KR20090056922A - Genetically-modified cell line for producing cloned miniature pigs for xenotransplantation and method for preparing the same - Google Patents

Abstract

A transformed cell line for xeno-transplantation is provided to produce a reproduction pig for xeno-transplantation by controlling the expression of genes relating to immune rejection response. A gene targeting vector removes synthetic gene of heteroantigenic determinant in a cell and hits a gene which encodes complement suppression protein or thrombosis suppression protein. The vector comprises: a homologous first area to an epitope synthesis gene; a positive selection marker gene; a internal ribosome entry site(IRES); a gene which encodes the complement suppression protein or thrombosis suppression protein; and a homologous second area to the epitope synthesis gene.

Description

Genetically-modified cell line for producing cloned miniature pigs for xenotransplantation and method for preparing the same}

This application enjoys the benefit of priority to Korean Patent Application No. 2007-123823, filed November 30, 2007, the entire application of which is incorporated herein by reference.

The present invention relates to a genetically engineered cell line for producing miniature pigs for xenotransplantation and a method of producing the same.

According to the National Organ Transplantation Center, more than 20,000 patients in Korea are waiting for organ transplants, but the number of donations is reported to be less than 10% of the demand. In addition, in the United States, there is an additional waiting time every 16 minutes for the need for organs. However, 11 of the waiting days die without surgery. Together with this situation, the development of life science and technology is the background for the development of xenotransplantation technology.

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 infection, 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 embryonated nucleus (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 production rate of transformed litter derived from fertilized eggs with foreign DNA inserted by microinjection was very low (Clark et al ., Transgenic Res. , 9: 263-275, 2000). It is not possible to control the insertion position of genes and to remove certain internal genes.

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 a foreign gene into a specific position and to remove a specific internal gene. 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, thereby enabling production of engineered litters of specific genes. Using gene targeting methods on these 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, diseases that can be used for cross-organ transplantation by eliminating genes involved in animal bioreactors or immune rejection reactions that produce large amounts of therapeutic proteins or by overexpression at specific sites Model animal production is possible, which is expected to 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, the possibility of normal somatic cells as nuclear donor cells can be used for gene targeting, thus 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).

On the other hand, as an optimal source of xenotransplantation, mini pigs, which are similar to humans in organ size and physiological characteristics and which can be supplied in large quantities by fertility, are considered.

Successful transplantation of porcine organs relies on overcoming a series of immune rejection reactions (super acute, acute vascular, cell mediated, and chronic immune rejection). It has been reported that hyperacute rejection reactions occurring within minutes after transplantation can be overcome by removing genes involved in the synthesis of alpha-1,3-galactosyl epitopes and overexpressing human complement regulatory genes.

Specifically, in 2002, PPL of the UK, the world's first alpha-1,3-galactosyltransferase (hereinafter, abbreviated as 'GT') gene was heterozygous (heterozygous) Removed somatic cloned pigs were produced (Yifan Dai et al ., Nat. Biotechnology , 20: 251-255, 2002). The GT 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 where the biorejection response is removed. In 2003, the company succeeded in replicating somatic cells from which the GT gene was homozygous removed, resulting in breakthroughs in the production of disease model animals for organ transplantation that could address the current shortage of organs (Carol J. Phelps, Science , 299: 411-414, 2003).

In 2005, GT-derived cloned pig-derived organs were transplanted into monkeys, and as a result, survival was delayed until 2-6 months after transplantation without acute immune rejection (Kenji Kuwaki et al ., Nature Medicine , 11 (1). ): 29-31, 2005). However, even though the GT gene has been eliminated, it has been reported that the activation of human complement genes by heterologous antigens via other pathways can lead to severe rejection in organ transplantation (Tanemura, M. et al. , Biochem. Biophys. Res). Commun ., 235: 359-364, 1997; Komoda, H. et al ., Xenotransplantation , 11: 237-246, 2004). In order to overcome such obstacles, along with GT gene removal, decay-accerating factor (abbreviated as 'DAF'), membrane co-factor protein (abbreviated as 'MCP') ), A method of producing cloned pigs that overexpress human complement inhibitory genes such as CD59 was used (Yoichi Takahagi, Molecular Reproduction and Development 71: 331-338, 2005 Cozzi, Eb et al ., Transplant Proc. , 26: 1402). -1403, 1994; Fodor, WL et al ., Proc. Natl. Acad. Sci. USA 91: 11153-11157, 1994; Adams, DH et al ., Xenotransplantation , 8: 36-40, 2001).

Meanwhile, N-glycolylneuraminic acid (hereinafter abbreviated as 'Neu5Gc') antigenic determinant present in most mammals except humans has also been reported to cause immunorejection reactions during xenotransplantation. (WO20061133356A; Pam Tangvoranuntakul, Proc. Natl. Acad. Soc. USA 100: 12045-12050, 2003; Barbara Bighignoli, BMC genetics , 8: 27, 2007). Neu5Gc is converted from N-acetylneuraminic acid (hereinafter abbreviated as 'Neu5Ac') by CMAH.

Complement is a protein complex (C1-C9) consisting of proteins involved in immunity in vivo. When an antigen-antibody complex is formed, the complement binds to the bacterial cell membrane and punctures the complement or the antigen-antibody complex. There is opsonization that is combined to promote phagocytosis. A number of regulatory proteins have been found that can modulate the activity of complement, and regulate the actions of complement by preventing the activation of complement or by promoting the degradation of activated complement. DAF in the cell membrane of the host can interfere with the binding of C2 and C4b, MCP promotes the degradation of C4b to prevent the complement from being activated in the host cell to prevent host cell destruction by complement. CD59 on the surface of the host cell can interfere with the binding of C7, C8 and C5b6 to prevent the formation of membrane attack complexes.

