform nuclear transfer into the oocyte cytoplasm Cells can be modified by well-known genetic modification techniques.

[0019] For example, methods for genetically modifying the genome of cells include methods of editing by genome editing techniques such as TALEN, methods of disrupting genes and introducing foreign genes by homologous recombination, and methods of modifying genes on the genome such as methods of introducing foreign genes using viral vectors. Modification of cells may also be performed by methods such as introducing DNA, RNA or protein into cells without modifying the genome.

[0020] TALEN is a hybrid enzyme that fuses the DNA-binding domain consisting of the highly conserved amino acids 33 to 35 of the TALE (Transcription Activator-Like Effector) protein derived from the plant pathogen Xanthomonas with the nuclease domain of the FokI protein, and is well-known as a tool for genome editing. That is, the cell may be modified to be transgenic by introducing a foreign gene, or may be knocked out by partially or completely destroying the function of the gene.

[0021] (a), the genetic modification causes a genetic disease. The gene to be introduced or destroyed to cause a genetic disease can be appropriately selected by those skilled in the art according to the disease model to be produced.

[0022] When producing a diabetic model pig, although not particularly limited, for example, a dominant negative mutant HNF-1α can be introduced into the cell. As the dominant negative mutant HNF-1α, human or porcine dominant negative mutant HNF-1α can be used, and HNF-1αP291fsinsC can be used. HNF-1αP291fsinsC was discovered as a causative gene for maturity-onset diabetes of the young and encodes a protein having the nucleotide sequence of SEQ ID NO: 3 and the amino acid sequence of SEQ ID NO: 4. Human HNF-1α is a protein having 631 amino acids, but HNF-1αP291fsinsC has a mutation in which one cytosine is inserted into eight consecutive cytosines at nucleotide numbers 1091 to 1098 of human HNF-1α. As a result, as shown in SEQ ID NO: 3, the 316th codon becomes a stop codon, and a protein shorter than the original protein is produced. This dominant negative mutant inhibits the normal function of HNF-1α by overexpression and exhibits a diabetic phenotype.

[0023] When producing a hyperammonemia model pig, although not particularly limited, for example, the ornithine transcarbamylase (OTC) gene can be disrupted. The OTC gene is present on the X chromosome, which is a sex chromosome, and is thought to consist of 10 exons. The coding region of the OTC gene, in the case of pigs, is as shown in positions 42 to 1106 of SEQ ID NO: 5, and the amino acid sequence is as shown in SEQ ID NO: 6. Disruption of the OTC gene causes hyperammonemia in males, and females that are heterozygous for the wild type and the mutant become carriers or develop the disease due to additional factors. Disruption of the OTC gene can be performed, for example, by modifying the gene so that a nonsense codon occurs. For example, disruption of the OTC gene may be achieved by modifying the first to third exons, preferably the second exon, to generate a nonsense codon. Such nonsense codons can be generated using gene modification techniques such as TALEN and site-directed mutagenesis. TALEN is said to have a lower off-target effect of unexpectedly cleaving DNA compared to other mutagenesis techniques (methods using zinc finger nucleases (ZFNs) or CRISPR / Cas9) (Ain et al., J Control Release (2015)). The sequence of the second exon in which a nonsense codon has occurred, in the case of pigs, is, for example, as shown in SEQ ID NO: 8. Here, due to the deletion of the bases at positions 109 to 113 of the base sequence of SEQ ID NO: 7 in a part of the second exon, the 64th codon in the base sequence of SEQ ID NO: 5 changes to a nonsense codon, and the resulting amino acid has a sequence as shown in SEQ ID NO: 10.

