JPWO2020085788A5 - - Google Patents

Description

The present invention relates to an IGF-1 gene mutation animal model for dwarfism and a method for producing the same.

The present invention was carried out with the support of the Korean government under Project No. KGM4251824 of the Korean Ministry of Science, ICT and Future Planning, "General-purpose/order-made artificial blood development project for utilization of minipig resources".

The main features of Laron syndrome are abnormally short stature (dwarfism) and physical symptoms include prominent protruding forehead, small skull, underdeveloped mandible, trunk obesity, and lack of hair. , bone development abnormalities (teeth, skull, thighs), saddle nose, double jaw, small genitalia, achondroplasia dwarfism (small limbs) and delayed puberty. indicates The etiology of such conditions is primarily associated with mutations in the growth hormone (GH) and GH receptor genes, resulting in decreased or deficient IGF-1. Such symptoms are known to occur. Therefore, in the development of therapeutic agents for Laron's syndrome, it is necessary to develop an animal model that can reflect a phenotype similar to that of Laron's syndrome so that the efficacy and safety of therapeutic agents can be evaluated.

Until now, most rodents have been used as disease models for drug therapy and mechanistic studies of genetic diseases, but the pathological aspects and symptoms of animal disease models differ greatly from those observed in humans. However, there have been many problems when conducting clinical trials based on results from rodent disease models.

In other words, the current situation is that an animal model that expresses all the symptoms of human genetic diseases to the extent that it can be used as an effective disease model has not yet been established. Therefore, problems due to certain differences in anatomy/physiology, reproduction, lifespan, and behavioral modalities from humans have raised the need for disease animal models using species that are more closely related to humans, and pigs that can be used to study intractable diseases. There is an increasing demand for using them as new model animals in the field of biopharmaceuticals.

An IGF-1 knockout mouse is an animal model of Laron's syndrome that has been developed so far, and it has been reported that the IGF-1 knockout mouse dies due to dyspnea one day after birth. To overcome this, mice were developed in which IGF-1 was specifically knocked out in the liver, which is responsible for 75% of IGF-1 production, using the Cre/loxp conditional system, and said mice had Although circulating IGF-I levels were reduced by more than 75%, there was a problem that the phenotype was not different from normal mice.

Recently, transgenic cloned minipigs were produced by knocking out the growth hormone receptor gene with genetic scissors, but only some phenotypes (dwarfism, obesity) observed in patients with Laron syndrome were observed. observed. In other words, the current situation is that a model animal that exhibits all the symptoms of Laron's syndrome to the extent that it can be used as an effective disease model has not yet been established.

Accordingly, the present inventors conducted research to develop transgenic animals having various phenotypes that appear in dwarfism such as Laron's syndrome, and completed the present invention.

KR10-2009-0051716A KR10-2018-0091291A WO01-72119A2

One object of the present invention is a nucleotide sequence encoding gRNA (guide RNA) that hybridizes to the DNA encoding the IGF-1 (insulin-like growth factor 1) gene; a nucleotide sequence encoding the Cas9 protein; and said nucleotide sequence is to provide a recombinant expression vector comprising a promoter operably linked to

Another object of the present invention is to provide a transformed cell line for producing an animal model of dwarfism into which a recombinant expression vector has been introduced.

Still another object of the present invention is to form a nuclear-transferred egg by transplanting a transformed cell line for preparing an animal model of dwarfism into an enucleated egg obtained from a non-human animal, and the nuclear-transferred egg. into the oviduct of a non-human animal surrogate mother.

Yet another object of the present invention is to provide an IGF-1 gene mutation animal model for dwarfism.

One aspect of the present invention is a nucleotide sequence encoding gRNA (guide RNA) that hybridizes to the DNA encoding the IGF-1 (insulin-like growth factor1) gene; a nucleotide sequence encoding the Cas9 protein; A recombinant expression vector is provided that includes a promoter operably linked thereto.

According to one embodiment of the present invention, the gRNA may comprise a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO:2.

Another aspect of the invention provides a transformed cell line for producing an animal model of dwarfism into which the recombinant expression vector has been introduced.

Still another aspect of the present invention is a step of forming a nuclear-transferred egg by transplanting a transformed cell line for preparing an animal model of dwarfism into an enucleated egg obtained from an animal other than a human, and the nuclear-transferred egg. into the oviduct of a non-human animal surrogate mother.

According to one embodiment of the invention, said animal may be a minipig.

According to one embodiment of the invention, said dwarfism may be due to IGF-1 knock-out.

According to a specific embodiment of the present invention, the knockout is generated by mutating the base sequence corresponding to SEQ ID NO: 2 to any one base sequence selected from the group consisting of SEQ ID NO: 5 to SEQ ID NO: 9. It may be

According to one embodiment of the invention, the dwarfism may be Laron's syndrome.

Yet another aspect of the invention provides an insulin-like growth factor 1 (IGF-1) gene mutation animal model of dwarfism.

According to one embodiment of the present invention, the animal model is an IGF-1 gene mutant dwarfism animal model in which the IGF-1 gene is knocked out.

In claim 10, the knockout is generated by mutating the base sequence corresponding to SEQ ID NO: 2 to any one base sequence selected from the group consisting of SEQ ID NO: 5 to SEQ ID NO: 9. good too.

According to one embodiment of the invention, said animal model may have reduced expression of one or more genes selected from the group consisting of EIF4H, CLIP2, ELN, LIMK1 and RFC2.

According to one embodiment of the invention, said animal model may be for Laron syndrome or Williams-Beuren syndrome model.

According to one embodiment of the invention, said animal may be a minipig.

According to one embodiment of the present invention, said mini-pigs may be pets.

According to one embodiment of the invention, said minipig may be for xenotransplantation.

According to one embodiment of the invention, said minipigs may be for artificial blood development.

The IGF-1 gene mutation animal model of dwarfism and its production method not only ameliorate the problem of death immediately after birth and allow observation of the majority of phenotypes seen in patients with Laron's syndrome. , which shows reduced expression of personality genes, can be usefully exploited for use in dwarfism-related disease models.