In addition to the complement-regulatory gene, overexpression of human CD39 gene has been reported to inhibit thrombosis caused by xenotransplantation (US20080003212A, Karren, MD, The Journal of Clinical Investigation , 113: 1440-1446, 2004). .

Existing reports suggest that off-regular expression of foreign genes causes disturbances in embryonic development and is fatal to the nervous system, which develops most during the late embryonic and early postnatal periods (Gao et al ., Neurochem. Res. , 24 (9): 1181-1188, 1999).

Therefore, in order to overcome the above problems, the present inventors have made efforts to remove the GT gene involved in the synthesis of alpha-1,3-galactosyl epitopes or the CMAH gene involved in the synthesis of Neu5Gc epitopes. A targeting vector capable of overexpressing the gene was prepared by targeting a human complement regulatory gene or a thrombus suppressor gene capable of suppressing side effects caused by heterologous transplantation such as a heterologous immune response or thrombosis. Even when the GT gene or CMAH gene was knocked out, the somatic cell line was successfully overexpressed with the complement regulatory gene or the thrombus suppressor gene inserted into the knocked out position.

The targeting vector and the transformed cell line prepared by the present invention can be effectively used for the production of cloned pigs for xenotransplantation by efficiently controlling the expression of genes involved in immunorejection.

It is a first object of the present invention to provide a gene targeting vector capable of knocking out a heterologous antigenic determinant synthetic gene and inserting a complement action regulatory gene or an antithrombogenic gene.

A second object of the present invention is transformed with the gene targeting vector, the heterologous antigenic determinant synthetic gene is knocked out, and the complement action regulatory gene or antithrombosis gene is inserted at the locus where the heterologous antigenic determinant synthetic gene is present. It is to provide a transgenic somatic cell line characterized in that.

It is a third object of the present invention to provide a non-human clone produced by nuclear transfer of the transgenic somatic cell line.

A fourth object of the present invention is to provide a method for producing a heterologous organ for transplantation, wherein the immune rejection reaction including the step of extracting the organ after breeding the non-human clone animal is removed.

Definition of Terms

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 the specific gene to be targeted for homologous recombination to occur. The homologous base sequence is included in the former. The terms "vector" and "vector cassette" are used in the same sense and may be of circular or linear form.

As used herein, the term "homologous" refers to a degree of identity between a first region or a second region and a nucleic acid sequence of a gene corresponding thereto, and is at least 90% or more identical, and preferably 95% or more identical.

The term "antigen determinant" of the present invention refers to a site recognized as an antigen by the immune system of the receptor during transplantation of heterologous organs, representatively galactose-alpha 1,3-galactose (galactose-α 1,3) -galactose, hereafter abbreviated as 'AGal' and N-glycolylneuraminic acid (hereinafter abbreviated as 'Neu5Gc'), and the "antigen determinant synthetizing gene" refers to biosynthesis of the antigenic determinant. A gene encoding an enzyme, which includes alpha-1,3-galactosyltransferase (hereinafter, abbreviated as GT) and CMP-acetylneuraminin responsible for biosynthesis of Neu5Gc. Acid hydroxylases (hereinafter abbreviated as 'CMAH') are representative.

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.

The term "positive selection marker" refers to a marker that allows only positive cells to express cells in the environment in which the selective agent is treated to enable positive selection, and "positive selection marker gene" refers to the positive selection marker. Refers to a gene coding for, for example, neomycin phosphotransferase (hereinafter abbreviated as 'neo') endows antibiotic resistance to allow eukaryotic cells to survive in a medium to which the antibiotic neomycin is added. Thus, it is used to select stable transfected cells in eukaryotic cells.

The term “negative selection marker gene” is a marker gene that enables negative selection to select and remove cells in which random insertion has occurred, and by selectively killing only cells expressing the gene, Serves to prevent transduction by

The term “internal ribosome entry site (hereinafter abbreviated as 'IRES')” refers to a nucleic acid sequence that functions to allow translation from an intermediate mRNA region instead of the 5'-cap structure in eukaryotic protein synthesis. By means of this, it is possible to produce a protein which performs a plurality of different functions from one mRNA.

The term "complement inhibitory protein" refers to a protein that inhibits the complement activated by a chain reaction of complement activating proteins acting as a determinant disorder of xenograft to the host cell's membrane to induce acute immune rejection.

The term "thrombosis inhibiting protein" refers to platelets in the endothelial of the transplanted organ after the platelet of the receptor begins to interact with the endothelial or subendothelial cells of the transplanted organ, after activation, after allograft or xenograft. As a result of this aggregation, a blood clot is formed, and thus, a protein capable of inhibiting thrombosis that causes organ transplantation to fail, typically ATP-diphosphohydrolase (abbreviated as 'NTPDase') is present. do.

The term "transformation" means the introduction of DNA into a host such that the DNA is replicable as an extrachromosomal factor or by chromosomal integration. Transformation includes any method of introducing a nucleic acid molecule into an organism, cell, tissue, or organ, and can be carried out by selecting appropriate standard techniques according to the host cell as known in the art. In order to distinguish eukaryotic transformation by plasmid or non-plasmid naked DNA from transformation as a means of tumorigenization of cells, it is also referred to as 'transfection'. Used.