[0024] When producing an enlarged cardiomyopathy model pig, although not particularly limited, for example, the γ-sarcoglycan (SGCD) gene can be modified. The SGCD gene is present on chromosome 16 and is considered to consist of 8 exons. The coding region of the SGCD gene, in the case of pigs, is as shown in 77 - 946 of SEQ ID NO: 11, and the amino acid sequence is as shown in SEQ ID NO: 13. Disruption of the SGCD gene can be carried out, for example, by modifying the gene so that a nonsense codon occurs. For example, disruption of the OTC gene can be achieved by modifying the 1st - 3rd exons, preferably the 2nd exon, to generate a nonsense codon. Such a nonsense codon can be generated, for example, using gene modification techniques such as TALEN and site-directed mutagenesis. The sequence of the 2nd exon where the nonsense codon occurs, in the case of pigs, is, for example, as shown in SEQ ID NO: 13. Here, due to the deletion of the bases at positions 208 - 211 of the base sequence of SEQ ID NO: 11 in a part of the 2nd exon, the 50th codon in the amino acid sequence of SEQ ID NO: 11 changes to a nonsense codon, and the resulting amino acid has a sequence as shown in SEQ ID NO: 14. Alternatively, 1859 bases upstream from the 147th position of the 2nd exon of the SGCD gene shown in SEQ ID NO: 15 are deleted to generate the sequence shown in SEQ ID NO: 16, and thereby, the region containing the start codon may be deleted.

[0025] In addition, gene modification is not limited to TALEN, and other well-known techniques such as homologous recombination and site-directed mutagenesis methods may be used.

[0026] The cells thus genetically modified are not particularly limited, but for example, they can be selected by sequencing the modified gene or the expression of a selection marker. The selected cells are preferably cloned.

[0027] Thereafter, the nuclei of the genetically modified cells can be used for somatic cell cloning.

[0028] (b) Allowing the obtained cloned embryo to develop in the uterus of a female pig to obtain offspring The obtained cloned embryos can be developed in the uterus of a female pig, whereby offspring (genetically modified pigs) composed of genetically modified cells can be obtained. By synchronizing the estrus of the female pig and then introducing the cloned embryos into the uterus, pregnancy can be established in its uterus, and by developing the cloned embryos, offspring can be obtained. The pigs obtained in (b) are sometimes referred to as first-generation pigs.

[0029] (c) Mating the obtained offspring or subjecting them to sexual reproduction to obtain further offspring with the genetic modification as disease model pigs When the offspring are capable of sexual maturity, they can be sexually matured and then mated or subjected to sexual reproduction to obtain the next generation of genetically modified pigs (sometimes referred to as "second-generation pigs"). Also, when the offspring die before sexual maturity, germ cells (sperm, eggs, and their progenitor cells) or gonads can be obtained by well-known methods, and then the next generation of genetically modified pigs can be obtained by methods such as artificial insemination or in vitro fertilization.

[0030] Sperm can be collected, for example, from the testis or obtained by inducing sperm with fertilizing ability from testicular tissue. The method for inducing sperm with fertilizing ability from testicular tissue is not particularly limited, but for example, it is known that sperm with fertilizing ability can be obtained by transplanting testicular tissue subcutaneously into immunodeficient mice.

[0031] In this way, the offspring obtained in (c) will develop the disease and become disease model pigs when the modified gene is a dominant trait (for example, when a dominant negative mutant is introduced). On the other hand, when the modified gene is a recessive trait (including the case where the gene is present on the sex chromosome and the individual is female), the individual will not develop the disease and will become a carrier of the mutant gene. Also, when the genetically modified gene is recessive and present on the sex chromosome, males will develop the disease and become disease model pigs, while females will not develop the disease and will become carriers of the modified gene. And carriers of the modified gene can be mated with each other to obtain disease model pigs. The above relationships are shown in Table 1.

[0032]

Table 1

[0033] Therefore, when the obtained offspring develop a disease, they can be used as disease model pigs. When they become carriers, the carrier offspring can be subjected to sexual reproduction to obtain offspring that develop the disease.