FIG. 2 shows the IGF-1 nucleotide sequence to which the CRISPR/Cas9 gene pincer guide RNA (gRNA) located in porcine IGF-1 exon 2 is hybridized . FIG. 2 shows the structure of the porcine IGF-1 reporter vector in the IGF-1 knockout gene scissors Surrogate Reporter System. FIG. 3 shows the structure of the porcine IGF-1 gRNA vector in the IGF-1 knockout gene scissors Surrogate Reporter System. FIG. 2 shows the structure of the Cas9 vector in the IGF-1 knockout gene scissors Surrogate Reporter System. It is a fluorescence microscopic photograph verifying the presence or absence of introduction and operation of the IGF-1 knockout gene scissors expression vector system. 1 is a graph showing the results of flow cytometry to verify the presence or absence of introduction and operation of an IGF-1 knockout gene scissors expression vector system. FIG. 3 shows mutations (#3 to #21) in the IGF-1 base sequence to which gRNA is hybridized in a normal minipig donor cell line (WT) into which the IGF-1 knockout gene scissors expression vector system was introduced. FIG. 2 shows changes in amino acid codons of the IGF-1 gene due to insertion of a base sequence in IGF-1 knockout (KO) transformant donor cell #7. The portion displayed in red is the stop codon. FIG. 2 shows changes in the codons of the amino acids of the IGF-1 gene due to the insertion of the base sequence in IGF-1 knockout transformant donor cell #15. The portion displayed in red is the termination codon. FIG. 2 shows the results of karyotype analysis of IGF-1 knockout (KO) transformed donor cell #7. FIG. 2 shows the results of karyotype analysis of IGF-1 knockout (KO) transformed donor cell #15. Schematic diagram showing the production process of IGF-1 knockout transgenic minipigs by somatic cell replication and surrogate mother transplantation. Fig. 10 is a photograph of a fetus of an IGF-1 knockout transgenic minipig that aborted on day 35 of gestation and an IGF-1 knockout transgenic minipig that was crushed to death by a surrogate mother on day 2 of birth. The yellow circle indicates the site crushed by the surrogate mother. Photographs comparing IGF-1 knockout (KO) transgenic minipigs and normal (WT) minipigs around day 56 of life. Identical IGF-1 base sequences to which gRNAs hybridize in Aborted, Died and Alive individuals of IGF-1 knockout transgenic minipigs and transgenic donor cell line #15. It is a figure which shows that it is. This is the result of nucleotide sequence analysis confirming that no off-target mutation occurs in nucleotide sequences similar to the IGF-1 target sequence other than the IGF-1 target sequence. This is the result of nucleotide sequence analysis confirming that no off-target mutation occurs in nucleotide sequences similar to the IGF-1 target sequence other than the IGF-1 target sequence. 2 is a graph showing the relative mRNA expression levels of IGF-1, IGF-2, IGFBP1, IGFBP2 and IGFBP3 genes between IGF-1 knockout transgenic clone minipigs that died by surrogacy to death around day 2 after birth and normal minipigs. FIG. 4 is a graph comparing hematologic alteration levels of growth hormone (GH), insulin (INS) and IGF-1 hormones in live IGF-1 knockout transgenic clone minipigs and normal minipigs. Figure 2 is a graph showing body weights from birth to postnatal day 27 for normal minipigs and IGF-1 knockout transgenic clone minipigs. Fig. 2 is a photograph comparing sizes after euthanizing IGF-1 knockout transgenic cloned minipigs at around 20 days after birth and normal minipigs at the same time. Fig. 3 is a photograph comparing the sizes of 3-month-old IGF-1 knockout transgenic cloned minipigs and normal minipigs. Fig. 3 is a photograph comparing trunk obesity in 3-month-old IGF-1 knockout transgenic cloned minipigs and normal minipigs. A graph showing the results of correcting and comparing the waist circumference (girth) of a 3-month-old IGF-1 knockout individual with the waist circumference of a normal individual (100%) as the standard (length) and height (height). be. Fig. 3 is a photograph comparing CT photographs of the head of a 3-month-old IGF-1 knockout transgenic cloned minipig and a normal minipig. Fig. 3 is a photograph comparing the presence or absence of stitching of the skulls of 3-month-old IGF-1 knockout transgenic cloned minipigs and normal minipigs. A graph showing the results of correcting and comparing the head circumference of a 3-month-old IGF-1 knockout individual with the head circumference of a normal individual as the reference (100%) using horizontal and vertical. is. Fig. 3 is a photograph comparing hair on the occipital side of 3-month-old IGF-1 knockout transgenic cloned minipigs and normal minipigs. Fig. 3 is a photograph comparing the thickness of the forefoot ankles of 3-month-old IGF-1 knockout transgenic cloned minipigs and normal minipigs. Fig. 3 is a photograph comparing IGF-1 knockout transgenic cloned minipigs (intraperitoneal or cryptic testicles; cryptorchism) and normal minipig testicles (normal descending) around 20 days after birth. Fig. 3 is a photograph comparing the testis thickness of 3-month-old IGF-1 knockout transgenic cloned minipigs and normal minipigs. Brain, heart, lung, liver, spleen, pancreas, kidney of 20 day old IGF-1 knockout transgenic clone minipigs and normal minipigs. ), femur and testes. 2 is a photograph comparing the mRNA expression levels of EIF4H, CLIP2, ELN, LIMK1 and RFC2 genes, which are personality genes, between an IGF-1 knockout transgenic clone minipig that died by surrogate mother's death around 2 days after birth and a normal minipig.

One aspect of the present invention is a nucleotide sequence encoding gRNA (guide RNA) that hybridizes to the DNA encoding the IGF-1 (insulin-like growth factor1) gene; a nucleotide sequence encoding the Cas9 protein; A recombinant expression vector is provided that includes a promoter operably linked thereto.

Herein, the IGF-1 gene is knocked out using IGF-1-specific gene scissors utilizing the CRISPR/Cas9 system, and by applying a transformant cell replication technique, dwarfism, specifically Laron Methods are provided for producing transgenic animals with a variety of phenotypes manifested in (Laron) syndrome.

The term "CRISPR / Cas9 system" used in the present invention is a third-generation genetic scissors composed of Cas9 protein and guide RNA, and is known as the immune system of microorganisms CRISPR (Clustered Regularly An artificial restriction enzyme designed to cleave a desired gene base sequence using the Interspaced Short Palindromic Repeats system.

As used herein, the term "Cas9 protein" refers to two RNAs called CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA), which are essential protein components in the CRISPR/Cas9 system. can form a complex with and act as an active endonuclease or nickase. The Cas9 protein may be from the genera Staphylococcus, Streptococcus, Neisseria, Pasteurella, Francisella, Campylobacter .

The term used in the present invention, "guide RNA (guide RNA, gRNA)" is a target DNA-specific RNA, gRNA can form a complex with Cas9 protein, Cas9 protein is brought into the target DNA be able to. The gRNA may be crRNA and tracrRNA, or may be sgRNA (single guided RNA) in which crRNA and tracrRNA are linked together.

By transforming a cell with the recombinant expression vector of the present invention, a guide RNA fragment can be transferred into the cell, and the transferred guide RNA fragment can recognize the IGF-1 gene. The recombinant expression vector can further comprise a tracerRNA. Therefore, by transforming a cell with a recombinant expression vector, a guide RNA fragment and a tracrRNA fragment, or an sgRNA in which crRNA and tracrRNA are linked together can be transferred into the cell. The fragment or portion can serve to form a complex or associated structure with the crRNA fragment or portion to form a structure recognizable by the Cas9 protein.

IGF-1 is a 70 amino acid with an 84 pi (isoelectric point) belonging to the somatomedin A system that has insulin-like and mitogenic biological activities that modulate the action of growth hormone. ) is a 7649 Da protein. Growth promotion by IGF-1 is the most important way that growth hormone regulates growth, and even if the level of growth hormone is normal, if IGF-1 secretion is impaired, it is more serious than general dwarfism. Laron syndrome can occur. The recombinant expression vector of the present invention can transfer the IGF-1 gene from a nuclear transfer donor cell during the production of a transgenic animal model by somatic cell replication without the off-target problem. Since it is possible to specifically knock out (KO) only , it can be usefully utilized for the production of animal models of dwarfism, especially Laron's syndrome.

The term "knockout" used in the present invention means to modify or remove a specific gene in the nucleotide sequence so that it cannot be expressed.