Detailed description of the invention

In one embodiment, the present invention

(1) a first region having a nucleic acid sequence of 2 to 4 kb in length homologous to the antigenic determinant synthetic gene; (2) positive selection marker genes; (3) internal ribosome entry site (abbreviated as 'IRES'); (4) genes encoding complement inhibitory proteins or thrombosis inhibitory proteins; And (5) encoding a complement inhibitory protein or a thrombosis inhibitory protein while simultaneously removing a heterologous antigenic decider synthetic gene in the cell which includes a second region having a nucleic acid sequence of 6 to 8 kb in length homologous to the antigenic determinant synthetic gene. Provide gene targeting vectors capable of targeting genes.

In one embodiment of the gene targeting vector of the present invention, the first region corresponds to a left arm including a 5 'untranslated region, and the second region is an open reading frame (ORF). Corresponds to the right arm including the exon region, including). The first region preferably has a size of 2 to 4 kb, more preferably 2.5 to 3.5 kb. The second region preferably has a nucleic acid sequence of 5 to 8 kb in length, and more preferably has a nucleic acid sequence of 5.5 to 7.5 kb in length.

In one embodiment of the gene targeting vector of the present invention, the heterologous antigenic determinant synthetic gene is a gene encoding alpha-1,3-galactosyltransferase (hereinafter abbreviated as 'GT') or CMP-acetyl It is preferred that the gene encodes neuraminic acid hydroxylase (hereinafter abbreviated as 'CMAH'), but is not limited thereto.

When the antigenic determinant synthetic gene is GT, the first region preferably comprises part or all of intron 2 and exon 4 of the GT gene, more preferably has a nucleic acid sequence of SEQ ID NO: 1 of 3.1 kb in size, The second region preferably comprises some or all of Intron 4 and Exon 5 of the GT gene, more preferably has a nucleic acid sequence of SEQ ID NO: 2 of 6.9 kb in size. When the antigenic determinant synthetic gene is CMAH, the first region comprises a portion or all of intron 3 and exon 4 of the CMAH gene, more preferably, has a size of 3.5 kb, and the second region is preferably Preferably part of exon 6 and part or all of intron 6 of the CMAH gene, more preferably 6 kb in size.

The GT gene targeted by the vector is preferably a GT gene derived from a mammal such as a cow, a sheep, a goat, a pig, a horse, a rabbit, a dog, a monkey, more preferably a GT gene derived from a pig, and a miniature pig. The GT gene is most preferably, but is not limited thereto.

In addition, the CMAH gene targeted by the vector is preferably a CMAH gene derived from a mammal such as cow, sheep, goat, pig, horse, rabbit, dog, monkey, more preferably a CMAH gene derived from pig, and a miniature pig. The CMAH gene is most preferably, but is not limited thereto.

In addition, the vector of the present invention contains a positive selection marker gene.

Positive selectable marker genes include neomycin phosphotransferase (Neo), hygromycin phosphotransferase (Hyg), histidinol dehydrogenase (hiD), puro Mycin (puromycin, Puro) And guanine phosphosribosyltransferase (Gpt) and the like may be used, preferably Neo, but not limited thereto. The positive selection marker gene can be knock-in so that it can be translated by a promoter trap approach, ie without the promoter, using the initiation codon of the endogenous epitope synthesizing gene, or otherwise It may be operably linked to a homeostatic promoter, such as a cytomegalovirus (CMV) promoter.

In addition, the gene targeting vector of the present invention includes an internal ribosome entry site (IRES). The IRES element independently functionally activates the translational initiation of the 5'-terminal methylguanosnium cap (CAP structure) upstream of the gene, and in animal cells encodes a sequence that reads two cystrons (open reading frame) from a single transcript. Include. IRES elements are known to provide independent ribosomal entry sites for the reading of open reading frames located immediately downstream. Neo positive gene of the present invention and human complement inhibitory gene or thrombosis inhibitory gene inserted into MCS is expressed as one mRNA, but may be expressed as two independent proteins by ribosomes bound to IRES.

The vector of the present invention is a knock-in gene, which includes a gene encoding a complement inhibitory protein and / or a thrombosis inhibitory protein, wherein the complement inhibitory protein is CD59, DAF, MCP or CD46. Preferably, it is more preferable that it is CD59, but it is not limited to this. The human complement regulatory protein decay accelerating factor (DAF) inhibits the activation of complement by interfering with the binding of C2 and C4b in the complement response pathway, and the membrane cofactor protein (MCP) promotes C4b degradation to activate complement in host cells. CD59 (homologous restriction factor) is known to inhibit the formation of membrane attack complex (MAC) by inhibiting the binding of C7, C8 and C5b6. On the other hand, as a thrombosis inhibiting protein, ATP-diphosphohydrolase (abbreviated as 'TPDase') is preferable, and CD39 is more preferable, but is not limited thereto (which can suppress thrombosis). Please let me know if there is anything else). The vector of the present invention can be prepared such that the gene encoding the complement inhibitory protein and the thrombosis inhibitory protein is simultaneously knocked-in through one vector, for the production of more efficient organ transplant animals. In this case, the complement inhibitory protein and the thrombosis inhibitory protein may be prepared in the form of a fusion protein, and may be transcribed into one cystron and translated into two independent proteins using IRES.