[0034] Examples of dominant gene mutations include disease model pigs into which dominant negative mutations have been introduced and disease model pigs having gene mutations that exhibit dominant traits. Offspring having such dominant gene mutations become model pigs that develop diseases. Examples of gene mutations that exhibit such dominant genetic traits include dominant negative mutants of HNF-1α. Transgenic animals into which this mutant has been introduced become model pigs that develop diabetes. Also, in disease model pigs in which at least part of the gene function existing on the X chromosome has been lost, the offspring become model pigs that develop diseases. Examples of genes that exist on the X chromosome and cause diseases when at least part of their function is lost include the ornithine transcarbamylase (OTC) gene. Model pigs into which at least part of the function of this gene has been lost develop hyperammonemia.

[0035] When the offspring become carriers of recessive gene mutations, (c) is (c) mating or subjecting the obtained offspring to sexual reproduction to obtain further genetically modified offspring that are heterozygous for the gene mutation (i.e., carriers of recessive gene mutations), and then further (d) subjecting carriers of recessive gene mutations to sexual reproduction to obtain offspring that are homozygous for the gene mutation as disease model pigs can be achieved.

[0036] Also, when the offspring become carriers of gene mutations on the X chromosome, (c) is (c) Breed the resulting offspring or subject them to sexual reproduction to obtain additional genetically modified offspring that are heterozygous for the gene mutation (i.e., carriers of the recessive gene mutation). and then further (d) Subject carriers of the recessive gene mutation to sexual reproduction, and subject female carriers having a gene mutation on the X chromosome to sexual reproduction to obtain male offspring as disease model pigs. This is possible.

[0037] Genes that are present on autosomes and cause diseases when at least part or all of their functions are lost include the γ-sarcoglycan (SGCD) gene. When homozygous knockout occurs, pigs that develop dilated cardiomyopathy are produced.

[0038] Here, when a pig obtained by somatic cell cloning dies before sexual maturity due to the influence of a disease, testicular tissue can be collected from a pig that survived before sexual maturity, and sperm can be obtained therefrom and used for mating. The method of obtaining sperm from testicular tissue is well known and is not particularly limited. For example, it is known that when testicular tissue is transplanted subcutaneously into immunodeficient mice, sperm with fertilizing ability can be obtained. In the present invention, sperm with fertilizing ability can be obtained from testicular tissue by such a method. Therefore, in this specification, "subjecting to sexual reproduction" is not particularly limited as long as offspring are obtained sexually, and includes methods of obtaining offspring from male and female by various methods.

[0039] In the above (c), among the offspring that are heterozygous for the gene mutation (i.e., carriers of the recessive gene mutation), females can be obtained as carriers. And the female carriers and the disease model pigs obtained as their offspring by the above (d) can be models that faithfully reproduce the phenotypes of human patients born from carrier mothers in humans. In the present invention, the above carriers and / or the disease model pigs obtained as their offspring by the above (d) are provided.

[0040] In addition, in the above (c), a female having a gene mutation on the X chromosome can be obtained as a carrier. And the female carrier and the male disease model pig obtained as its offspring by the above (d) can be a model that faithfully reproduces the phenotype of a human male patient born from a human carrier mother. In the present invention, the above carrier and / or the male disease model pig obtained as its offspring by the above (d) are provided.

[0041] Also, even when a pig obtained by somatic cell cloning dies before obtaining testes or ovaries due to the influence of a disease, an embryo of a pig obtained by somatic cell cloning and an embryo of a pig that does not develop the genetic disease are made into a chimeric state, transplanted into a synchronized surrogate female, and a chimeric individual is produced. After reducing the influence of the disease, it can be sexually matured, or reproductive glands (for example, testes and ovaries) or germ cells (sperm, eggs, and their progenitor cells) can be collected and matured as necessary, and used for sexual reproduction. Thus, as long as it develops and grows in a chimeric state, it can be sexually matured, or reproductive glands can be collected and matured as necessary, and a disease model pig or a carrier pig can be obtained by sexual reproduction. The disease model pig or carrier pig obtained in this way is considered not to be affected by epigenetic effects due to somatic cell cloning.