As used herein, the term "recombinant expression vector" refers to a recombinant DNA molecule containing a desired coding sequence and the proper nucleic acid sequences necessary to express the operably linked coding sequence in a particular host organism. means Promoters, enhancers, termination and polyadenylation signals available in eukaryotic cells are known.

As used herein, the term "operably linked" means a functional linkage between a gene expression regulatory sequence and another nucleotide sequence. The gene expression regulatory sequence may be one or more selected from the group consisting of a replication origin, a promoter, a transcription termination sequence (terminator), and the like. The transcription termination sequence may be a polyadenylation sequence (pA), the origin of replication may be the f1 origin of replication, the SV40 origin of replication, the pMB1 origin of replication, the adeno origin of replication, the AAV origin of replication or the BBV origin of replication, etc. Good, but not limited to this.

As used herein, the term "promoter" refers to a region of DNA upstream from a structural gene and refers to a DNA molecule to which RNA polymerase binds to initiate transcription.

A promoter according to one embodiment of the present invention is one of the transcriptional regulatory sequences that regulate the initiation of transcription of a particular gene, and may be a polynucleotide fragment of about 100bp to about 2500bp in length. Promoters can be used without limitation as long as they can regulate transcription initiation in cells such as eukaryotic cells (eg, plant cells or animal cells (eg, mammalian cells such as humans and mice)). For example, promoters include CMV promoter (cytomegalovirus promoter (e.g., human or mouse CMV immediate-early promoter), U6 promoter, EF1-alpha (elongation factor 1-a) promoter, EF1-alpha short (EFS) promoter, SV40 promoter, Adenovirus promoter (major late promoter), pLλ promoter, trp promoter, lac promoter, tac promoter, T7 promoter, vaccinia virus 7.5K promoter, HSV tk promoter, SV40E1 promoter, respiratory syncytial virus; RSV) promoter, metallothionin promoter, β-actin promoter, ubiquitin C promoter, human IL-2 (human interleukin-2) gene promoter, human lymphotoxin gene promoter and human GM-CSF (human granulocyte-macrophage colony stimulating factor) gene promoter, but not limited thereto.

A recombinant expression vector according to one embodiment of the invention is selected from the group consisting of viral vectors such as plasmid vectors, cosmid vectors and bacteriophage vectors, adenoviral vectors, retroviral vectors and adeno-associated viral vectors. There may be. Vectors that can be used as recombinant expression vectors include plasmids used in the industry (for example, pcDNA series, pSC101, pGV1106, pACYC177, ColE1, pKT230, pME290, pBR322, pUC8/9, pUC6, pBD9, pHC79, pIJ61, pLAFR1, pHV14, pGEX series, pET series, pUC19, etc.), phages (eg, λgt4λB, λ-Charon, λΔz1, M13, etc.) or virus vectors (eg, adeno-associated virus (AAV) vectors, etc.), etc. However, it is not limited to this.

A recombinant expression vector of the invention can further comprise one or more selectable markers. Said marker is a nucleic acid sequence that has a selectable property, usually by chemical means, and corresponds to any gene that allows transfected cells to be distinguished from non-transfected cells. For example, herbicide resistance genes such as glyphosate, glufosinate ammonium or phosphinothricin, ampicillin, kanamycin, G418, Bleomycin, hygromycin ), antibiotic resistance genes such as chloramphenicol, but not limited thereto.

The recombinant expression vector of the present invention can be produced using gene recombination technology well known in the art, and site-specific DNA cleavage and ligation are common in the art. It is carried out using enzymes known to

According to one embodiment of the present invention, the gRNA may comprise a base sequence complementary to the base sequence of SEQ ID NO:2.

By using gRNA comprising a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 2, it is possible to efficiently change the codon of the amino acid encoding IGF-1 and / or generate a premature stop codon. can be induced.

Other aspects of the invention include a nucleotide sequence encoding a gRNA that hybridizes to DNA encoding an insulin-like growth factor 1 (IGF-1) gene; a nucleotide sequence encoding a Cas9 protein; Transformed cell lines for producing animal models of dwarfism into which a recombinant expression vector containing a linked promoter has been introduced are provided.

As used herein, the term "dwarfism" refers to a condition that indicates a state of being small relative to the age-appropriate body size of an individual. The animal model of dwarfism of the present invention is "Laron's syndrome," a genetic disease in which the biological effects of growth hormone are reduced or lost despite normal or high production and secretion of growth hormone. phenotype can be reflected in a particularly similar manner.

Methods known in the art for introducing nucleic acid molecules into organisms, cells, tissues or organs can be used to produce transformed cell lines into which a recombinant expression vector according to one embodiment of the invention has been introduced. can be performed by selecting standard techniques suitable for the host cell, as known in the art. Such methods include, for example, electroporation, calcium phosphate (CaPO ₄ ) precipitation, calcium chloride (CaCl ₂ ) precipitation, microinjection, polyethylene glycol (PEG) method, DEAE-dextran method, Examples include, but are not limited to, cationic liposome method and lithium acetate-DMSO method.

The cell types used as transformed cell lines may be animal cells or cells derived from animal cells, preferably somatic cells derived from mammals, most preferably pigs or minipigs. When minipig-derived somatic cells are used as a transformed cell line, not only can the problem of minipig death immediately after birth be improved, but the phenotype of dwarfism, especially Laron's syndrome, can be similarly reflected. .
Yet another aspect of the present invention provides a nucleotide sequence encoding a gRNA that hybridizes to DNA encoding an insulin-like growth factor 1 (IGF-1) gene; a nucleotide sequence encoding a Cas9 protein; A transformed cell line for producing a dwarfism animal model into which a recombinant expression vector containing a promoter linked to a Provided is a method for producing a dwarfism animal model, comprising the steps of forming and transplanting the nuclear-transplanted egg into the fallopian tube of a surrogate mother, which is an animal other than a human.

The method for producing the dwarfism animal model may be by somatic cell nuclear transfer (SCNT). "Somatic cell nuclear transfer" is a genetic manipulation technology that allows offspring to be born without going through meiosis and haploid-bearing germ cells, which are generally performed in the reproductive process, and is a genetic manipulation technique that allows the generation of offspring without going through meiosis and haploid-bearing germ cells that are commonly performed in the reproductive process. It is a method of producing a fertilized egg by transplanting it into an egg from which the nucleus has been removed, and then transplanting the fertilized egg into a living body to generate a new individual.

The term "nuclear transfer egg" used in the present invention refers to an egg into which a nuclear donor cell has been introduced or fused, and "fusion" means binding of the nuclear donor cell and the lipid membrane portion of the egg. For example, the lipid membrane may be the plasma or nuclear membrane of a cell. Fusion is the application of electrical stimulation when the donor cell and egg are located adjacent to each other or when the donor cell is located within the perivitelline space of the recipient egg. can occur due to The transformed cell line is a nuclear donor cell and refers to a cell or a nucleus of a cell that transfers the nucleus to the nuclear receptor, the ovum. The ovum is preferably a matured ovum that has reached metaphase secondary meiosis, and preferably a porcine ovum.

According to one embodiment of the invention, said animal may be a minipig.