In addition, the vector of the present invention may further include a negative selection marker. The negative screening markers include Herpes simplex virus-thymidine kinase (HSV-tk), hypoxanthine phosphoribosyl transferase (Hprt), and cytosine deaminase (cytosine). deaminase) and Diphtheria toxin (DT) and the like and are preferably, but are not limited to, thymidine kinase or diphtheria toxin. The negative selection marker may be located at the 5 'end of the first region or at the 3' end of the second region.

When the gene targeting vector of the present invention is targeted into a host cell, homologous recombination occurs between the endogeneous epitope synthesizing gene on the host cell genome and the targeting vector, replacing the base sequence. A positive selection marker gene and a gene encoding a complement inhibitory protein or a thrombosis inhibitory protein on the vector may be a promoter of an endogenous antigenic determinant synthetic gene or a promoter inserted at the 5'-end of the positive selection marker gene for expression of the positive selection marker gene. In the former case, it can be referred to as a promoter trap vector. In addition to a promoter trap, there are an enhancer trap, an exon trap, and the like as a method of capturing and trapping genes (expressed genes) that are transcribed and function in cells.

In another embodiment, the present invention provides a kit comprising: (1) a first region having a nucleic acid sequence of 2 to 4 kb in length homologous to an antigenic determinant synthetic gene; (2) genes encoding complement inhibitory proteins or thrombosis inhibitory proteins; (3) a positive selection marker gene operably linked to a promoter; And (4) encoding a complement inhibitory protein or a thrombosis inhibitory protein while simultaneously removing the heterologous antigenic receptor synthetic gene in the cell which includes a second region having a nucleic acid sequence of 5 to 8 kb in length homologous to the antigenic determinant synthetic gene. Provide gene targeting vectors capable of targeting genes.

In one embodiment of the gene targeting vector, the first region corresponds to a left arm including a 5 'untranslated region, and the second region includes a right arm including an exon region. Corresponds to The first region preferably has a size of 2 to 4 kb, more preferably 2.5 to 3.5 kb. The second region preferably has a nucleic acid sequence of 5 to 8 kb in length, and more preferably has a nucleic acid sequence of 5.5 to 7.5 kb in length.

In one embodiment of the gene targeting vector, the heterologous antigenic determinant synthetic gene is a gene encoding alpha-1,3-galactosyltransferase (hereinafter abbreviated as 'GT') or CMP-acetylneuraminin Preferred is a gene encoding acid hydroxylase (hereinafter abbreviated as 'CMAH'), but is not limited thereto.

When the antigenic determinant synthetic gene is GT, the first region preferably comprises part or all of intron 2 and exon 4 of the GT gene, more preferably has a nucleic acid sequence of SEQ ID NO: 1 of 3.1 kb in size, The second region preferably comprises some or all of Intron 4 and Exon 5 of the GT gene, more preferably has a nucleic acid sequence of SEQ ID NO: 2 of 6.9 kb in size. When the antigenic determinant synthetic gene is CMAH, the first region comprises a portion or all of intron 3 and exon 4 of the CMAH gene, more preferably, has a size of 3.5 kb, and the second region is preferably Preferably part of exon 6 and part or all of intron 6 of the CMAH gene, more preferably 6 kb in size.

The GT gene targeted by the vector is preferably a GT gene derived from a mammal such as a cow, a sheep, a goat, a pig, a horse, a rabbit, a dog, a monkey, more preferably a GT gene derived from a pig, and a GT of miniature pigs. Most preferably, the gene is not limited thereto.

In addition, the CMAH gene targeted by the vector is preferably a CMAH gene derived from a mammal such as cow, sheep, goat, pig, horse, rabbit, dog, monkey, more preferably a CMAH gene derived from pig, and a miniature pig. The CMAH gene is most preferably, but is not limited thereto.

The vector of the present invention is a knock-in gene, which includes a gene encoding a complement inhibitory protein and / or a thrombosis inhibitory protein, wherein the complement inhibitory protein is CD59, DAF, MCP or CD46. Preferably, it is more preferable that it is CD59, but it is not limited to this. The human complement regulatory protein decay accelerating factor (DAF) inhibits the activation of complement by interfering with the binding of C2 and C4b in the complement response pathway, and the membrane cofactor protein (MCP) promotes C4b degradation to activate complement in host cells. CD59 (homologous restriction factor) is known to inhibit the formation of membrane attack complex (MAC) by inhibiting the binding of C7, C8 and C5b6. On the other hand, as a thrombosis inhibiting protein, ATP-diphosphohydrolase (abbreviated as 'TPDase') is preferable, and CD39 is more preferable, but is not limited thereto (which can suppress thrombosis). Please let me know if there is anything else). The vector of the present invention can be prepared such that the gene encoding the complement inhibitory protein and the thrombosis inhibitory protein is simultaneously knocked-in through one vector, for the production of more efficient organ transplant animals. In this case, the complement inhibitory protein and the thrombosis inhibitory protein may be prepared in the form of a fusion protein, and may be transcribed into one cystron and translated into two independent proteins using IRES.

At this time, the 5'-terminus of the gene encoding the complement inhibitory protein and / or thrombosis inhibitor protein may be additionally included, the promoter used at this time is a conventional eukaryotic promoter such as CMV, EF1a, SV40 early promoter Preferably, the SV40 initial promoter is used, and if the promoter is not additionally included, transcription may be achieved through a promoter of an endogenous antigenic determinant synthetic gene.

In addition, the vector of the present invention contains the positive selection marker gene described above.