[0042] Therefore, according to the present invention, A method for producing a gene disease model pig, comprising: (i) performing nuclear transplantation into oocyte cytoplasm using the nucleus of a genetically modified cell; (ii) mixing the cloned embryo obtained by nuclear transplantation with an embryo of a pig that does not develop the genetic disease to make it chimeric, and allowing it to develop in the uterus of a female pig to obtain offspring; (iii) mating the obtained offspring or subjecting the germ cells of the obtained offspring to sexual reproduction to obtain offspring having the genetic modification. A method is provided.

[0043] Genetic diseases can be fatal diseases. Examples of fatal diseases include hyperammonemia and dilated cardiomyopathy.

[0044] Hereinafter, the above (i) to (iv) will be described for each step.

[0045] (i) Using the nucleus of a genetically modified cell to perform nuclear transfer into the oocyte cytoplasm (i) is the same as the above (a), so refer to that and the description here will be omitted.

[0046] (ii) Mixing the cloned embryo obtained by nuclear transfer with the embryo of a pig that does not develop a genetic disease to make it in a chimeric state, and allowing it to develop in the uterus of a female pig to obtain offspring Embryos obtained by somatic cell nuclear transfer (somatic cell cloning) can be mixed with embryos of pigs that do not develop genetic diseases, made into a chimeric state, and allowed to develop in the uterus of a female pig to obtain offspring. When using embryos in the early cleavage stage (1-cell stage to morula stage) for embryo mixing, blastomeres can be mixed with the other embryo, and when using embryos at the blastocyst stage, the inner cell mass can be mixed with the other embryo. In certain embodiments, the embryos of pigs that do not develop the genetic disease can be cloned embryos obtained by somatic cell cloning from labeled cells. In this specific embodiment, the label is not particularly limited, and can be, for example, a label with a fluorescent protein such as calcein orange and green fluorescent protein (GFP). The chimeric embryos can be, for example, transplanted into estrus-synchronized surrogate females, develop, and offspring can be obtained as chimeric individuals. The present invention also provides chimeric individuals obtained in this way.

[0047] The technique for making embryos into a chimeric state is well-known and can be used in the present invention. For example, Step (x): Prepare a nuclear transfer embryo (cloned embryo) having a lethal gene mutation and a nuclear transfer embryo or a healthy embryo without the gene mutation, respectively. Here, any of the nuclear transfer embryos may have been cryopreserved once. Step (y): When a blastomere obtained from one embryo of the other in the same developmental stage is injected into one embryo during the initial segmentation stage (1-cell stage to morula stage), the blastomeres of both can be aggregated inside the zona pellucida to produce a chimeric state. In step (y), embryos at the blastocyst stage can also be used. When using embryos at the blastocyst stage, a chimeric state can be produced by injecting all or part of the inner cell mass separated from one embryo into the other blastocyst. Step (z): After confirming the progression of the chimeric state by in vitro culture for several hours to about several days, it is transplanted into the oviduct or uterus of a surrogate mother for development.

[0048] The offspring is a chimeric individual of cells from a pig that develops a genetic disease and cells from a pig that does not develop the genetic disease, so the symptoms of the genetic disease can be alleviated. And thereby, it becomes possible to grow the chimeric individual until sexual maturity, or to grow it to the extent that gonads (for example, testicular tissue and ovarian tissue) and germ cells (sperm, eggs, and their precursors) are formed.

[0049] In one aspect of the present invention, germ cells (sperm, eggs, and their precursor cells) and gonads (testes and ovaries) obtained from the chimeric individual obtained by step (ii) are provided. Such germ cells and gonads have been difficult to obtain when the gene modification is lethal. Also, such gonads are chimeras of cells derived from a cloned embryo having a modified gene and a healthy embryo. In one aspect, the germ cells (sperm, eggs, and their precursor cells) and gonads (testes and ovaries) of the present invention have a gene modification that makes it difficult or impossible for the individual to survive.