Pigs are recognized to be anatomically and physiologically similar to humans and have already been used for research into the pathological mechanisms and treatments of various diseases. Ethical problems can be avoided compared to using large animals as disease models, and there are advantages in that a stable breeding system has been established and maintenance and management are easy during the development of experimental animal models.

According to one embodiment of the invention, said dwarfism may be due to IGF-1 knock-out.

An animal model of dwarfism can be produced by knocking out IGF-1 by partial substitution, deletion, or insertion of some bases in the bases constituting the IGF-1 gene.

According to a specific embodiment of the present invention, the knockout is generated by mutating the base sequence corresponding to SEQ ID NO: 2 to any one base sequence selected from the group consisting of SEQ ID NO: 5 to SEQ ID NO: 9. It may be

SEQ ID NO: 5 is a base sequence in which the 5th to 7th bases “GGC” in the base sequence of SEQ ID NO: 2 are deleted. SEQ ID NO: 6 is a base sequence in which "C" is added next to the 7th base of the base sequence of SEQ ID NO: 2. SEQ ID NO: 7 is a base sequence in which "CT" is added next to the eighth base of the base sequence of SEQ ID NO: 2. SEQ ID NO: 8 is a base sequence in which the 7th base "C" is deleted from the base sequence of SEQ ID NO: 2. SEQ ID NO: 9 is a nucleotide sequence obtained by adding "CTG" next to the ninth nucleotide in the nucleotide sequence of SEQ ID NO: 2.

According to one embodiment of the invention, the dwarfism may be Laron's syndrome.

In conventional growth hormone receptor knockout transgenic minipigs, size reduction and obesity-related parts have been mainly reported, and although they are born with normal size at birth, problems occur later in the growth process, resulting in size/ A weight difference was noted and no delayed puberty was reported. In contrast, the animal model of the present invention exhibits various phenotypes seen in patients with Laron's syndrome. , protruding forehead, unclosed skull sutures, short nose length, small skull, missing teeth, missing hair, achondroplasia dwarfism and delayed puberty. Therefore, the animal model of the present invention is suitable as a model for dwarfism, particularly Laron's syndrome.
Yet another aspect of the invention provides an insulin-like growth factor 1 (IGF-1) gene mutation animal model of dwarfism.

As used herein, the term "animal model" refers to an animal that has a form of the disease that closely resembles that of humans. The significance of disease model animals in human disease research is due to the physiological or genetic similarity between humans and animals. Biomedical disease model animals in disease research can provide materials for research on various causes, pathogenic processes, and diagnosis of diseases. It is possible to obtain basic data for judging the feasibility of practical application from the actual efficacy and toxicity tests of the developed new drug candidates.

In the dwarfism animal model of the present invention, the parts overlapping with the above contents can be used with the same meanings as above.

The term "mutation" used in the present invention refers to a state in which hereditary traits are changed due to changes in genetic information due to changes in the base sequences of genes. Such mutations include point mutations, deletions, Mutations, insertion mutations, missense mutations and nonsense mutations are included. The present invention provides a dwarfism animal model with reduced expression of the IGF-1 gene due to mutation of the IGF-1 gene by the CRISPR/Cas9 system.

According to one embodiment of the present invention, the animal model may have an IGF-1 gene knock-out.

According to a specific embodiment of the present invention, the knockout is generated by mutating the base sequence corresponding to SEQ ID NO: 2 to any one base sequence selected from the group consisting of SEQ ID NO: 5 to SEQ ID NO: 9. It may be

According to one embodiment of the invention, said animal model may have reduced expression of one or more genes selected from the group consisting of EIF4H, CLIP2, ELN, LIMK1 and RFC2.

As used herein, the term "personality gene" is a gene for hyper-intimacy/hyper-sociality that is down-regulated in Williams-Beuren syndrome in humans. This confirms that it is related to personality.

According to one embodiment of the invention, said animal model may be for Laron syndrome or Williams-Beuren syndrome model.

The term used in the present invention, "Williams-Beuren syndrome", is excessively kind to humans, not only does not shy away from strangers when meeting strangers, but also has very good sociality. , refers to the symptoms of moderately low intelligence and impaired health and appearance. The animal model of dwarfism of the present invention has decreased expression of the personality genes EIF4H, CLIP2, ELN, LIMK1 and RFC2, and exhibits similar features to Williams-Boylen syndrome.

According to one embodiment of the invention, said animal may be a minipig.

According to one embodiment of the present invention, said mini-pigs may be pets.

Since the miniature pig of the present invention is smaller in size and fatter than normal miniature pigs, it can provide cuteness in appearance, and since the expression of personality genes is reduced, it is friendly and useful as a pet animal. It is possible.

According to one embodiment of the invention, said minipig may be for xenotransplantation.

Since the minipigs of the present invention are smaller in size than normal minipigs, they can be reared in a limited space in a larger number than general pigs, and their organs are larger than normal minipigs. Due to their small size, they can be bred and utilized as donor animals for xenogenic organ transplantation for patients requiring small organs.

According to one embodiment of the invention, said minipigs may be for artificial blood development.

The term "artificial blood" used in the present invention refers to a substitute for human blood that can perform functions such as oxygen supply, waste transport, antibacterial action and/or nutrient transport in the human body. , artificial red blood cells with genetically engineered recombinant human hemoglobin, and the like.

Since the minipigs of the present invention are smaller in size than normal minipigs, they can be reared in a limited space with a larger number of individuals than general pigs. It can be usefully utilized as a transgenic animal for production or as a surrogate mother for human hematopoietic mother transplantation.

The present invention will now be described in greater detail through one or more examples. However, these examples are for illustrative description of the present invention, and the scope of the present invention is not limited to these examples.

Example 1. Construction of IGF-1 knockout gene scissors expression system
1-1. IGF-1 knockout gene scissors expression vector system
CRISPR/Cas9 gene scissors were designed targeting exon number 2 among the four exons of the porcine IGF-1 gene. A specific porcine IGF-1 locus is shown in FIG. sequence and complementary sequence) are shown in Table 1.

The IGF-1 knockout gene scissors system was made with a total of 3 vectors Surrogate Reporter System.

Specifically, as shown in FIG. 2, the mRFP1 (monomeric red fluorescent protein 1) coding sequence is located upstream of the IGF-1 base sequence to which the sgRNA hybridizes , and the IGF-1 to which the sgRNA hybridizes . A reporter vector was constructed with an EGFP (enhanced green fluorescent protein) coding sequence located one base downstream. Alternatively, as shown in FIG. 3, an sgRNA vector is produced by inserting a nucleotide sequence encoding sgRNA, and as shown in FIG. A Cas9 vector was generated by inserting a sequence encoding a Cas9 protein to recognize the bound sgRNA and cleave the target sequence of IGF-1.

The IGF-1 gene-targeted sgRNA vector uses CRISPR to target four target-specific sites with higher out-of-frame scores to achieve maximal knockout efficiency in the IGF-1 coding region using published design tools. (http://crispr.mit.edu; https://crispr.cos.uni-heidelberg.de/; http://www.rgenome.net/cas-designer/) and thus designed Cas9, sgRNA and RGS reporter vectors were synthesized and purchased from ToolGen, Inc. Seoul, Korea.