In addition, the vector of the present invention may further include the negative selection marker described above.

In a specific embodiment of the present invention, the inventors target an alpha 1,3-galactosyltransferase gene that can target the human complement inhibitory gene while simultaneously removing the porcine alpha 1,3-galactosyltransferase gene. A pGTKOIRESCD59KITK vector was prepared as a vector, and pGTKOneoTK and pGTKOIRESKITK vectors capable of alpha 1,3-galactosyltransferase gene targeting were prepared in the preparation of the vector.

First, in order to obtain a portion homologous to the pig alpha 1,3-galactosyltransferase nucleic acid sequence included in the pGTKOIRESCD59KITK vector, a miniature that is hit by the alpha 1,3-galactosyltransferase gene of the miniature pig Some nucleotide sequences of the alpha 1,3-galactosyltransferase gene region in pigs were identified.

In order to determine the alpha 1,3-galactosyltransferase gene portion of the miniature pig defined as the first region and the second region on the vector, a previously reported pig sequence (Kihiro Koike et al ., Transplantation , 70: 1275-1283, 2000) to prepare a primer (primers) and the PCR reaction (Taq polymerase) with a proof (proofreading) function.

The pGTKOneoTK vector comprises a first region of about 3.1 kb in size consisting of a portion of the intron 2 and a full base of exon 4 of the alpha 1,3-galactosyltransferase gene. It also includes a second region of about 6.9 kb in size, consisting of all of Intron 4 and some of the exon 5 nucleotide sequences. The vector has a neo gene region as a positive selection marker, a polyA gene region and a TK gene region as a negative selection marker.

The pGTKOIRESKITK vector contains the IRES gene region and multicloning site (MCS) in addition to the construction of the pGTKOneoTK vector. The gene encoding the protein of interest can be cloned into the MCS of the pGTKOIRESKITK vector for expression in the host cell. In the present invention, it is preferable to insert a gene encoding a complement inhibitory protein.

The pGTKOIRESCD59KITK vector is a form in which the CD59 gene, which is a type of gene encoding a complement inhibitory protein, is inserted into the MCS of the pGTKOIRESKITK vector. The pGTKOIRESCD59KITK vector is a vector capable of removing the porcine alpha 1,3-galactosyltransferase gene and simultaneously targeting the human complement inhibitory gene. The pGTKOIRESCD59KITK vector is the vector most suitable for the purpose of the present invention. , A positive region marker of approximately 3.1 kb, consisting of a portion of intron 2 and exon 4 of the porcine alpha 1,3-galactosyltransferase gene from the 5′-end, positive selection marker, acting on the SV40 early promoter Enabled neo The gene region, a multicloning site having restriction enzyme positions of IRES, Sac I and Not I, and a second region of about 6.9 kb in size consisting of poly A, all of Intron 4, and a part of exon 5, are included. In addition, a gene encoding a complement inhibitory protein was inserted into the restriction enzyme position (FIG. 8).

The vector construction can be prepared using genetic recombination techniques well known in the art, site-specific DNA cleavage and ligation can be used enzymes generally known in the art.

In another specific embodiment of the present invention, the present inventors prepared a pCMAHKOCD39KIDT vector as a CMAH gene targeting vector capable of simultaneously removing a pig CMAH gene and targeting a gene encoding a human thrombosis inhibitory protein. A pCMAHKOneoDT vector capable of CMAH gene targeting was prepared.

First, in order to obtain a part homologous to the porcine CMAH gene nucleic acid sequence included in the pCMAHKOCD39KIDT vector, a partial base sequence of the CMAH gene region of the miniature pig that was hit by the CMAH gene of the miniature pig was identified.

In order to determine the CMAH gene portion of the miniature pig defined as the first region and the second region on the vector, the previously reported pig sequence (Kihiro Koike et al ., Transplantation , 70: 1275-1283, 2000) Primers were prepared and PCR reacted with Taq polymerase with proofreading.

The pCMAHKOneoDT vector comprises a first region of about 3.5 kb size comprising a portion of intron 3 and a portion of exon 4 of the CMAH gene. In addition, it comprises a 6.0 kb sized second region comprising a portion of exon 6 and a portion of intron 6. The vector is a positive screening marker neo Gene region, polyA gene region and TK gene region as negative selection marker.

Next, a pCMAHKOCD39KIDT vector was prepared by inserting a gene encoding CD39, a thrombosis inhibitory protein, in the neo gene at the front of the pCMAHKOneoDT vector. In this case, the gene encoding the CD39 overlaps the CMAH gene and the start codon, and is designed to express the CD39 gene through a promoter of the CMAH gene.

The pCMAHKOCD39KIDT vector is a vector capable of hitting the human CD39 gene while removing the porcine CMAH gene. The pCMAHKOCD39KIDT vector can be referred to as a vector meeting the object of the present invention. A first region of about 3.5 kb in size, a gene encoding the CD39 protein framed to the start codon of the CMAH gene, a neo gene region operably linked to the SV40 early promoter, poly A, a portion of exon 6 and intron 6 A second region of 6.0 kb size comprising a portion of the bun and a DT gene as a negative selection marker.

The vector construction can be prepared using genetic recombination techniques well known in the art, site-specific DNA cleavage and ligation can be used enzymes generally known in the art.

In another aspect, the present invention relates to a transformant into which the gene targeting vector is introduced.

