KR101068479B1 - alpha 1,3-galactosyltransferase gene targeting vector and uses thereof - Google Patents

Abstract

The present invention relates to an alpha 1,3-galactosyltransferase gene targeting vector capable of removing porcine alpha 1,3-galactosyltransferase gene by homologous recombination, and specifically (1) Localization signal (NLS) regions; (2) part of the intron 8 and exon of the swine alpha 1,3-galactosyltransferase gene as a 4-6 kb long nucleic acid sequence homologous to the swine alpha 1,3-galactosyltransferase nucleic acid sequence A first region comprising a portion of nine; (3) positive selectable marker regions; (4) a portion of exon 9 of the pig alpha 1,3-galactosyltransferase gene as a nucleic acid sequence 0.5 to 2 kb long that is homologous to the pig alpha 1,3-galactosyltransferase nucleic acid sequence A second region comprising; And (5) a negative selection marker region, wherein the (1) to (5) regions are configured in order from the 5 ′ end, and the alpha 1,3-galactosyltransferase gene targeting vector, wherein the vector And transgenic animals knocked out by the vector, and a method for producing the same.

Alpha 1,3-galactosyltransferase, homologous recombination, gene targeting vector

Description

Alpha 1,3-galactosyltransferase gene targeting vector and uses approximately}

The present invention relates to an alpha 1,3-galactosyltransferase gene targeting vector capable of removing porcine alpha 1,3-galactosyltransferase gene by homologous recombination, and specifically, a nuclear localization signal. The present invention relates to an alpha 1,3-galactosyltransferase gene targeting vector having an increased targeting efficiency, a cell into which the vector is introduced, and a transgenic animal knocked out by the vector, and a method of preparing the same.

Steady progress in animal genetics Removal or insertion of specific genes enabled the identification of each gene's role and enabled the production of commercially useful transgenic animals. Methods for preparing transformed animals include random gene manipulation using microinjection or viral transfection and specific genes using embryonic stem cells or somatic cells. There is a gene targeting method that can target.

Microinjection has been widely used to produce transgenic animals by the classical method of inserting foreign DNA into the pronucleus of fertilized eggs (Harbers et al., Nature., 293 (5833); 540-2, 1981: Hammer et al. , Nature., 315 (6021); 680-683, 1985; van Berkel et al., Nat Biotechnol., 20 (5); 484-487, 2002; Damak et al., Biotechnology (NY), 14 (2 ; 185-186, 1996). However, the rate of production of transformed eggs derived from fertilized eggs with foreign DNA inserted by microinjection was very low (Clark et al., Transgenic Res., 9; 263-275, 2000). The insertion position of the gene and removal of certain internal genes are impossible.

Viral infections are also widely used for genetic manipulation of animals (Soriano et al., Genes Dev., 1 (4); 366-375, 1987; Hirata et al., Cloning Stem Cells., 6 (1); 31 -36, 2004). Viral infection is more efficient than microinjection because the gene to be inserted is introduced into the animal's gene through the viral vector, but it is still impossible to insert foreign genes into specific positions and to remove specific internal genes. In addition, the maximum size of the gene to be inserted is limited to 7 kb, the protein expressed by the virus is a problem (Wei et al., Annu Rev Pharmacol Toxicol., 37; 119-141, 1997; Yanez et al., Gene Ther., 5 (2); 149-159, 1998).

To overcome the problems mentioned above, gene targeting techniques can be used that can remove or insert a particular gene. Gene targeting techniques were used for the first time in the study of gene function using mouse embryonic stem cells. By using homologous recombination, mouse embryonic stem cells targeted to specific genes are inserted into embryos in the blastocyst stage. Using gene targeting to such mouse embryonic stem cells, a large number of specific gene targeted mice have been produced (Brandon et al., Curr Biol., 5 (6); 625-634, 1995; Capecchi et al., Science, 244 (4910); 1288-1292, 1989; Thompson et al., Cell, 56 (2); 313-321, 1989; Hamanaka et al., Hum Mol Genet., 9 (3); 353-361, 2000; Thomas et al, Cell, 51 (3); 503-512, 1987; te Riele et al., Proc Natl Acad Sci USA, 89 (11); 5182-5132, 1992; Mansour et al., Nature, 336 (6197), 348-352, 1988; Luo et al., Oncogene, 20 (3); 320-328, 2001). When genetic targeting is applied to livestock, it is possible to produce animal bioreactors that produce large quantities of therapeutic proteins or disease model animals that can be used for cross-transplantation, which will bring significant economic benefits.

In order to produce genetically targeted animals, the use of embryonic stem cells has been considered an essential factor. However, although embryonic stem cell-like cell lines have been reported in livestock, including pigs and cattle, the use of embryonic stem cells in livestock is currently limited (Doetschman et al., Dev Biol., 127 (1); 224-227). , 1988; Stice et al., Biol Reprod., 54 (1); 100-110, 1996; Sukoyan et al., Mol Reprod Dev., 36 (2); 148-158, 1993; Iannaccone et al., Dev Biol., 163 (1); 288-292, 1994; Pain et al., Development, 122 (8); 2339-2348, 1996; Thomson et al., Proc Natl Acad Sci USA., 92 (17) 7844- 7848, 1995; Wheeler et al., Reprod Fertil Dev., 6 (5); 563-568, 1994). Instead, it has been suggested that normal somatic cells can be used for gene targeting as nuclear donor cells, enabling the production of transformed cloned livestock (Brophy et al., Nat Biotechnol., 21 (2); 157-162, 2003; Cibelli et al., Science, 280 (5367); 1256-1258, 1998; Campbell et al., Nature, 380 (6569); 64-66, 1996; Akira Onishi et al., Science, 289; 1188- 1190, 2000 Denning et al., Cloning stem cells, 3 (4); 221-231, 2001 McCreath et al., Nature, 405 (6790); 1066-1069, 2000).

In addition, knock-out of genes has been reported as a technique for introducing mutations into specific genes in mouse-specific locations as one of gene targeting techniques (Thomas, KR and Capecchi, MR Cell, 51, 3, 503-512 (1987). This technique has been used in the production of a variety of genetically modified mice (Fujino, T et al., Proc Natl Acad Sci US A. 100, 1, 229-234 (2003)), and these techniques are used for livestock sheep (McCreath, KJ et al. 405, 1066-1067 (2000).