[0050] (iii) Mating the obtained offspring or subjecting the germ cells of the obtained offspring to sexual reproduction to obtain offspring with the genetic modification (iii) is the same as (c) above, so reference is made to (c) above and the description here is omitted. If the offspring develop a disease, the offspring can be obtained as a disease model pig. On the other hand, if the offspring do not develop a disease, the offspring can be obtained as a carrier. The present invention also provides a carrier (for example, female or male) having a gene mutation. When the offspring obtained by (iii) become a carrier, the following (iv) can be further performed.

[0051] (iv) Mating the obtained offspring or subjecting them to sexual reproduction to obtain disease model pigs with the genetic modification (iv) is the same as (d) above. That is, carriers of recessive gene mutations are subjected to sexual reproduction, or female carriers having a gene mutation on the X chromosome are subjected to sexual reproduction, and offspring having a homozygous gene mutation can be obtained as a disease model pig.

[0052] In the above (iii), among the offspring having a heterozygous gene mutation (that is, a carrier of a recessive gene mutation), a female can be obtained as a carrier. And the female carrier and the disease model pig obtained as its offspring by the above (iv) can become a model that faithfully reproduces the phenotype of a human male patient born from a human carrier mother. Therefore, the present invention provides a female carrier and / or a disease model pig obtained as its offspring by the above (iv).

[0053] Also, in the above (iv), a female having a gene mutation on the X chromosome can be obtained as a carrier. And the female carrier having a gene mutation on the X chromosome and the male disease model pig obtained as its offspring by the above (iv) can become a model animal that faithfully reproduces the phenotype of a human male infant patient born from a human carrier mother. There is provided a model pig having a gene mutation on the X chromosome, a female carrier and / or a disease model pig obtained as its offspring by the above (iv).

[0054] In another aspect of the present invention, the present invention provides a population of genetically modified pigs obtained by the method of the present invention or genetically modified pigs with a constant phenotype.

[0055] Hereinafter, for the purpose of explanation, specific examples will be shown, but the present invention is not limited to the examples and is specified within the scope of the invention described in the claims.

Example

[0056] Example 1: Preparation of diabetic model pigs and analysis of their phenotypes In this example, based on the description of Umeyama K. et al., Transgenic Res. 18:697-706 (2009), an attempt was made to produce a diabetic model pig and its phenotype was analyzed in detail.

[0057] First, a diabetic model pig was produced based on the description of Umeyama K. et al., (2009). Specifically, the cDNA of the dominant negative mutant of human HNF-1α (HNF-1αP291fsinsC), which is known as the causative gene of type 3 juvenile-onset adult diabetes, was operably linked to the enhancer of the human CMV immediate early gene and the porcine insulin promoter, and the human HNF-1αP291fsinsC, polyA addition signal, and chicken β-globin insulator were linked downstream thereof to construct an expression unit, and a human mutant HNF-1α expression plasmid was obtained.

[0058] The human mutant HNF-1α expression unit was excised from the expression plasmid, and its DNA was contacted with the sperm surface and introduced into the in vitro matured eggs by microinjection-mediated gene transfer. The eggs were returned to the pig, and cells were obtained from the pig's kidney, lung, and muscle and their nuclei were transplanted into the egg cytoplasm (for details of the method, see Kurome K. et al., Transgenic Research 15:229-240 (2006)).

[0059] When the obtained pigs were analyzed, those showing the phenotype of diabetes were obtained. The results of analyzing a plurality of the obtained pigs are shown in Fig. 1.

[0060] In Fig. 1, the blood glucose levels and their changes are shown for five pigs. Each individual is a clone individual derived from the same human HNF-1α P291fsinsC gene-introduced cells. However, as shown in Fig. 1, the changes in blood glucose levels were significantly different for each of the obtained pigs, and it was found that their lifespans were also significantly different. Therefore, it became clear that further improvement was required for this diabetes model.