The transformation may include any method of introducing a nucleic acid molecule into an organism, cell, tissue or organ, and may be carried out by selecting a suitable standard technique according to the host cell as known in the art. These methods include electroporation, calcium phosphate (CaPO ₄ ) precipitation, calcium chloride (CaCl ₂ ) precipitation, microinjection, polyethylene glycol (PEG) method, DEAE-dextran method, cationic liposome method, and Lithium acetate-DMSO method and the like, but are not limited thereto.

Two targeting cell lines (CMAHKI # 1, CMAHKI # 2) were prepared by introducing pCMAHKOCD39KIDT, a targeting vector prepared in a specific example of the present invention, into miniature porcine ear tissue-derived fibroblasts (pESF), of which CMAHKI # 1 The cell line was deposited with KCTC 11433BP on November 24, 2008 to KCTC (Korean Collection for Type Cultures, Korea Research Institute of Bioscience and Biotechnology, 111, Gwahak-ro, Yuseong-gu, Daejeon, Korea).

The trustee is a trustee designated by the Budapest Treaty on the Deposition of Microorganisms, and the inventors ensure that the deposit meets the conditions laid down in the Butapest Treaty.

The present invention also provides a non-human clone produced by nuclear transfer of the transgenic somatic cell line.

The non-human clone may be a mammal similar in size to a human such as sheep, goats, pigs, and dogs, but more preferably pigs, and most preferably, miniature pigs.

The nuclear transfer method used for the production of the cloned animal can be used in the art well known in the art, more preferably US6,781,030B, US6,603,059B, US6,235,969B, US7,355,094B, US7 , 071,372B, KR862298B, KR500412B, KR807644B, JP4153878B, US6,700,037B, US7,291,764B, US6,258,998B, US6,548,741B, WO03 / 089632A, US7,371,922B and the like can be used. More preferred is the use of the methods described in KR500412B, KR807644, JP4153878B, US6,700,037B, US7,291,764B, US6,258,998B, US6,548,741B, WO03 / 089632A or US7,371,922B. All of these patent documents are incorporated herein by reference.

In addition, the present invention provides a method for producing a heterologous organ for transplantation, comprising the step of extracting the organ for transplantation after breeding the non-human clone animal. The organs may be harvested by adjusting the breeding time in consideration of the sex, age, weight, height of the recipient, breeding donor clone animals, and then performing a conventional surgical operation. Or can be refrigerated quickly.

The gene targeting vector of the present invention targets an endogenous antigenic determinant synthetic gene of an organ donor animal and simultaneously encodes a complement inhibitory protein and / or a thrombosis inhibitory protein to be expressed at the locus of the endogenous antigenic determinant synthetic gene. By knock-in can solve the problems such as gene silencing following the random insertion of the gene to be introduced, cancer-causing caused by the activation of the germination gene, it can be very effectively used to prepare donor animals for safer cross-organ transplantation have.

Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are provided to illustrate the present invention, but the content of the present invention is not limited thereto.

Example 1: GT  Gene Targeting  Vector manufacturing

1-1: Identification of the base sequence of the entire alpha 1,3-galactosyltransferase gene exon 4 and part of the intron 2 in miniature pigs

The swine alpha 1,3-galactosyltransferase gene consists of nine exons, and the entire sequence of the exon and a portion of the intron has been reported (Kihiro Koike et al ., Transplantation , 70: 1275-). 1283, 2000). In the present invention, in order to target the alpha 1,3-galactosyltransferase gene of the miniature pig, some base sequences of the alpha 1,3-galactosyltransferase gene region of the miniature pig that were hit were identified. A previously reported pig sequencing (Kihiro Koike et al.) To determine the base sequences of a portion of the intron 2 and exon 4 of the miniature pig. al ., Transplantation , 70: 1275-1283, 2000) to prepare a forward primer of SEQ ID NO: 3 (5'-TCCATGAACAACTTCGATTGCATG-3 ') and a reverse primer of SEQ ID NO: 4 (5'-TGGCTGATAACTAGGAGATTAGAGGAG-3') Taq DNAase (Taq polymerase, Intron Biotechnology, Korea) with proofreading, preheated at 94 ° C for 2 minutes, 94 ° C 20 seconds, 60 ° C 45 seconds and 68 ° C 4 minutes 35 times and 68 ° C 10 minutes PCR reactions were carried out under the conditions.

The synthesized PCR product was cloned into a T-easy vector (Promega, USA) and sequenced for a total of four clones to identify common nucleotide sequences (SEQ ID NO: 5 and Figure 1). The intron 2 region of the alpha 1,3-galactosyltransferase gene of miniature pigs is shown in SEQ ID NO: 6 and the intron 4 region is shown in SEQ ID NO: 7.

The left arm portion to be used in the targeting vector is a reverse primer of SEQ ID NO: 8 (5'-GAATTCATGATTATTATCCTCCCAAGC-3 ') and SEQ ID NO: 9 (5'-CATGAATTCCATTATTTTCTCCTGGG-3') linked to the Eco RI restriction enzyme position. A primer was prepared to pre-correct Taq DNA polymerase (Intron Biotechnology, Korea) for 2 minutes at 94 ° C, 94 ° C 20 seconds, 60 ° C 45 seconds and 68 ° C 3 minutes 30 seconds 35 times and 68 ° C 10 minutes. PCR reactions were carried out under the conditions.

The synthesized PCR product was cloned into a T-easy vector (Promega, USA), and the obtained clone was cut with the restriction enzyme Eco RI, and then left arm that constitutes the first region (SEQ ID NO: 1) of the targeting vector. Was used (FIGS. 5 and 6).