Meanwhile, alpha 1,3-galactosyltransferase gene is a gene that causes a rapid immune rejection reaction between xenotransplantation. If this gene is removed, it is possible to develop a disease model animal for xenotransplantation which has been rejected. Do. In 2002, UK-based PPL produced the world's first somatic cloned pig that had been heterozygous for its removal of the alpha 1,3-galactosyltransferase gene (Yifan Dai et al., Nat Biotechnology, 20; 251). -255, 2002). In 2003, the company succeeded in replicating somatic cells in which the alpha 1,3-galactosyltransferase gene was homozygous removed, producing a disease model animal for organ transplantation that can solve the current organ shortage. Has led to breakthroughs for the future (Carol J. Phelps, Science, 299; 411-414, 2003). Later, targeting methods using the promoter trap principle, which can more efficiently remove the alpha 1,3-galactosyltransferase gene, were introduced (Sharon Harrison et al., Cloning and stem). cells, 6 (4); 327-331, 2004). In 2005, cloned pig-derived organs from which alpha 1,3-galactosyltransferase was removed were transplanted into monkeys, resulting in delayed survival until 2-6 months after transplantation without acute immune rejection (Kenji Kuwaki). et al., Nature medicine, 11 (1); 29-31, 2005).

Gene targeting is to introduce a gene targeting vector into a cell using homologous recombination phenomena, a positive screening marker capable of selecting cells where homologous gene recombination has occurred at a desired gene position, and a homologous gene recombination without random homologous recombination. It is common to use negative screening markers to select cells into which cells have been inserted (Suzanne, L. et al., Nature, 336, 348-352 (1988)). The method using the positive and negative screening markers is mainly used for the development of gene targeting mice using mouse stem cells, and its efficiency is known to be high (Templeton, NS et al., Gene Therapy, 4, 700-709 (1997)). ).

However, gene targeting efficiencies in livestock sheep and pigs have been reported to vary according to gene type and animal species. In experiments knocking out positive α1 (I) procollagen gene positions, promoter-less neo selection markers yielded 7.1-13.8% efficiency and positive beta-lactoglobulin in the same vector. The vector inserted after the neo selection marker to express human α1-antitrypsin in the promoter showed a gene targeting efficiency of 67% (McCreath, KJ et al., Nature, 405, 1066-1069 (2000)). However, when the alpha 1,3-galactosyltransferase gene and the prion protein gene were genetically targeted using neo selection markers without the same promoter, the efficiency was very low (Denning, C). Et al., Nature Biotechnology, 19, 559-562 (2001).

However, to date, the efficiency of knocking out alpha 1,3-galactosyltransferase gene by gene targeting method in somatic cells of pigs is known to be less than 5% (Denning, C. and Priddle, H. Reproduction, 126, 1-11 (2003). In addition, the gene efficiency of alpha 1,3-galactosyltransferase gene using only a positive selection marker in pigs has been reported to be 1.5 to 5% (Denning, C. and Priddle, H). Reproduction, 126, 1-11 (2003).

Accordingly, the inventors of the present invention have prepared a gene targeting vector including a nuclear localization signal (NLS), a positive selection marker, and a negative selection marker at the same time, and when using such a vector, alpha 1,3-galacto The present invention has been completed by discovering that the targeting efficiency of siltransferase gene can be significantly increased.

It is an object of the present invention to provide a (1) nuclear localization signal (NLS) region; (2) part of the intron 8 and exon of the swine alpha 1,3-galactosyltransferase gene as a 4-6 kb long nucleic acid sequence homologous to the swine alpha 1,3-galactosyltransferase nucleic acid sequence A first region comprising a portion of nine; (3) positive selectable marker regions; (4) a portion of exon 9 of the pig alpha 1,3-galactosyltransferase gene as a nucleic acid sequence 0.5 to 2 kb long that is homologous to the pig alpha 1,3-galactosyltransferase nucleic acid sequence A second area comprising; And (5) having a negative selection marker region, wherein (1) to (5) regions are configured in sequence from the 5 ′ end to provide an alpha 1,3-galactosyltransferase gene targeting vector. will be.

Still another object of the present invention is to provide a cell genetically targeted with the vector.

Still another object of the present invention is to provide a transgenic animal in which the vector is introduced and knocked out of the alpha 1,3-galactosyltransferase gene.

Still another object of the present invention is to provide a method for producing somatic cells of pigs targeted to alpha 1,3-galactosyltransferase gene using the vector.

Another object of the present invention to provide a method for producing a transgenic pig knocked out alpha 1,3-galactosyltransferase gene using the vector.

In one aspect, the present invention provides a kit comprising: (1) a nuclear localization signal (NLS) region; (2) part of the intron 8 and exon of the swine alpha 1,3-galactosyltransferase gene as a 4-6 kb long nucleic acid sequence homologous to the swine alpha 1,3-galactosyltransferase nucleic acid sequence A first region comprising a portion of nine; (3) positive selectable marker regions; (4) a portion of exon 9 of the pig alpha 1,3-galactosyltransferase gene as a nucleic acid sequence 0.5 to 2 kb long that is homologous to the pig alpha 1,3-galactosyltransferase nucleic acid sequence A second region comprising; And (5) a negative selection marker region, wherein the regions (1) to (5) are configured in order from the 5 ′ end. The present invention relates to an alpha 1,3-galactosyltransferase gene targeting vector. .

As used herein, the term "gene targeting vector" refers to a vector capable of removing or inserting a gene of interest into a specific gene position of a genome, and to target a gene such that homologous recombination occurs. Contains homologous base sequences. In the gene targeting vector of the present invention, an alpha 1,3-galactosyltransfer in which a gene constituting the vector is inserted in a 5 '→ 3' array into an alpha 1,3-galactosyltransferase gene on the genome by homologous recombination. Laase targeting vector, which may be circular or linear.

The alpha 1,3-galactosyltransferase gene targeting vector of the present invention comprises a nuclear localization signal (NLS) region.

The nuclear localization signal region is characterized by being included at the 5 'end of the vector. Influx of transcription factors into the nucleus is a process that occurs in response to a variety of external and internal stimuli as well as signals of occurrence. Therefore, the nuclear localization signal inserted into the vector assists the movement of the vector of the present invention into the nucleus after the vector has been introduced into the cell, thereby allowing alpha 1,3-galactos on the genome present in the nucleus. Targeting efficiency for the siltransferase gene can be improved. In addition, when a gene targeting vector is introduced into a cell to target an intracellular gene, direct electroporation is used to increase the efficiency of influx into the nucleus, and the vector of the present invention includes a nuclear localization signal region. Since it is easy to transport to the nucleus in the inside, the gene can be targeted by introducing the vector efficiently even in the liposome method, which is a transformation method of conventional cells. In particular, the inventors demonstrate that when the nuclear localization signal region is included at the 5 'end of the vector, the gene targeting efficiency is significantly higher than when the nuclear localization signal region is not included or at the 3' end. (FIG. 5).