[0061] Example 2: Preparation of model pigs showing a stable diabetic phenotype In this example, the offspring obtained from the diabetes model pigs obtained in Example 1 were analyzed.

[0062] The diabetes model pigs obtained in Example 1 were sexually matured by insulin administration, sperm were collected and used for artificial insemination, and then transplanted into estrus-synchronized surrogate females to establish pregnancy and obtain offspring. The results of analyzing the diabetes phenotype of the obtained offspring are shown in Fig. 2. Fig. 2 shows the diabetes phenotype in representative offspring among a plurality of offspring.

[0063] As shown in Fig. 2, it became clear that the random blood glucose levels of the obtained offspring were stable throughout the week of age and that they became a good diabetes model.

[0064] Also, when the offspring of individuals showing different phenotypes among the diabetes models obtained in Example 1 were analyzed in the same way, it was also found that they were stable throughout the week of age and became a good diabetes model (data publication omitted).

[0065] It is considered possible that differences in the epigenetic state of the cells used for nuclear transfer are involved in the differences in phenotypes among individuals, and the reason why the phenotypes were stable in these offspring is considered to be that the above epigenetic state is eliminated by fertilization.

[0066] According to this example, it was revealed that by obtaining offspring after nuclear transfer, it is possible to stabilize the phenotype of the animal obtained by nuclear transfer (for example, a disease model).

[0067] From the above results, it became clear that the epigenetic state of the cells used for somatic cell cloning greatly affects the phenotype of the offspring obtained from somatic cell cloned embryos, and it became clear that this epigenetic effect can be eliminated by obtaining offspring of the next generation.

[0068] Example 3: Preparation of hyperammonemia model pigs In this example, an attempt was made to produce a hyperammonemia model pig.

[0069] Gene disruption of ornithine transcarbamylase (OTC) was performed as follows. As shown in Figure 3, OTC is present on the X chromosome and is considered to consist of 10 exons. OTC-TALEN targeting the second exon was constructed (Figure 3).

[0070] mRNA encoding TALEN was introduced into male porcine fetal fibroblasts by electroporation. Subsequently, when the sequences of the DNA targeted by TALEN were analyzed for 176 clones obtained by limiting dilution of the cells, mutations were confirmed in 43 clones (Figure 4). Among the obtained clones, clone No. 69 had a 5-base deletion in the second exon. Hereinafter, a model pig with hyperammonemia was produced using this clone. In clone No. 69, it was considered that the above 5-base deletion produced a stop codon, thereby disrupting the OTC gene function.

[0071] Using the nuclei of the No. 69 OCT gene disrupted clones, somatic cell cloning was performed by the method described in Matsunari et al., Cloning and Stem Cells, Vol. 10, No. 3, 313 - 323, 2008. The somatic cell cloned embryos were transferred to estrus synchronized surrogate females to establish pregnancy. OTC gene knockout pigs were obtained from the pregnant females by natural delivery and cesarean section.

[0072] When the obtained offspring were analyzed, these offspring developed hyperammonemia (Table 2). The general symptoms of the offspring showed more severe symptoms (such as convulsions) than those estimated from the symptoms of normal OTC gene deficiency (OTCD) patients (Table 2). When multiple offspring were analyzed, their phenotypes varied greatly among individuals (Table 2).

[0073]

Table 2

[0074] Next, embryos (female) were created by somatic cell cloning from cells expressing humanized kusabira orange, and were combined in a chimeric state with the somatic cell cloned embryos (male) obtained using the nuclei of the No. 69 OCT gene disrupted clones, and transferred to estrus synchronized surrogate females to produce chimeric individuals. The obtained chimeric individuals became male.

[0075] These chimeric individuals reached sexual maturity and produced sperm with OTC gene deficiency.