Since mRFP (red fluorescence) is expressed in cells into which the genetic scissors system of the present invention has been introduced, it can be verified whether or not the genetic scissors system of the present invention has been introduced into the cells. On the other hand, when the IGF-1 target sequence contained in the reporter vector is cleaved by the Cas9 protein expressed in the Cas9 vector, EGFP (green fluorescence) is expressed in cells into which the genetic scissors system of the present invention has been introduced. Therefore, by confirming the presence or absence of green fluorescence, it is possible to verify whether the gene scissors system of the present invention operates normally.

1-2. Validation of the IGF-1 Knockout Gene Scissors Expression Vector System--Fluorescence Microscopy Analysis IGF-1 Knockout Transformed IGFs produced in Example 1-1 into a normal minipig donor cell line for somatic cell replication for minipig production. After the introduction of the scissors expression vector system for the -1 knockout gene, the presence or absence of the introduction and operation of the vector system was confirmed by fluorescence microscope analysis.

Specifically, donor cells were obtained from kidney tissue of KSP minipigs (2 days old, male) and were supplemented with 10% (v/v) Penicillin/Streptomycin (Invitrogen) until kidney tissue isolation. Stored in the included DPBS wash buffer and refrigerated. Thereafter, the kidney tissue was minced using a sterilized surgical blade, placed on a 100 mm culture dish pretreated with 0.1% gelatin (Sigma-Aldrich), and added with 10% FBS. (fetal bovine serum) (16000-044, GIBCO, Carlsbad, CA, USA), DMEM (Dulbecco's Modified Eagle's Medium) containing 10 ng/ml basic fibroblast growth factor (bFGF) and 1% penicillin/streptomycin (Invitrogen) until sufficient donor cells were obtained.

2×10 ⁶ donor cells were premixed with 1 μg Cas9, 1 μg sgRNA and 2 μg RGS reporter plasmid DNA in 100 μl buffer of Amaxa ^™ P3 primary cell 4D Nucleofector ^™ X Kit L (Lonza) before 4D Nucleofector ^™ (Lonza). The vector system was introduced by electroporation using the program EH-113, and after 24 hours, cells expressing fluorescence were confirmed under a fluorescence microscope (Leica).

As a result, red fluorescence was detected in the donor cell line into which only the reporter vector was introduced, confirming that the reporter vector was introduced into the cells. On the other hand, both red fluorescence and green fluorescence were detected in the donor cell line into which the IGF-1 knockout gene scissors expression vector system produced in Example 1-1 was introduced. Therefore, it was confirmed that the vector system of the present invention was introduced into the donor cell line and was operable in the donor cell line (Fig. 5).

1-3. Validation of IGF-1 Knockout Gene Scissors Expression Vector System-Flow Cyt Analysis IGF-1 Knockout Transformed Clones For Minipig Production, To Normal Minipig Donor Cell Lines for Somatic Cell Replication, IGFs Produced in Example 1-1 After introduction of the scissors-1 knockout gene expression vector system, flow cytological analysis was performed to quantitatively identify cells expressing red fluorescence and green fluorescence among the whole cells in order to confirm the introduction and operation of the vector system. confirmed to

Specifically, for flow cytometry, donor cells were transfected with red-fluorescent, green-fluorescent, and empty vectors to form positive and negative control groups, respectively, followed by wavelength bands where red and green fluorescence overlap. (wavelength band) was removed by a compensation process to obtain the setting values at which each intrinsic fluorescence was measured. Thereafter, fluorescence expression was confirmed using donor cells introduced with the gene scissors vector system under the set conditions.

As a result, little red and green fluorescence was detected in the untransformed donor cell line, whereas red fluorescence was detected in the donor cell line transfected with the reporter vector alone, indicating that the cells had been transfected with the reporter vector. confirmed. On the other hand, both red fluorescence and green fluorescence were detected in the donor cell line into which the IGF-1 knockout gene scissors expression vector system produced in Example 1-1 was introduced. Therefore, it was confirmed that the vector system of the present invention was introduced into the donor cell line and was operable in the donor cell line (Fig. 6).

Example 2. Generation of IGF-1 Mutant Transformed Donor Cell Lines
2-1. Validation of IGF-1 Knockout Transformed Donor Cell Lines - Sequence Analysis
After introducing the IGF-1 knockout gene scissors vector system into a normal minipig donor cell line for somatic cell replication, sequencing was performed on the transformed minipig donor cell line cultured in single cells. analysis) was performed to confirm whether mutation occurred in the IGF-1 base sequence to which the sgRNA is hybridized by the IGF-1 knockout gene scissors vector system.

Specifically, cells expressing green fluorescence were sorted into single cells in 96-well plates containing growth medium using a FACSArial III sorter (BD Biosciences). Two weeks after sorting, single colony cells selected from the 96-well plate were cultured in a 6-well plate by a subculture process, and then a portion of each clonal cell line was subjected to genomic DNA analysis using the DNeasy Blood & Tissue Kit (Qiagen). Extracted. Primers were designed so that the size of the PCR product would be about 500 bp around the sgRNA of the IGF-1 gene, and the gene was amplified using a PCR (Applied Biosystems) device. After confirming the amplified PCR product by electrophoresis, it was collected by the gel elution process. In order to confirm the form of Indel (mutation, insertion, etc. in the base sequence), the base sequence of the DNA recovered by gel elution was confirmed using the primers (Table 2) used for PCR amplification.

As a result, as shown in Table 3 and FIG. 7, in the donor cell lines #3, #7, #15, #18 and #21, the nucleotide sequences corresponding to the IGF-1 nucleotide sequences to which the sgRNAs hybridized confirmed that a mutation occurred in

2-2. Verification of IGF-1 Knockout Transformed Donor Cell Lines--Amino Acid Sequence Analysis How mutations in the nucleotide sequence of the IGF-1 nucleotide sequence to which the sgRNA is hybridized confirmed in Example 2-1 affect the structure of the IGF-1 protein. Amino acids encoded by the coding sequence of the mutant IGF-1 in the transformed donor cell line were analyzed to see if they were affected.

As a result, the insertion of the base sequence in #7 (+1 bp) (Fig. 8) and #15 (+2 bp) (Fig. 9) transformation donor cell lines in which the IGF-1 base sequence to which the sgRNA is hybridized was mutated. confirmed that the codon of the amino acid encoding IGF-1 was changed to generate a premature stop codon. Termination codons are indicated in red in FIGS.

2-3. Analysis of Karyotype of IGF -1 Mutated Transformed Donor Cell Line In order to confirm whether the chromosomes and sex chromosomes were affected, the karyotypes of the chromosomes of the transformed donor cell lines were analyzed by the G-banding karyotyping method to examine the stability of the chromosomes.

Specifically, when the cells reached 70-80% confluency, they were treated with 0.1 mg/ml colcemid (Sigma-Aldrich) for 50 minutes in a 5% CO ₂ incubator at 37° C. Cell division was arrested at the stage. Arrested cells were treated with 0.06 M KCl (Sigma-Aldrich) hypotonic solution for 10 min at 20° C. to swell the cells and fixed in methanol/acetic acid (3:1) fixative for 30 min. After that, it was centrifuged at 150 g for 10 minutes. After repeating the fixation procedure three times, fixed cells were dropped onto glass slides and air-dried at 65° C. overnight. Finally, after pretreatment with 0.25% trypsin, chromosomes were stained by Giemsa staining to confirm the karyotype.