The alpha 1,3-galactosyltransferase gene targeting vector of the present invention includes a first region and a second region having a sequence homologous to the nucleic acid sequence of the alpha 1,3-galactosyltransferase gene.

"First region" of the present invention is characterized by having a nucleic acid sequence of 4 to 6 kb in length homologous to the nucleic acid sequence of the alpha 1,3-galactosyltransferase gene. The length of the first region is an important factor in determining gene targeting efficiency, and preferably has a length of 4.8 kb. In addition, the first region preferably comprises a portion of intron 8 and a portion of exon 9 in the porcine alpha 1,3-galactosyltransferase gene. Herein, a part of the restriction enzyme SmaI recognition site of the 5'-terminal intron 8 to the restriction enzyme EcoRV recognition site of the 3'-terminal exon 9 is preferable.

“Second region” of the invention is characterized by having a nucleic acid sequence of 0.5 to 2 kb in length that is homologous to the alpha 1,3-galactosyltransferase gene nucleic acid sequence. Preferably it has a length of 1.9kb. In this case, the first region corresponds to the upstream of the 5 ′ → 3 ′ sequence of the nucleic acid sequence of the alpha 1,3-galactosyltransferase gene, and the second region corresponds to the downstream. In addition, the second region preferably comprises a portion of exon 9 of the porcine alpha 1,3-galactosyltransferase gene. Herein, a part of the restriction enzyme EcoRV recognition region at the 5 'end of exon 9 to the restriction enzyme PstI at the 3' end of exon 9 is preferable. Porcine alpha 1,3-galactosyltransferase genes are known to have exon 1 to exon 9 and some introns, most of which are associated with exon 9 sites, so if these sites are removed by targeting, It is expected to be able to remove the activity.

As used herein, the term “homologous” refers to a degree of identity between a first region or a second region and a corresponding nucleic acid sequence of an alpha 1,3-galactosyltransferase gene, which is at least 90% identical. Preferably, it is 95% or more the same.

In addition, the alpha 1,3-galactosyltransferase gene targeted by the vector is not particularly limited, but preferably alpha 1,3- such as cattle, sheep, goats, pigs, horses, rabbits, dogs, and monkeys. It is a galactosyltransferase gene, and more preferably the alpha 1,3-galactosyltransferase gene of a miniature pig.

In addition, the vector of the present invention includes a selection marker region, and preferably includes a positive selection marker and a negative selection marker at the same time.

As used herein, the term "selection marker" is for selecting cells transformed with a gene targeting vector into cells, and selecting such drugs as drug resistance, nutritional requirements, resistance to cytotoxic agents or expression of surface proteins. Markers that confer a possible phenotype can be used, with positive and negative screening markers.

In the present invention, the term "positive selection marker" refers to a marker that allows only positive cells to express selection markers in an environment in which a selective agent is treated to enable positive selection. Neomycin (Neomycin: Neo), hygromycin (hygromycin: Hyg), histidinol dehydrogenase gene (hiD), and guanine phosphosribosyltransferase (Gpt), and are preferably neomycin markers, but are not limited to these. .

The positive selection marker of the present invention is preferably located between the first region and the second region, and is connected to the 3 'end of the first region, and may be positioned toward the upper portion 5' of the second region. By inserting at such a position, when the vector of the present invention is targeted into a cell to homologous recombination, and when recombined with a target gene on the host cell genome, insertion into the genome is facilitated, and their expression identifies gene targeting. This becomes possible.

In addition, in the present invention, the term "negative selection marker" refers to a marker that enables negative selection to select and remove cells in which random insertion has occurred, and the herpes simplex virus-thymidine kinase (Herpes simplex virus-thymidine). kinase (HSV-tk), hypoxanthine phosphoribosyl transferase (Hprt), cytosine deaminase, Diphtheria toxin, and the like, preferably diphtheria toxin (DT). -A), but is not limited to this. Diphtheria toxin markers have several advantages over thymidine kinases. First, thymidine kinases are convenient in that they require gancyclovir (GANC) treatment, while diphtheria toxin does not require any treatment. In addition, it has already been reported that the thymidine kinase / gancyclovir (GANC) system is likely to kill cells without the thymidine kinase gene (Yoshiyasu Kaneko et al., Cancer Letters, 96; 105-110, 1995). In this respect, diphtheria toxin is advantageous as a negative selection marker.

The negative selection marker of the present invention may be located at the 5 'end side of the first region or the 3' end side of the second region, but is preferably the 3 'end side of the second region, and the second region on the vector of the present invention. More preferably connected to the 3 'end of the vector and positioned at the 3' end of the vector.

In addition, the selection marker of the present invention may include a separate promoter in the vector for transcription of the selection marker gene. Promoters that can be used for the selection marker transcription of the present invention include a phosphoglycerate kinase (PGK) promoter, monkey virus 40 (SV40), mouse breast tumor virus (MMTV) promoter, long terminal repeats of HIV ( LTR) promoter, molony virus, cytomegalovirus (CMV) promoter, Epstein bar virus (EBV) promoter, Loews sacoma virus (RSV) promoter, RNA polymerase II promoter, β-actin promoter, human heroglobin promoter and Human muscle creatine promoters, and the like, but is not limited thereto. The promoter for transcription of the positive selection marker of the present invention is preferably inserted into the vector so that the phosphoglycerate kinase (PGK) promoter is linked to the upstream of the positive selection marker.

In a specific embodiment of the present invention, a vector is constructed using mini-pig alpha 1,3-galactosyltransferase gene homology region, nuclear localization signal (NLS), positive selection marker and negative selection marker. It was. In the present invention, the nuclear localization signal (NLS), which is a 72 bp tandem repeat enhancer sequence included in the Simian virus 40 genome, is placed at the front and then exon 9 of the alpha 1,3-galactosyltransferase gene. The front 4.8 kb (first region) was connected to the 5 'homology region from the EcoRV restriction enzyme position of the SmaI position, and the neo ^r gene expressed by the pohosphoglycerine kinase (PGK) promoter was connected as a positive selection marker. Next, the homologous region of the alpha 1,3-galactosyltransferase gene connects the rear 1.9 kb (second region) from the EcoRV restriction enzyme position of exon 9 to the PstI position. The final vector was constructed by inserting it into the pMCDT-A (A + T / pau) vector containing the negative selection marker. The constructed vector of the present invention is the first region-positive screening marker region-mini piglet alpha 1,3 homologous to the nuclear localization signal region-mini piglet alpha 1,3-galactosyltransferase gene from the 5 'end. -Second region-negative selection marker region homologous to the galactosyltransferase gene. Such vectors can be used by linearizing with SalI restriction enzymes when introduced into cells.