[0076] The obtained chimeric individuals were mated with wild - type females to obtain second - generation offspring. Among the offspring, female individuals had OTC gene deficiency on one of the X chromosomes and became OTCD carriers, or developed OTCD due to the influence of other inducing factors.

[0077] Thus, cloned embryos were created from the cells of a somatic cell cloned individual with a lethal OTC gene deficiency, and by making it chimeric with a healthy embryo, a chimeric individual with an avoided lethal phenotype was obtained. As a result of subjecting this chimeric individual to sexual reproduction, we succeeded in producing pigs that faithfully reproduced the female OTC gene deficiency.

[0078] It is possible to obtain third-generation individuals by subjecting female pigs that faithfully reproduce the OTC gene deficiency to mating or other sexual reproduction. Among the third-generation individuals, male individuals with the OTC gene deficiency become model animals that faithfully reproduce the phenotype of male human patients born from a mother who is a carrier of the OTC gene deficiency.

[0079] In addition, the obtained third-generation individuals become model animals that faithfully reproduce the pathological condition excluding the epigenetic effects present in the first generation, that is, the phenotype of male human patients born from a carrier mother.

[0080] Example 4: Preparation of dilated cardiomyopathy model pigs In this example, we attempted to produce a dilated cardiomyopathy model pig.

[0081] Disruption of the δ-sarcoglycan gene (SGCD) was performed as follows. Porcine SGCD is thought to be located on chromosome 16 and consists of 8 exons. In this example, a TALEN targeting the second exon including the start codon was constructed (Figure 5).

[0082] mRNA encoding TALEN was introduced into male porcine fetal fibroblasts by electroporation. Subsequently, 185 clones obtained by limiting dilution of the cells were analyzed for the sequence of the DNA targeted by TALEN, and mutations were confirmed in 67 clones (Figure 6). Among the clones obtained, clone No. 26 had a 4-base deletion creating a nonsense mutation in exon 2 and a 1,859-base deletion significantly deleting the transmembrane domain important for the function of SGCD. Also, clone No. 49 had a 5-base deletion creating a nonsense mutation in exon 2 and a 713-base deletion completely deleting exon 2. In these clones, the function of the SGCD gene was considered to be disrupted.

[0083] Using the nuclei of these clones, somatic cell cloned embryos were obtained in the same manner as in Example 3, and SGCD gene knockout pigs were obtained. These offspring developed symptoms characteristic of dilated cardiomyopathy at 4 weeks after birth. The general symptoms of the offspring were more severe than those estimated from patients with normal SGCD gene deficiency, and some individuals died of heart failure within a few weeks after birth. When multiple offspring were analyzed, their phenotypes varied greatly from individual to individual (Table 3).

[0084]

Table 3

[0085] In the same manner as in Example 3, the obtained embryos and somatic cell cloned embryos obtained from cells expressing Kusabira Orange were made into a chimeric state and transplanted into estrus-synchronized surrogate females, and chimeric individuals were produced. The obtained chimeric individuals became male. However, this male died before reaching sexual maturity. The cause of death was presumed to be arrhythmia due to stress associated with drug administration such as sedatives. Before the male died, testicular tissue was collected and cryopreserved.

[0086] It is known that frozen testis tissue can be transplanted subcutaneously into immunodeficient mice to obtain fertile sperm. Therefore, if the obtained sperm is fertilized with unfertilized eggs of wild-type females to obtain second-generation offspring, females with a deficiency in the SGCD gene on one of their chromosomes should be obtained among the offspring. Such females become carriers of the SGCD gene deficiency.

[0087] It is possible to obtain third-generation individuals by subjecting the unfertilized eggs obtained from the carrier to sexual reproduction with the sperm obtained above. Among the third-generation individuals, male individuals with an SGCD gene deficiency become model animals that faithfully reproduce the phenotype of human male patients born to mothers who are carriers of the SGCD gene deficiency.