1-2: miniature  Alpha 1,3- pigs Galactosyltransferase  gene Intron  Sequence identification of whole 4 and parts of exon 5

Miniature Pig Alpha 1,3-galactosyltransferase Sal I and Xho I based on previously reported pig sequencing (Kihiro Koike et al ., Transplantation , 70: 1275-1283, 2000) to determine the nucleotide sequence of the entire intron 4 and exon 5 portions of the gene. A reverse primer of SEQ ID NO: 10 (5'-GTCGACCTGTCAATGCTGCTTG-3 ') and a reverse primer of SEQ ID NO: 11 (5'-CTCGAGCCAGAACAAAGAACCTTC-3'), each having a restriction enzyme position, were prepared, thereby correcting Taq DNA polymerase (Intron). Biotechnology, Korea) was subjected to a PCR reaction at 94 ° C for 2 minutes, 94 ° C 20 seconds, 60 ° C 45 seconds and 68 ° C 7 minutes 35 times and 68 ° C 10 minutes.

The synthesized PCR product was cloned into a T-easy vector (Promega, USA), and the sequencing of a total of four clones was able to identify a common base sequence (SEQ ID NO: 12 and Figure 2). The obtained clone was cut with restriction enzymes Sal I, Xho I and used as a right arm constituting the second region of the targeting vector (SEQ ID NO: 2) (FIGS. 5 and 6).

1-3: Construction of pGTKOneoTK Vector

The left arm having the Eco R I restriction enzyme positions prepared in Examples 1-1 and 1-2 and the light arm having the Sal I and Xho I restriction enzyme positions had the thymidine kinase (TK) gene, which is a negative screen And inserted into the Eco R I and Sal I positions, respectively, of the pBst-TK vector (FIG. 5). When the TK gene is present in the medium called gancyclovir, it breaks down to produce enzyme products that are toxic to cells. Homologous recombination occurs so that the cell line from which the TK gene of the targeting vector has been eliminated (FIGS. 3, 4 and 8) can be grown in a medium to which gancyclovir is added, but the randomization of the targeting vector causes the targeting vector to be located at an arbitrary position of the chromosome. When inserted into the TK gene is expressed together to die in the growth medium containing gancyclovir (gancyclovir). The neo gene, a positive selection marker, has a forward primer of SEQ ID NO: 13 (5'-CTAGGAATTCCTTCGCGATGTACGGGCC-3 ') linked with the Eco R I, Eco RV restriction enzyme position in the pcDNA3.1 vector (Invitrogen, USA) with a promoter and poly A moiety. Reverse primer of SEQ ID NO: 14 (5'-CAGTGATATCCTTATCGCTATCGATTCACAC-3 ') was prepared and pre-warmed with Taq DNA polymerase (Intron Biotechnology, Korea) for 2 minutes at 94 ° C for 20 minutes, 60 ° C for 45 seconds, and PCR reaction was performed at 68 ° C for 1 minute 30 seconds 35 and 68 ° C for 10 minutes.

The synthesized PCR product was inserted into the Eco R I and Eco R V positions of the pBst-TK vector, respectively (FIG. 5). As a result, the pGTKOneoTK vector is complete and can be linearized by restriction enzyme Not I or Xho I and then introduced into somatic cells. When homologous recombination occurs, the TK gene is eliminated. Targeting was carried out with a primer pair consisting of a forward primer of SEQ ID NO: 15 (5'-GGTCTGCCTACATCTCTCTGATGAAC-3 ') and a reverse primer of SEQ ID NO: 16 (5'-GGCATCAGAGCAGCCGATTG-3') and SEQ ID NO: 17 (5'-CCTTCTATCGCCTTCTTGACGAG-3 '). ) Was identified as Taq DNA polymerase (Takara, Japan) by a pair of primers consisting of a forward primer of s) and a reverse primer of SEQ ID NO: 18 (5'-CTAGAGATATTTGGAATTCAAAGCACTTAC-3 '). PCR reactions using primer pairs as set forth in SEQ ID NOs: 15 and 16 used conditions of preheating at 94 ° C. for 2 minutes, 94 ° C. 20 seconds, 60 ° C. 45 seconds, and 68 ° C. 3 minutes 35 times and 68 ° C. 10 minutes. PCR reactions using primer pairs described in Nos. 17 and 18 were preheated at 94 ° C. for 2 minutes, 94 ° C. 20 seconds, 60 ° C. 45 seconds, and 68 ° C. 8 minutes 35 times and 68 ° C. 10 minutes conditions (FIG. 3). ).

Internal ribosome entry site (IRS), multicloning site (MCS, Sal I, Not I), and Poly A (pA) moiety are located in Eco R V and Xho I restriction enzymes in pIRES (Clontech, USA) vector. A forward primer of linked SEQ ID NO: 19 (5'-GATATCCGCGTCGAGCATGC-3 ') and a reverse primer of SEQ ID NO: 20 (5'-CTCGAGTCGCCATTCAGGC-3') were prepared to provide a calibration of Ta q DNA polymerase (Intron Biotechnology, Korea) PCR reaction was carried out under the conditions of preheating at 94 ° C. for 2 minutes, 94 ° C. 20 seconds, 60 ° C. 45 seconds, and 68 ° C. 1 minute 30 seconds 35 times, and 68 ° C. 10 minutes. The neo gene, which is a positive selection marker by the IRES sequence, and the human complement inhibitory gene inserted into the MCS can be expressed as one mRNA but expressed as two independent proteins by ribosomes bound to IRES (Louis Marie). Houdebine et al ., Transgenic research , 8: 157-177, 1999).