The vector of the present invention was confirmed to have higher gene targeting efficiency than a vector containing no nuclear localization signal region or a vector including a nuclear localization signal region at the 3 'end (FIG. 5).

In another aspect, the present invention relates to cells genetically targeted with the alpha 1,3-galactosyltransferase gene targeting vector.

Cells that can be targeted with the vectors of the invention are somatic cells derived from animals, preferably pigs. Useful tissues that can separate or activate cells include liver, kidney, spleen, bone, bone marrow, thymus, heart, muscle, lungs, brain, testes, ovaries, islets, intestines, bone marrow, ears, skin, bones, Gallbladder tissue, prostate, bladder, embryo (embryo), immune system and hematopoietic system and the like, but is not limited thereto. Cell types include, but are not limited to, fibroblasts, epithelial cells, neurons, embryonic cells, liver cells, and follicle cells. Preferably fibroblasts or ear tissue cells.

The method of introducing the vector of the present invention into the cell may use a known method for introducing a nucleic acid into the cell, and suitable standard techniques, such as electroporation, calcium phosphate, as known in the art Calcium phosphate co-precipitation, retroviral infection, microinjection, DEAE-dextran and cationic liposome methods can be used, including but not limited to It doesn't work. At this time, it is preferable to cut the circular vector with an appropriate restriction enzyme and introduce the linear vector.

In another embodiment, the present invention relates to a transgenic animal in which the vector is introduced to knock out the alpha 1,3-galactosyltransferase gene.

In the present invention, the term "knock-out" refers to modifying or removing a specific gene from a nucleotide sequence so that it cannot be expressed, and using the somatic cell into which the targeting vector of the present invention is introduced. Embryos can be nuclear transplanted and the transplanted embryos can be implanted to produce transgenic individuals knocked out of the alpha 1,3-galactosyltransferase gene. These individuals can be said to be genetically identical clones because the genetic material of the cell has been transferred to the nuclear donor cytoplasm.

Animals capable of introducing and knocking out the vector of the present invention are preferably pigs, more preferably mini pigs, and traits in which the alpha 1,3-galactosyltransferase gene has been knocked out through the vector of the present invention. Transgenic pigs do not cause acute immune responses and can be effectively used for xenotransplantation.

In still another aspect, the present invention provides a method for preparing a pig alpha 1,3-galactosyltransferase gene targeting vector of claim 1; (2) separating and culturing somatic cells from pigs; (3) introducing the vector of the constructed step (1) into the somatic cells isolated in the step (2) to induce homologous recombination; And (4) selecting the somatic cells of the pigs to which the pig alpha 1,3-galactosyltransferase gene is targeted by homologous recombination, wherein the alpha 1,3-galactosyltransferase gene is targeted. It relates to a method for producing somatic cells of pigs.

Hereinafter, a step-by-step method for producing somatic cells of pigs targeted to the alpha 1,3-galactosyltransferase gene of the present invention will be described.

First, step (1) is a step of constructing a pig alpha 1,3-galactosyltransferase gene targeting vector of the present invention, as described above, a nuclear localization signal (NLS) region − 4 to 6 kb long nucleic acid sequence homologous to porcine alpha 1,3-galactosyltransferase nucleic acid sequence, part of intron 8 and exon 9 of porcine alpha 1,3-galactosyltransferase gene A first region comprising a positive-selective marker region-a pig's alpha 1,3-galactosyltransfer as a nucleic acid sequence 0.5 to 2 kb in length homologous to the pig's alpha 1,3-galactosyltransferase nucleic acid sequence It has a second region including a part of exon 9 of the lyase gene-negative selection marker region, the regions can be constructed to be configured in order from the 5 'end. The targeting vector of the present invention thus constructed can be targeted to the intrinsic alpha 1,3-galactosyltransferase gene in the cell to destroy these genes.

Step (2) is a step of isolating and culturing somatic cells from pigs, and isolating and culturing somatic cells of pigs for introducing the targeting vector of step (1) and producing somatic cells. Preferably, the somatic cells of the pig are separated from the pig's ear tissue, and the ear tissue may be cultured by cutting the tissue about 1 to 3 mm using the 10-day-old ear tissue.

When culturing these tissues, it is desirable that the ear tissues are not completely submerged into the culture medium. In addition, the medium for somatic cell isolation and culture is to isolate somatic cells from the ear tissues using a medium containing high concentration of glucose and FBS, to produce somatic cells through passage in a medium containing FBS, non-essential amino acids and antibiotics Preferably, the ear tissue of the pig is cultured in Dulbecco's Modified Eagle Medium (DMEM) medium containing high concentration of glucose and 15% fetal bovine serum (FBS) to isolate somatic cells, 20% FBS, 1X non-essential amino acids. , 1X sodium pyruvate, 10 ^-4 M beta-mercaptoethanol meoreu, 100Unit / ml penicillin and may be sub-cultured in DMEM medium containing 100㎍ / ㎖ streptomycin.

Step (3) is a step of inducing homologous recombination by introducing the constructed vector of step (1) into the somatic cells isolated in step (2), wherein the somatic cells of the pig produced in step (2) of the targeting vector of the present invention. Inducing homologous recombination to disrupt the alpha 1,3-galactosyltransferase gene on the pig somatic genome.

The method of introducing the vector of the present invention into somatic cells of pigs is a known method for introducing nucleic acids into cells, as described above, for example, electroporation, calcium phosphate co-precipitation. ), Retroviral infection, microinjection, DEAE-dextran and cationic liposomes, and the like, but are preferably electroporated, but are not limited thereto. no. In addition, conventional gene targeting vectors are large in gene size and are mainly introduced into somatic cells using electroporation. However, since the vector of the present invention includes a nuclear localization signal region, the vector is easily introduced into the nucleus. Even using the cationic liposome method, it can be efficiently introduced into the cell.