As a result, the autosomes and sex chromosomes of transformation donor cell lines #7 (FIG. 10) and #15 (FIG. 11) in which the IGF-1 base sequence to which the sgRNA hybridizes were mutated show normal chromosome images. It was confirmed that a normal diploid karyotype was maintained in the transformed minipig donor cell line.

Example 3. Production of IGF-1 Knockout Transformed Cloned Minipigs In the IGF-1 knockout transformed donor cell line prepared in Example 2, when somatic cell nuclear transfer (SCNT) was performed using embryos, embryos (embryo ) IGF-1 knockout transgenic cloned minipigs were produced using #15 donor cell line which was confirmed to have superior developmental rate.

FIG. 12 is a schematic diagram showing the production process of IGF-1 knockout transgenic minipigs by somatic cell replication and surrogate mother transplantation.

First, the somatic nuclei of the IGF-1 knockout transformed #15 donor cell line were transformed into enucleated eggs.

Specifically, after in vitro maturation (IVM), mature oocytes with visible first polar bodies for SCNT were selected. Cumulus cell-depleted oocytes were slit by pipetting in DPBS containing 4 mg/ml BSA, 75 μg/ml penicillin G, 50 μg/ml streptomycin sulfate and 7.5 μg/ml CB. were made to remove cytoplasm and first polar bodies from oocytes under an automated phase-contrast microscope (DMI 6000B, Leica) fitted with approximately 10% (NT-88-V3, Nikon-Narishige). Donor cells were cultured for 3 days without changing the medium to induce cell cycle synchronization in the G0-G1 stage, and oocytes were placed in IVC medium in 5% CO ₂ until electrofusion,38. Maintained in an incubator at 5°C. Single donor cells/oocytes were allowed to equilibrate in fusion medium composed of 280 mM mannitol (Sigma _- Aldrich) containing 0.1 mM _MgSO4.7H2O and 0.01% PVA. It was then placed between two attached parallel electrodes (100 μm diameter, CUY5100-100, Nepagene). After activation by one DC pulse of 23 V for 50 μsec using an electrocell fusion generator, they were placed in an incubator at 5% _CO2 and 38.5°C. _Two hours later, pairs of fully fused oocytes were selected under a phase _- contrast microscope and added to 0.1 mM _MgSO4.7H2O , 0.1 mM _CaCl2.2H2O , 0.5 mM HEPES and 0.01 mM HEPES. 10 μl 280 mM mannitol solution containing % PVA was transferred to an overlapping 1 mm gap wire chamber and then activated with an electro cell fusion generator at 110 V DC for 50 μsec. To chemically activate the electrofusion-activated oocytes, place them in medium supplemented with 50 nM Trichostatin A (TSA) for 4 h at ₅ % CO and 38.5 °C. Maintained in an incubator. After 4 hours, activated embryos were transferred to IVC medium supplemented with 50 nM TSA and culture continued for 20 hours. After 20 h, SCNT embryos were washed with IVC medium and placed in an incubator at 5% _CO2 and 38.5 °C. Division and blastocyst stage formation were then assessed on days 2 and 6, respectively, to prepare for transfer to surrogate mothers.

After that, the ovum in which the somatic cell nucleus of the IGF-1 knockout-transformed donor cell line #15 was transformed was transplanted into the surrogate mother.

Specifically, the fused cloned eggs were cultured in a 4-well plate containing 500 μl of culture medium for 1 to 2 days, then placed in a freezing tube containing 2 ml of the same culture medium and heated to 39°C. The fertilized eggs were transported to the place of transplantation using a fertilized egg transporter (Embryo transfer kit, Minitube, USA). A surrogate mother for cloned egg transplantation was prepared by selecting an individual in which estrus had started. let me After fixing the anesthetized surrogate mother to the operating table, inhalation anesthesia was performed by supplying 5% Isoflurane. The anesthetized surrogate mother was prepared by incising about 5 cm along the midline of the abdomen and exposing the uterus and ovaries to the outside of the body. At this time, the cloned egg was implanted by inhaling the cloned egg with a catheter (Tom cat catheter, Monoject, USA) and pushing it into the isthmus of the fallopian tube. A total of 600 cloned eggs were implanted into two foster mothers for the production of IGF-1 knockout transgenic cloned pigs. The surrogate mothers who completed the implantation of the cloned eggs underwent pregnancy test using ultrasound for 30 days, and the animals confirmed to be pregnant were induced to give birth after 114 days.

The surrogate mothers are raised in a SPF (Special Pathogen Free) facility, taking into account the characteristics of the minipigs, and the floor material uses plastic to facilitate the movement and flow of the minipigs, and the floor of the breeding cage is equipped with a temperature-maintaining pad. provided to help maintain a constant temperature of the minipig piglets. IGF-1 knockout-transformed cloned minipigs were artificially fed with a feed containing 70 g of safe milky, 1 g of colostrum stick and 1 g of vitamin in 500 ml of water at intervals of 2 hours from 2 days after birth. An iron injection inoculation was performed. IGF-1 knockout minipigs were systematically maintained by microbiological monitoring, with individual body weights and feed amounts established for 3 months after birth of the minipigs.

As can be seen from FIG. 13, one IGF-1 knockout individual miscarried on the 35th day after transplantation into the surrogate mother, and one IGF-1 knockout individual was crushed to death by the surrogate mother on the second day after birth. It was confirmed that the part crushed by the 2 is the yellow circle part in FIG. 13 .

On the other hand, other IGF-1 knockout individuals survived, and photographs comparing IGF-1 knockout minipigs and normal minipigs at around 56 days after birth are shown in FIG.

Example 4. Confirmation of IGF-1 knockout in transgenic cloned minipigs
4-1. Nucleotide Sequence Analysis of IGF-1 Knockout Transformed Cloned Minipigs
To confirm whether the cloned minipigs produced were IGF-1 knockout transformed minipigs, the nucleotide sequences of the IGF-1 knockout transformed cloned minipigs were analyzed.

Specifically, except that cells from aborted, crushed, and surviving IGF-1 knockout-transformed cloned minipigs and IGF-1 knockout-transformed #15 donor cell lines were used. The nucleotide sequence was analyzed in the same manner as in Example 2-1.

As a result, as can be seen from FIG. 15, the IGF-1 base sequence to which the gRNA is hybridized was found in the aborted individual, the crushed individual and the surviving IGF-1 knockout transformed clone minipig. It was confirmed that the nucleotide sequence was identical to #15, and the produced cloned minipigs were confirmed to be IGF-1 knockout transformed cloned minipigs.

4-2. Verification of off-target of IGF-1 knockout transformed clone minipig The IGF-1 knockout gene scissors expression vector system prepared in Example 1-1 targets other base sequences than the IGF-1 target sequence Off-target in IGF-1 knockout transgenic cloned minipigs was examined to see if it did.

Specifically, the IGF-1 target sequence (sgRNA hybridized with the IGF-1 target sequence (sgRNA Sequences for genes containing similar sequences (Table 4) were analyzed.