[0088] In addition, the obtained third-generation individuals become model animals that faithfully reproduce the pathological condition from which the epigenetic effects present in the first generation have been excluded, that is, the phenotype of human male patients born to mothers who are carriers.

[0089] There is also a high possibility that the pathological condition of dilated cardiomyopathy will appear in second-generation pigs with a heterozygous deficiency in the above SGCD gene. Second-generation pigs are considered to exhibit a pathological condition closer to that of human patients with familial dilated cardiomyopathy because the epigenetic modifications of somatic cells have been reset as shown in Example 2.

Claims

Claim 1 (A) A cloned porcine embryo obtained by somatic cell cloning using the nucleus of a porcine somatic cell having a genetic modification is mixed with a porcine embryo to be in a chimeric state, and is allowed to develop in the body of a female pig to obtain a pig having a genetic modification. The obtained pig is grown to sexual maturity, or the obtained pig is grown to obtain a gonad or germ cell derived from the pig, wherein the cloned porcine embryo has a genome having an epigenetic state derived from the somatic cell, (B) subjecting the obtained pig, or the germ cell obtained from the pig or its gonad, to sexual reproduction to obtain a further pig having a genetic modification, comprising (i) In step (A), the obtained pig is grown to obtain a gonad or germ cell derived from the pig. In step (B), the germ cell obtained from the obtained pig or the gonad derived from the pig is subjected to sexual reproduction to obtain a further pig having a genetic modification. Here, in the obtained further pig, the epigenetic state based on the nucleus of the porcine somatic cell is eliminated. As a result, the obtained further pig exhibits a phenotype based on the genotype of the nucleus of the porcine somatic cell, and the epigenetic influence on the phenotype is eliminated. Method. Claim 2 (A) A cloned porcine embryo obtained by somatic cell cloning using the nucleus of a porcine somatic cell having a genetic modification is mixed with a porcine embryo to be in a chimeric state, and is allowed to develop in the body of a female pig to obtain a pig having a genetic modification. The obtained pig is grown to sexual maturity, or the obtained pig is grown to obtain a gonad or germ cell derived from the pig, (B) subjecting the obtained pig, or the germ cell obtained from the pig or its gonad, to sexual reproduction to obtain a further pig having a genetic modification, comprising In step (A), a pig grown to sexual maturity is obtained. Here, the cloned porcine embryo has a genome having an epigenetic state derived from the somatic cell. In step (B), the obtained pig is subjected to sexual reproduction to obtain a further pig having a genetic modification. Here , in the obtained further pig, the epigenetic state based on the nucleus of the porcine somatic cell is eliminated. As a result, the obtained further pig exhibits a phenotype based on the genotype of the nucleus of the porcine somatic cell, and the epigenetic influence on the phenotype is eliminated. Method. Claim 3 (A)A cloned porcine embryo obtained by somatic cell cloning using the nucleus of a porcine somatic cell having a genetic modification is mixed with a porcine embryo to be in a chimeric state and allowed to develop in the body of a female pig to obtain a pig having the genetic modification, and the obtained pig is grown to sexual maturity, or the obtained pig is grown to obtain a gonad or germ cell derived from the pig, and (B)subjecting the obtained pig, or the germ cell obtained from the pig or its gonad, to sexual reproduction to obtain a further pig having the genetic modification, and comprising In step (A), the cloned porcine embryo is lethal before sexual maturity, but the lethal symptoms are alleviated by being in a chimeric state. Method.

4. (a)The genetic modification is autosomal dominant, or (b)The genetic modification is X-chromosomal recessive, and the further pig is male. The method according to any one of claims 1 to 3.

5. The method according to claim 4, wherein the genetic modification is a modification by introduction of a foreign gene.

6. A composition comprising a gonad or germ cell of a pig obtained by step (A) in the method according to claim 1 or 2, and the composition is used for subjecting the gonad or the germ cell to sexual reproduction to obtain a further pig having the genetic modification.

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