In addition, step (3) of the present invention may further comprise the step of treating thymidine to the somatic cells of the pig before introducing the vector. Cymidine can block the cycle of the cell in the S phase (synchronized to the S phase), which allows the cells synchronized with the S group can significantly increase the targeting efficiency of the gene targeting vector. The concentration of the thymidine to be treated in the porcine somatic cells of the present invention is preferably about 1 to 5 mM, more preferably 2 mM, the thymidine treatment time is preferably about 15 to 18 hours, more preferably 16 It's time.

Step (4) is a step of selecting the somatic cells of pigs targeted to the pig alpha 1,3-galactosyltransferase gene by homologous recombination, which is present on the genome of the somatic cells of the pig by homologous recombination of step (3). This step is to select the somatic cells of pigs in which the intrinsic alpha 1,3-galactosyltransferase gene is destroyed.

The somatic cell selection may use a selection marker gene inserted into the vector. In particular, when a positive selection marker existing on the vector is introduced into and expressed on the porcine somatic cell genome, a treatment agent capable of recognizing the expression of these positive selection markers may be used. It can be used to identify whether targeting. As the selection marker of the present invention, a neomycin marker may be preferably used, and an antibiotic, G418, may be used as a treatment agent for selection. The concentration of G418 used for selection is preferably 200 to 400 µg / ml, more preferably 300 µg / ml.

In addition, the culture that can be used for selection of clonal somatic cells into which the gene targeting vector has been introduced may use DMEM including 15% to 20% FBS, and preferably 20% FBS. This is because promoting the proliferation of cells at the time of selection can result in faster cell selection. More preferably, DMEM medium containing 20% of FBS (fetal bovine serum, FBS), non-essential amino acids, sodium pyruvate, and beta-mercaptoethanol may be used. In addition, the pig somatic cells induced homologous recombination through step (3) is preferably incubated in G418 selection medium for 11 to 12 days and then selected.

In addition, PCR and Southern blots can be used to analyze whether the vectors of the present invention are properly targeted to the alpha 1,3-galactosyltransferase gene in somatic cells.

The PCR assay can analyze efficiently targeted cells through primary and secondary PCR. First, the primary PCR is a 3 'homologous region of the alpha 1,3-galactosyltransferase gene (second region). By using the outer primer and the primer on the neo ^r gene, the vector is introduced into the cell. Since gene targeting occurs in one of the homologous chromosomes, homologous chromosomes and homologous chromosomes that are not present in the cells are present in the cells, so that PCR can be analyzed at the same time, so that they can be analyzed more accurately. To this end, secondary PCR may be performed on cells selected by primary PCR. As a primer for the secondary PCR, a primer outside the 3 'homology region (second region) and the front primer of exon 9 EcoRV position may be used.

Porcine somatic cell lines genetically targeted with the vector of the present invention by the method of producing a gene-targeted somatic cell was named GTKO 21, 2008 in KCTC (Korean Collection for Type Cultures, 111, Sciro-ro, Yuseong-gu, Daejeon, Korea) The deposit was made on July 24 with accession number KCTC 11369BP.

In another aspect, the present invention comprises the steps of (1) obtaining a somatic cell of a pig targeted to the alpha 1,3-galactosyltransferase gene by the somatic cell production method; (2) removing the nucleus of the pig egg and introducing the gene-targeted cell to produce a somatic cell nuclear transfer embryo; And (3) relates to a method for producing a transgenic pig knocked out alpha 1,3-galactosyltransferase gene, comprising the step of implanting the fertilized egg.

Since the vector of the present invention is a vector optimized for targeting to the alpha 1,3-galactosyltransferase gene of pigs, it can be used for the production of pigs knocked out of the alpha 1,3-galactosyltransferase gene. The somatic cells targeted by the vector may be nuclear-transplanted into a fertilized egg of a pig, and the nuclear-transplanted fertilized egg may be implanted to produce a pig knocked out of the alpha 1,3-galactosyltransferase gene.

In order to remove the nucleus of the egg, that is, the genetic material, physical methods, chemical methods, and centrifugation using Cytochalasin B can be used. Preferably, the physical properties using glass micropipettes are used. Phosphorus nucleation can be used.

The method of introducing the genetically targeted somatic cells into the nucleus from which the nucleus has been removed may use cell membrane fusion, intracellular microinjection, etc. The cell membrane fusion method is simple and suitable for large-scale fertilized egg production. Has the advantage of maximizing contact between the nucleus and the material in the egg.

Fusion of the somatic cell and the nucleus from which the nucleus has been removed can be recombined by changing the viscosity of the cell membrane through electric stimulation and fusing. In this case, an electric fusion device capable of freely adjusting the microcurrent and voltage can be used.

Nuclear transplanted embryos are preferably activated and developed to the implantable stage and implanted into surrogate mothers. Here, activation of a replication fertilized egg means reactivating a cell cycle that is temporarily stopped during maturation so that the fertilized egg can divide. In order to activate the fertilized eggs, it is desirable to reduce the activity of cell signaling materials such as MPF and MAP kinases, which are cell cycle arrest elements. To this end, the cell membrane permeability is modified to increase calcium ions in the cloned fertilized eggs or extracellular. Or a method of rapidly inhibiting the influx of calcium, or directly inhibiting cell cycle arrester activity using chemicals such as inomycin and 6-DMAP.

Pigs produced through this method are deficient in the cellular intrinsic alpha 1,3-galactosyltransferase gene, and thus acute immune responses may be inhibited during organ transplantation.

The alpha 1,3-galactosyltransferase gene targeting vector of the present invention includes a nuclear localization signal region, so that targeting cells can be easily introduced into the nucleus and have high targeting efficiency. The transgenic animals knocked out of the alpha 1,3-galactosyltransferase gene group produced using such a targeting vector can be usefully used for organ transplantation between xenografts due to suppression of rapid immune rejection.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, these examples are only for carrying out the present invention by way of example, but the scope of the present invention is not limited to these examples.

Example  1.Mini Pig Alpha 1,3- Galactosyltransferase  gene Targeting  Vector building

The alpha 1,3-galactosyltransferase gene in pigs consists of nine exons and some sequences of exons 1 to exon 9 and introns are known (Kihiro Koike et al., Transplantation, 70; 1275-1283). , 2000). Since most of the enzymatic activity of the alpha 1,3-galactosyltransferase gene is present in exon 9 (Yifan Dai, Nature, 20; 251-255, 2002), in this example alpha 1,3-galactosyl A gene targeting vector was constructed to remove exon 9 using a transferase gene region, a nuclear localization signal (NLS), a positive selection marker, and a negative selection marker.