As a result, as can be seen from FIGS. 16 and 17, it was confirmed that mutations did not occur in base sequences similar to the IGF-1 target sequence other than the IGF-1 target sequence, and the off-target problem occurred. Confirmed not to.

4-3. Analysis of mRNA Expression Levels in IGF-1 Knockout Transformed Clone Minipigs IGF-1 and IGF-2 are regulated by proteins known as IGFBPs. Such proteins not only promote possible IGF action by aiding transduction to the receptor and increasing IGF half-life, but also inhibit IGF action by preventing binding to the IGF-1 receptor. It helps regulate the actions of IGF in a complex way that involves doing everything.

Therefore, in order to confirm the expression of IGF-1, IGF-2, IGFBP1, IGFBP2 and IGFBP3, cDNA was synthesized by extracting mRNA from the liver tissue of a normal individual and an IGF-1 knockout individual who died by surrogacy. Afterwards, the expression of these genes was analyzed using the probes in Table 4 below.

Specifically, according to the method provided by the manufacturer, total RNA was extracted from cells and tissues using RNeasy plus mini kit (QIAGEN). Total RNA (1 μg) was used for cDNA synthesis using PrimeScript® RT reagent kit (TAKARA), and relative expression levels of genes were measured by real-time RT-PCR using SYBR Premix Ex Taq II (TAKARA). , StepOnePlus Real-Time PCR System (Applied Biosystems). The reaction variables for quantitative RT-PCR analysis were 95°C for 10 minutes, 95°C for 30 seconds, 62°C for 30 seconds, 72°C for 30 seconds, and a final cycle step of 72°C for 5 minutes repeated 40 times. . For comparative analysis, mRNA expression levels were quantified with GAPDH followed by fold-change. WT was used as the control group ΔCt (CΔCt) value and the sample delta Ct (SΔCt) value was calculated from the difference between the Ct values of GAPDH and the target gene. Relative gene expression levels between sample and control groups were determined using the 2-(SΔCt-CΔCt) formula, and for amplification of porcine cDNA, PCR primers were used as shown in Table 5 below using Primer3 software. designed to.

As a result, little expression of IGF-1 was observed in IGF-1 knockout-transformed cloned minipigs, and IGF-2 was compensatory increased by the deficient IGF-1 gene, suggesting the opposite effect of IGF-1. It was confirmed that functional IGFBP1 (IGF binding protein 1) increased to a large level (Fig. 18).

4-4. Analysis of Altered Levels of Hematologic Hormones in IGF-1 Knockout Transformed Clone Minipigs Since IGF-1 suppresses growth hormone (GH) with negative feedback and insulin (INS) shares receptors with IGF-1, It is known to increase in the absence of IGF-1 to compensate for its function.

Therefore, to confirm the expression of IGF-1, ELISA analysis was performed on the blood of normal and living IGF-1 knockout individuals to analyze the altered levels of these hormones.

Specifically, using MyBioSource's Porcine IGF-1 ELISA Kit (Cat.MBS761388), Porcine Growth Hormone ELISA Kit (Cat.MBS026403) and Porcine Insulin (INS) ELISA Kit (Cat.MBS2601735), The experiment was performed according to the instructions and the result values were derived.

As a result, it was confirmed that almost no IGF-1 expression was observed in IGF-1 knockout transgenic clone minipigs, INS was increased compensatory by IGF-1 deficiency, and GH levels were markedly increased. (Fig. 19).

Example 5. Confirmation of IGF-1 knockout phenotype in transgenic cloned minipigs
5-1. Confirmation of low birth weight
To confirm whether the IGF-1 knockout transgenic cloned minipigs exhibit the IGF-1 knockout phenotype, it was determined whether the IGF-1 knockout individuals had normal body weight from birth.

Specifically, the body weights of IGF-1 knockout and normal individuals were compared from birth to 27 days of age.

As a result, the weight at birth of the individuals was 835±91 g on average for the normal individuals, while the surviving IGF-1 knockout individuals weighed 400 g and the crushed IGF-1 knockout individuals weighed 273 g. confirmed. It was also confirmed that the weight of the individuals on day 27 after birth was 892 g for the surviving IGF-1 knockout individuals, while the average for normal individuals was 4608±256 g (FIG. 19).

Through these results, the difference in body weight of the IGF-1 knockout transformed clone minipigs occurs from birth, and the weight gain during growth is significantly decreased unlike normal individuals, so the IGF-1 knockout transformed clones Minipigs were confirmed to exhibit an IGF-1 knockout phenotype.

5-2. Confirmation of Small Size at Birth and Small Postnatal Stature To confirm whether IGF-1 knockout transgenic cloned minipigs exhibit the IGF-1 knockout phenotype, IGF-1 knockout individuals were small size from birth. and whether they have small stature after birth.

Specifically, the sizes of IGF-1 knockout individuals at birth and normal individuals were compared, and the sizes of IGF-1 knockout individuals and normal individuals at 3 months of age were compared.

As a result, compared to the normal individuals, the size of the crushed IGF-1 knockout individuals was smaller at birth (Fig. 20), and around 20 days after birth, a clear difference in development was confirmed compared to the control group (Fig. 21). ). We also confirmed the smaller length and height of the surviving IGF-1 knockout individuals at 3 months of age compared to normal individuals (FIG. 22).

Through these results, IGF-1 knockout-transformed cloned minipigs have a difference in size from birth, and the height increase during growth is significantly reduced unlike normal individuals, so IGF-1 knockout-transformed Cloned minipigs were confirmed to exhibit an IGF-1 knockout phenotype.

5-3. Confirmation of truncal obesity To confirm whether the IGF-1 knockout transgenic cloned minipigs exhibit the IGF-1 knockout phenotype, the presence or absence of truncal obesity in IGF-1 knockout individuals was confirmed.

Specifically, the width of the trunk (forefoot scapula/backfoot pelvic girdle baseline) of 3-month-old IGF-1 knockout individuals and normal individuals was compared.

As a result, truncal obesity was confirmed in surviving 3-month-old IGF-1 knockout individuals (FIG. 23). In addition, the waist circumference (girth) of the IGF-1 knockout individual was measured, and this was compared with the normal individual as the reference (100%), and the waist circumference was corrected using the body length and height. It was confirmed that the waist circumference was 216% of that of a normal individual (Fig. 24).
Through these results, it was confirmed that the IGF-1 knockout transgenic cloned minipigs exhibit the IGF-1 knockout phenotype, since they exhibit marked trunk obesity unlike normal individuals.

5-4. Identification of protruding forehead, unclosed skull sutures, short snout length, small skull and missing teeth. Knockout animals were checked for protruding forehead, unclosed skull sutures, short snout length, small skull and missing teeth.

Specifically, a 3-month-old IGF-1 knockout individual and a normal individual were subjected to computed tomography (CT), and the images were compared.

As a result, it was confirmed that IGF-1 has a protruding morphology and normal individuals have a flat forehead morphology. As can be seen from FIG. 25, unlike normal individuals whose skulls are closed from birth, surviving IGF-1 knockout individuals show unclosed skull sutures (arrows), and compared to normal individuals, surviving IGF-1 -1 knockout individuals confirmed a relatively short snout length (human saddle nose phenotype) and small skull size, and relatively few teeth (underdeveloped upper and lower anterior molars) (Fig. 25).