Specifically, gene fragments including exons 8 and 9 of the mini pig alpha 1,3-galactosyltransferase gene were identified by long PCR using the mini pig genomic DNA as follows. PCR was performed at 200 ° C genomic DNA, 1 XPCR buffer, 2.5 mM dNTP, 2.5 U i-Max II DNA polymerase, 10 pmole sense primer (AGGTCGTGACCATAACCAGATGGAAGGCTC, SEQ ID NO: 1) and antisense primer (CTTGCACCATGAAAGGTCTCTGCAACTCCAGAA, SEQ ID NO: 2). It carried out on the conditions of the second, 68 degreeC 40 second, and 72 degreeC 10 minutes. The amplified DNA fragments were subcloned into the pGEM T-easy vector and sequenced to confirm that the gene was finally a mini pig alpha 1,3-galactosyltransferase gene.

In addition, the nuclear localization signal (Nuclear localization signal, NLS) was cloned using the Clontech pEGFP-N3 vector (Clontech). DNA was diluted to 10 pg and used in 1 × PCR-buffer, 0.5 U tack polymerase (Taq polymerase, Promega) using 10 pmol sense primer (CGGAGTTAGGGGCGGGACTA, SEQ ID NO: 3) and antisense primer (CCAGCTGTGGAATGTGTGTC, SEQ ID NO: 4). PCR was performed under reaction conditions of denaturation at 94 ° C. 30 seconds, annealing at 60 ° C. 30 seconds, extension at 72 ° C. 30 seconds, and 40 cycles. PCR products were identified by electrophoresis on 2% agarose gel and subcloned into pGEM T-easy vector (Promega) for sequencing. Sequencing was determined using ABI PRISM 377 sequencer (Applied Biosystems). Analysis of the determined nucleotide sequence was analyzed using Genetyx-win (version 4.0).

Gene fragments comprising exons 8 and 9 of the mini pig alpha 1,3-galactosyltransferase gene obtained by the above method were digested with Not I restriction enzyme and subcloned into pBluscriptII SK (-) vector to obtain pBSK ( GT DNA was obtained.

PBSK (-) GT DNA was digested with Not I and EcoR V restriction enzymes to prepare 5 'homology region (first region) and 3' homology region (second region) of the targeting vector. Were subcloned into pBluscriptII SK (-) vectors, respectively, to obtain pBSK (-) GT5.4 DNA and pBSK (-) GT2.5 DNA. To construct the 5 'homology region, pBSK (-) GT5.4 DNA was digested with restriction enzyme SmaI and blunted to connect the Sal I linker to this position. The EcoR V site also blunted to connect the Not I linker. The 4.8 kb Sal I-Not I fragment thus prepared was subcloned to Sal I-Not I site of pBluscriptII SK (+) to prepare pBSK (+) GT4.8 DNA.

In addition, in order to construct 3 'homology region, pBSK (-) GT2.5 DNA was cut and blunted by Pst I restriction enzyme, and then Xho I linker was connected, and then, the subsited at EcoR V-Xho I site of pBluscriptII SK (-). Cloning to construct pBSK (-) GT1.9 DNA.

The neo gene, a positive selection marker, cleaved pKJ2-neo plasmid with EcoR I and BamH I and then linked Not I and EcoR V linker. The Not I-EcoR V fragment of the neo gene and the EcoR V-Xho I fragment of pBSK (-) GT1.9 DNA were subjected to 3 fragment ligation at the Not I-Xho I site of pBluscriptII SK (-). GTneo 1.9 DNA was constructed.

To connect the 5 'homology region, a 4.8 kb fragment of pBSK (+) GT4.8 DNA digested with Sal I-Not I restriction enzyme was placed at the Sal I-Not I restriction enzyme in pBSK (-) GTneo1.9 DNA. Subcloning. The DNA thus prepared was named pBSK (-) GT4.8neo1.9 DNA, and the fragment was treated with Sal I and Not I restriction enzymes, and then the fragment was prepared with pMCDT-A (A) containing a negative selection marker, the Dipreria toxin gene. + T / pau) constructs a basic mini-pig alpha 1,3-galactosyltransferase gene targeting vector by connecting to the Sal I-Not I site of the vector, DT-A / pGT5 '/ neo ^r / pGT3' We named it vector. In addition, the linkage of NLS is a vector DT- of the present invention by linking a fragment obtained by cutting NLS with Xho I and Sal I restriction enzymes to the Xho I-Sal I site of DT-A / pGT5 '/ neo ^r / pGT3' vector. A / NLS / pGT5 '/ neor ^r / pGT3' was constructed. The vector was linearized by cleavage with Sal I restriction enzyme and used for gene introduction into cells (FIG. 1, FIG. 2).

In addition, a gene targeting vector generally used to compare efficiency with the vector produced in the present invention was prepared.

As a first control vector, a vector having a positive selection marker and a negative selection marker was prepared, which does not include a nuclear localization signal region which is generally used. This vector is a Sal I and 5 I homologous region of the mini pig alpha 1,3-galactosyltransferase gene, Sal I, and EcoR V position of exon 9, as described above. Substituted with I was used. In addition, the EcoR V-Xho I 1.9 kb fragment prepared by replacing the PGK-neo ^r gene with the positive selection marker and Xho I at the 3st homology region with the position of Pst I restriction enzyme located at the position of exon 9 was used. The fragment 5 'homology region (Sal I-Not I, 4.8kb), the PGK-neo ^r gene, and the 3' homology region (EcoR V-Xho I) were obtained, and then the entire linked fragment was cut into Sal I and Xho I. The final control vector DT-A / pGT5 '/ neo ^r / pGT3 was inserted into the SalI and XhoI restriction enzyme positions of the pMCDT-A (A + T / pau) vector containing the negative selection marker (diphtheria toxin). 'Was built (FIG. 2). When introduced into the cells were cut and linearized with salI restriction enzyme.

As a second control vector, a vector consisting only of a positive selection marker and a nuclear localization signal region was constructed without using a negative selection marker. The vector is inserted into the pBluescript SK vector by connecting the 5 'homology region (Sma I-Not I, 4.8kb), the PGK-neo ^r gene, and the 3' homology region (EcoR V-Xho I) in the same manner as above. The NLS region was inserted at the 3 'terminal xhoI restriction site of the 3' vector to construct the final second control vector, pGT5 '/ neor ^r / pGT3' / NLS (Figure 2). When introduced into the cells were cut and linearized with ApaI restriction enzyme.

Example  2. Isolation and Culture of Somatic Cells from Mini Pig Ear Tissue

The ear of a 10-day-old male Chicago mini pig was aseptically removed, and then the skin and cartilage tissue were removed using a mass under a stereoscopic microscope, and the tissue was cut into 2 x 2 mm sections. The cleaved tissue was washed in the culture medium, and about 8 tissue sections were placed in a 6 cm dish containing 2 ml of culture solution, and cultured in 37 ° C., 5%, and CO ₂ incubator for 5 days. After 5 days, the cultured cells were passaged in 1 × 10 ⁶ cells / 10cm dishes. After the cells were grown properly, these cells were cryopreserved at 1 × 10 ⁶ cells / ml. Indicated.

The culture medium used was a medium containing 15% FBS (fetal bovine serum) based on Dulbecco's Modified Eagle's Medium (DMEM) containing high concentration of glucose. However, sub-culturing of the produced ear cells in DMEM 20% FBS base, 1 × nonessential amino acids, 1 × sodium pyruvate, 10 ^-4 M beta-mercaptoethanol meoreu, 100Unit / ml penicillin and 100㎍ / ㎖ streptomycin the The included medium was used (FIG. 3).

Example  3. Introduction of Gene Targeting Vectors to Mini-Pig Somatic Cells

Introduction of gene targeting vectors into mini pig somatic cells was performed using electroporation. In the electroporation method, the cultured cells were recovered by trypsin treatment, suspended in a cell count of 5 × 10 ⁶ cells / 0.4 ml F10, and then mixed with 10 µg linearized gene targeting vector dissolved in 0.1 ml F10 culture. The cell vector mixture was placed in a mm gap cuvette. The cuvette was mounted on a BTX Electro-cell manipulator (ECM 2001) and subjected to electric shock at 480 V, 4 pulses and 2 ms. After the electric shock, the cuvette was left on ice for 10 minutes, transferred to a 10 ml culture, suspended well, and then transferred to a 10 cm culture dish. The knock-out vector is mainly inserted by homologous recombination in the cell cycle S phase, so that the vector is treated with cells treated with 2 mM thymidine for 16 hours to synchronize the cell cycle with the S phase at the time of vector introduction. Introduction was carried out. Introduction conditions were validated using the GFP gene (Fig. 4).

Example  4. Gene Targeting  Screening and culturing cloned somatic cells

Three vectors prepared in the present invention were introduced into mini-pig somatic cells using the electroporation method to verify the introduction efficiency. In this example, the gene targeting vector was introduced into the cells, and after 48 hours, the cells were selected for 12 days with 300 µg / ml G418.

In particular, DMEM medium containing 20% FBS, 1 × sodium pyruvate, 1 × non-essential amino acids, 10 ⁻⁴ M beta-mercaptoethanol, 100 Unit / ml penicillin and 100 μg / ml streptomycin was used. On the 11th day of selection, cloned somatic cells showing normal morphology were cultured separately using cloning cylinders, and on day 12, the cloned somatic cells were trypsinized and passaged into 24-well plates. This passaged somatic cells were passaged into 12-well plates at 90% confluent after 3-4 days. Subsequent subcultured with 6-well, 60 mm culture dishes and 100 mm culture dishes every 3-4 days, cryopreservation or experiment was used.

The result of screening the knocked out somatic cells after introducing the vector prepared in the present invention into mini pig somatic cells was 8.56% of the gene vector of the present invention, DT-A / NLS / pGT 5 '/ neo ^r / pGT 3' vector. Gene targeting efficiency was obtained. This result is not only DT-A / pGT5 '/ neo ^r / pGT3' vector without nuclear localization signal, but also DT-A / pGT5 '/ neo ^r / pGT3' with nuclear localization signal inserted at 3 'end. It was confirmed that the targeting efficiency is higher than pGT5 '/ neo ^r / pGT3' / NLS vector (Fig. 5).

Example  5. Mini Pig Alpha 1,3- Galactosyltransferase  Gene Targeted  Analysis of Porcine Somatic Cells

The cloned somatic cells obtained in Example 4 were confirmed to be genetically targeted by primary PCR when passaged from a 24-well plate to a 12-well plate, and then passaged with a 100 mm culture dish, followed by secondary PCR and Southern Analysis via blotting (FIG. 6).

For primary PCR analysis, 200 μl of cell mixture suspended in 1 ml culture following trypsin treatment in 24-well plates was transferred to 0.5 ml tubes. These cells were centrifuged at 10000 rpm for 5 minutes to precipitate the cells, discard the supernatant, add 40 µl of sterile distilled water, mix well, and add 50 µg / ml proteinase K. The treated cells were treated with 130 minutes at 55 ° C. to destroy the cells, and then treated at 98 ° C. for 10 minutes to inactivate proteinase K. 20 μl of the cell disruption solution thus prepared, 10 p mole sense and antisense primer, 1 × PCR buffer, 2.5 mM dNTP, and 1.25 U Ex tack polymerase were added to prepare a total of 50 μl of PCR reaction solution. PCR reaction conditions were performed 38 cycles at the conditions of 94 ℃ 30 seconds, 68 ℃ 30 seconds, 72 ℃ 2 minutes 30 seconds. In addition, the sense primers used were TCGTGCTTTACGGTATCGCCGCTCCCGATT (SEQ ID NO: 5) array, which is present in the neo gene. In this way, when PCR was completed, 20 μl of the reaction solution was electrophoresed on an agarose gel to confirm that a band of about 2 kb was detected (FIG. 6A).

Secondary PCR analysis was performed by subcultured only cells where 2 kb of band was detected in the first PCR analysis, passaged with 100 mm culture dishes, and genomic DNA was isolated. PCR reaction solution was prepared by adding 20ng genomic DNA, 10 p mole sense and antisense primer, 1 × PCR buffer, 2.5mM dNTP, 2.5U i-MAXII DNA polymerase to make a total of 20µl. PCR reaction conditions were 33 cycles at 94 ℃ 30 seconds, 68 ℃ 30 seconds, 72 ℃ 3 minutes 30 seconds conditions. In addition, the sense primers used were CATACTTCATGGTTGGCCACAAAGTC (SEQ ID NO: 7) located in front of the exon 9 EcoR V position of the alpha 1,3-galactosyltransferase gene, and the antisense primers were used for the first PCR analysis. The same primers as the antisense primers were used. In this way, when PCR was completed, 5 μl of the reaction solution was electrophoresed on an agarose gel to confirm that a gene targeted band of about 3.4 kb and a normal band of about 2.1 kb were detected (FIG. 6B).