In addition, as can be confirmed from FIG. 26, when the skull was anatomically exposed, the sutures were not closed (red arrows) in GF-1 KO individuals (#002, #003), and WT individuals (#031, #031). #032) confirmed that the skull protruded.

Furthermore, the head circumference of the IGF-1 knockout individual was measured, and this was corrected using the horizontal and vertical head circumference as the normal individual standard (100%). As a result, IGF It was confirmed that the head circumference of the -1 knockout individual was 58% of that of the normal individual (Fig. 27).

Through these results, IGF-1 knockout transgenic cloned minipigs express IGF-1 knockout expression, as they exhibit prominent forehead, unclosed skull sutures, short snout length, small skull and missing teeth, unlike normal individuals. Confirmed to show type.

5-5. Confirmation of Missing Hair To confirm whether the IGF-1 knockout transgenic cloned minipigs exhibit the IGF-1 knockout phenotype, the presence or absence of missing hair in IGF-1 knockout individuals was checked.

Specifically, the hair on the occipital side of a 3-month-old IGF-1 knockout individual was compared with that of a normal individual.

As a result, we confirmed the severely deficient hair count and poor hair condition of the surviving IGF-1 knockout individuals (Fig. 28), through which the IGF-1 knockout transgenic cloned minipigs exhibited the IGF-1 knockout phenotype. Confirmed to show.

5-6. Confirmation of achondroplasia dwarfism, occult testicles and small genitalia The presence or absence of anthropomorphism and small genitalia was confirmed.

Specifically, the forefoot ankle thickness and testicle size of 20-day-old or 3-month-old IGF-1 knockout individuals and normal individuals were compared.

As a result, it was confirmed that the thickness of the forefeet ankle of the 3-month-old normal individual was 42.75 cm, while the thickness of the ankle of the surviving IGF-1 knockout individual was 27.16 cm (Fig. 29).

In addition, at around 20 days after birth, the surviving IGF-1 knockout individuals had cryptic testicles with testicles in their organs (Fig. 30). On the other hand, it was confirmed that the testicle size of the surviving IGF-1 knockout individual was 27.16 cm (Fig. 31).

Through these results, unlike normal individuals, achondroplasia dwarfism appears and delayed puberty is predicted due to delayed testicular development. It was confirmed that the phenotype was exhibited.

5-6. Confirmation of small organ size and weight To confirm whether the IGF-1 knockout transgenic cloned minipigs exhibit the IGF-1 knockout phenotype, the organ size and weight of the IGF-1 knockout individuals were confirmed.

Specifically, the heart, lung, liver, spleen, pancreas and kidney of 20 day old surviving IGF-1 knockout and normal individuals. and testes size and weight were compared.
As a result, it was confirmed that all the organs of the surviving IGF-1 knockout individuals were significantly smaller than those of the normal individuals around 20 days after birth, and the weights of all the organs were 16-36% of the weight of the normal individuals. (Table 6 and FIG. 32).

Through these results, it was confirmed that the IGF-1 knockout transgenic cloned minipigs exhibit the IGF-1 knockout phenotype, since they exhibit smaller organ size and weight than normal individuals.

Example 6. Confirmation of Altered Expression of Personality Gene Expression in Transformed Cloned Minipigs Personality genes are genes for hyper-sociality, whose expression is reduced in Williams-Beuren syndrome in humans. In 2010, it was published in the Nature journal that similarities between dogs and Williams-Boylen syndrome-inducing dielectrics were confirmed. rice field. Williams-Boylen syndrome is not only excessively kind to humans and unfamiliar with strangers, but also displays excessively good social skills, often accompanied by low intelligence and impaired health and appearance. .

To confirm whether the IGF-1 knockout transgenic cloned minipigs exhibit characteristics of Williams Boylen syndrome, the expression of personality genes in IGF-1 knockout individuals was analyzed.

Specifically, the method of Example 4-3 except that the probes in Table 7 below were used after mRNA was extracted from the liver tissue of a normal individual and an IGF-1 knockout individual who was crushed to death and cDNA was synthesized. The expression of the personality genes EIF4H, CLIP2, ELN, LIMK1 and RFC2 was analyzed in a similar manner.

As a result, it was confirmed that the expression of personality genes was largely decreased in IGF-1 knockout individuals compared to normal individuals (Fig. 33).

Through these results, it was confirmed that unlike normal individuals, IGF-1 knockout transgenic clone minipigs exhibit the characteristics of Williams-Boylen syndrome.
So far, the present invention has been described mainly with reference to its embodiments. Those skilled in the art to which the present invention pertains will appreciate that the present invention can be embodied in modified forms without departing from the essential characteristics of the invention. As such, the disclosed embodiments should be considered in an illustrative rather than a restrictive perspective. The scope of the invention is indicated in the appended claims rather than in the foregoing description, and all differences within the scope of equivalents thereof are to be construed as included in the present invention.

Claims (12)

A nucleotide sequence encoding gRNA (guide RNA) that hybridizes to DNA encoding the IGF-1 (insulin-like growth factor 1) gene;
a nucleotide sequence encoding a Cas9 protein; and a promoter operably linked to said nucleotide sequence, said gRNA comprising a base sequence complementary to the base sequence of SEQ ID NO:2. Recombinant expression vector. A transformed cell line for producing a minipig dwarfism animal model into which the recombinant expression vector according to claim 1 is introduced. a step of transplanting the transformed cell line according to claim 2 into an enucleated oocyte obtained from a non-human animal to form a nuclear transfer oocyte;
and implanting the nuclear transfer egg into the fallopian tube of a non-human animal surrogate mother. The method for producing a minipig dwarfism animal model according to claim 3, wherein said dwarfism is due to IGF-1 knock-out. 5. The knockout according to claim 4 , wherein the nucleotide sequence corresponding to SEQ ID NO: 2 is mutated to any one nucleotide sequence selected from the group consisting of SEQ ID NO: 5 to SEQ ID NO: 9. of the mini- pig dwarfism animal model. 4. The method for producing a minipig dwarfism animal model according to claim 3, wherein said dwarfism is Laron's syndrome. A minipig dwarfism animal model having an IGF-1 (insulin-like growth factor 1) gene mutation, wherein the animal model is knock-out of the IGF-1 gene, and the knockout is , the base sequence corresponding to SEQ ID NO: 2 is mutated to any one base sequence selected from the group consisting of SEQ ID NO: 5 to SEQ ID NO: 9, and has an IGF-1 gene mutation Minipig dwarfism animal model. 8. The minipig with IGF-1 gene mutation according to claim 7 , wherein said animal model has decreased expression of one or more genes selected from the group consisting of EIF4H, CLIP2, ELN, LIMK1 and RFC2 . Dwarfism animal model. The minipig dwarfism animal model with IGF-1 gene mutation according to claim 7 , wherein said animal model is for Laron syndrome or Williams-Beuren syndrome model. The minipig dwarfism animal model having an IGF-1 gene mutation according to claim 7 , wherein said minipig is for pets. The minipig dwarfism animal model with IGF-1 gene mutation according to claim 7 , wherein said minipig is for xenotransplantation. The minipig dwarfism animal model with IGF-1 gene mutation according to claim 7 , wherein said minipig is for artificial blood development.