Southern blotting digested 20 μg of genomic DNA with EcoR V or BstE II restriction enzymes and transcribed the DNA with an electrophoretic zeta-probe membrane on a 0.8% agarose gel. The membrane was hybridized at 42 ° C. for 16 hours in a solution containing 5 × SSPE, 5 × Denhardt's, 1% SDS (w / v), and 50% fromamide (w / v). Probes were prepared using a random labeling kit (Amersharm) and [α- ³² P] dCTP (110 TBq / mmol, Amersham) of about 0.5 kb and about 0.6 kb of DNA containing the exon 8 moiety. After hybridization, the membrane was washed three times at 68 ° C. for 30 minutes at 0.2% SSC and 0.1% SDS (w / v), followed by autoradiography (FIG. 6C).

The somatic cell line of pigs targeted to the DT-A / NLS / pGT5 '/ neo ^r / pGT3' vector of the present invention identified by the above analysis method was named GTKO 21, and KCTC (Korean Collection for Type Cultures, Yuseong-gu, Daejeon, Korea) It was deposited with KCTC 11369BP on July 24, 2008, at the Korea Research Institute of Bioscience and Biotechnology.

Porcine alpha 1,3-galactosyltransferase gene targeting vector having enhanced targeting efficiency according to the present invention can inhibit the activity of alpha 1,3-galactosyltransferase, a gene which causes rapid immunorejection reaction in xenotransplantation. Can be effectively removed, minimizing the immune response during organ transplantation.

In addition, the pig alpha 1,3-galactosyltransferase gene targeting vector of the present invention can be usefully used in the field of organ transplantation as well as in the production of cloned miniature pigs with suppressed immune rejection.

1 is a schematic diagram of a mini pig alpha 1,3-galactosyltransferase gene targeting vector according to the present invention.

2 is a schematic diagram of three types of mini-pig alpha 1,3-galactosyltransferase gene targeting vectors described in the present invention.

Figure 3 is a miniature pig somatic cell preparation and culture photograph used in the present invention.

Figure 4 is a photograph verified with the GFP gene in order to confirm the efficiency of gene introduction into somatic cells mini pigs in the present invention.

5 is a table comparing and comparing the results obtained by introducing the alpha 1,3-galactosyltransferase gene targeting vector and the control vector of the present invention into mini-pig somatic cells and knocking out mini-pig somatic cells.

6 is a PCR photograph and a Southern blot analysis result confirming knocked out mini pig somatic cells after introducing the alpha 1,3-galactosyltransferase gene targeting vector of the present invention into mini pig somatic cells.

<110> Korea Research Institute of Bioscience and Biotechnology <120> alpha 1,3-galactosyltransferase gene targeting vector and uses          the <130> PA080043 / KR <160> 7 <170> KopatentIn 1.71 <210> 1 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> sense primer for amplifying alpha 1,3-galactosyltransferase gene <400> 1 aggtcgtgac cataaccaga tggaaggctc 30 <210> 2 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> antisense primer for amplifying alpha 1,3-galactosyltransferase          gene <400> 2 cttgcaccat gaaaggtctc tgcaactcca gaa 33 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> sense primer for amplifying nls gene <400> 3 cggagttagg ggcgggacta 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> antisense primer for amplifying nls gene <400> 4 ccagctgtgg aatgtgtgtc 20 <210> 5 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> sense primer for identifying gene targeting <400> 5 tcgtgcttta cggtatcgcc gctcccgatt 30 <210> 6 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> antisense primer for identifying gene targeting <400> 6 gacacaaatg acctaaactg gaacacaagc 30 <210> 7 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> sense primer for identifying gene targeting <400> 7 catacttcat ggttggccac aaagtc 26  

Claims (10)

(1) a nuclear localization signal (NLS) region; (2) a 5 'terminal intron of the swine alpha 1,3-galactosyltransferase gene as a 4-6 kb long nucleic acid sequence homologous to the swine alpha 1,3-galactosyltransferase nucleic acid sequence A first region comprising a restriction enzyme SmaI recognition site of 8 to a restriction enzyme EcoRV recognition site of exon 9 at the 3 'end; (3) positive selection marker regions; (4) 5 ′ of the exon 9 of the pig alpha 1,3-galactosyltransferase gene as a nucleic acid sequence 0.5 to 2 kb long that is homologous to the pig alpha 1,3-galactosyltransferase nucleic acid sequence A second region comprising a terminal restriction enzyme EcoRV recognition region to a 3 'terminal restriction enzyme PstI recognition region of exon 9; And (5) a negative selection marker region, wherein the (1) to (5) regions are configured in order from the 5 ′ end. 5. The alpha 1,3-galactosyltransferase gene targeting vector. The vector of claim 1, wherein the first region has a length of 4.8 kb and the second region has a length of 1.9 kb. The method of claim 1, wherein the positive selection marker is Neomycin (Neoomycin: Neo), hygromycin (Hyg), histidinol dehydrogenase gene (hiD) and guanine phosphoribosyltransferase ( guanine phosphosribosyltransferase (Gpt). The vector of claim 1, wherein the pig is a mini pig. The method of claim 1, wherein the negative selection marker is Diphtheria toxin, Herpes simplex virus-thymidine kinase (HSV-tk), hypoxanthine phosphoribosyl transferase (hypoxanthine phosphoribosyl) transferase: Hprt) and a vector characterized in that it is selected from the group consisting of cytosine deaminase. The vector of claim 1, wherein the vector is DT-A / NLS / pGT'5 / neo ^r / pGT3 'disclosed in FIG. 2. Cells genetically targeted with the vector of claim 1. A transgenic pig, wherein the vector of claim 1 is introduced to knock out the alpha 1,3-galactosyltransferase gene. (1) constructing the pig alpha 1,3-galactosyltransferase gene targeting vector of claim 1; (2) separating and culturing somatic cells from pigs; (3) introducing the vector of the constructed step (1) into the somatic cells isolated in the step (2) to induce homologous recombination; And (4) selecting the somatic cells of the pigs to which the pig alpha 1,3-galactosyltransferase gene is targeted by homologous recombination, wherein the alpha 1,3-galactosyltransferase gene is targeted. How to make somatic cells of pigs. (1) obtaining somatic cells of pigs targeted to the alpha 1,3-galactosyltransferase gene by the method of claim 9; (2) removing the nucleus of the pig egg and introducing the gene-targeted cell to produce a somatic cell nuclear transfer embryo; And (3) implanting the fertilized egg, wherein the method of producing a transgenic pig knocked out of the alpha 1,3-galactosyltransferase gene.

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