The synthesized PCR product was inserted into a position where the pBst-TK vector was cut with Hin d III, treated with Klenow enzyme, and cut with Sal I again (FIG. 6). As a result, a pGTKOIRESKITK vector cassette having a MCS ( Sal I, Not I) into which a human complement inhibitory gene can be inserted is completed, linearized by restriction enzyme Xho I, and then introduced into somatic cells. When homologous recombination occurs, the TK gene is eliminated (FIG. 4).

1-4: Construction of pGTKOIRESCD59KITK Vector Cassette

The human complement inhibitory gene CD59 was inserted into the MCS ( Sal I, Not I) position of the pGTKOIRESKITK vector cassette (FIG. 7). The CD59 gene produced a forward primer of SEQ ID NO: 21 (5'-CTGAGTCGACATGGGAATCCAAGGAGGGTCTG-3 ') and a reverse primer of SEQ ID NO: 22 (5'-CTGAGCGGCCGCTTAGGGATGAAGGCTCCAGG-3') with Sal I and Not I restriction enzymes linked thereto. PCR was carried out in a human cDNA library with a tack polymerase (Intron Biotechnology, Korea) at 94 ° C for 2 minutes, 94 ° C 20 seconds, 60 ° C 45 seconds, and 68 ° C 40 seconds 35 times and 68 ° C 10 minutes.

The synthesized PCR product was inserted into the position where the MCS of the pGTKOIRESKITK vector cassette was cut into Sal I, Not I (FIG. 7). As a result, a pGTKOIRESCD59KITK vector was completed that could remove the alpha 1,3-galactosyltransferase gene and simultaneously hit the CD59 gene. The pGTKOIRESCD59KITK vector can be linearized by restriction enzyme Xho I and then introduced into somatic cells. When homologous recombination occurs, the TK gene is eliminated (FIG. 8).

Targeting is confirmed by Taq DNA polymerase (Takara, Japan) by the primer pair consisting of SEQ ID NOs: 17 and 18 and the primer pair consisting of SEQ ID NOs: 19 and 20 described in Example 1-3 (FIG. 8). ). The conditions of the PCR reaction were also the same as those described in Examples 1-3.

Example  2: CMAH  gene Targeting  Manufacture of vector

2-1: Construction of pCMAHKOneoDT Vector

Sac I and Not to use the intron 3 and exon 4 portions of the CMAH gene sequences of miniature pigs (www.ncbi.nlm.nih.gov, NW_001886419.1) as the left arm of the targeting vector. I Taq DNA polymer with proofreading by constructing a forward primer of SEQ ID NO: 23 (5'-GAGCTCCATGACAGGAACTCCTGAGATGAATATC-3 ') and a reverse primer of SEQ ID NO: 24 (5'-GCGGCCGCTCGTTGCCTCTCTCCAGGTATTAAG-3') with restriction enzyme positions PCR was performed with Laze (ExTaq polymerase, Takara, Japan) at 94 ° C. for 2 minutes, 94 ° C. 20 seconds, 60 ° C. 45 seconds, and 72 ° C. for 4 minutes 35 times and 72 ° C. for 10 minutes.

The synthesized PCR product was cut into restriction enzymes Sac I and Not I, and then inserted into the Sac I and Not I positions of the pBCKIDT (AU 2005256120 B2) vector (FIG. 10).

Specifically, in order to use as a right arm of the targeting vector, a part of exon 6 and a part of intron 6 in the CMAH gene sequence SEQ ID NO: 25 (5'- connected with Eco R V and Sal I restriction enzyme positions) A forward primer of GATATCACCATCAATACTGATCAATGTTTTCTG-3 ') and a reverse primer of SEQ ID NO: 26 (5'-GTCGACAGCAGGAAAAACTGAGTCTGCAGTTC-3') were prepared to provide Taq DNA polymerase (ExTaq polymerase, Takara, Japan) with proofreading at 94 ° C. PCR reaction was performed under conditions of preheating for 2 minutes, 94 ° C 20 seconds, 60 ° C 45 seconds, and 72 ° C 6 minutes 35 times and 72 ° C 10 minutes. The synthesized PCR product was cut with Eco R V and Sal I restriction enzymes, and then linked to the Eco R V and Sal I positions of the pBCKIDT vector to prepare a pCMAHneoDT vector (FIG. 10).

2-2: Construction of pCMAHKOCD39KIDT Vector

The human CD39 gene was linked to the pCMAHKOneoDT vectors Xho I and Pac I (FIG. 11). Human cDNA libraries cutting the CD39 gene in the (Openbiosystems, USA) with Sal I, Kpn I restriction enzymes, and at the same time forward primer sequence of the Kpn I, Bam HI position is SEQ ID NO: 27 (5'A-TGCAATTTCGCCTCTTGGC-3 ' ) connected Reverse primer of No. 28 (5'-GAGGATCCCTATACCATATCTTTCCAGAAATATGAAGG-3 ') was prepared and pre-warmed in a cDNA library at 94 ° C for 4 minutes in 94 ° C, 94 ° C, 20 seconds, 57 ° C and 45 seconds with Taq DNA polymerase (Genotech, Korea). And PCR reactions at 72 ° C. for 1 minute 35 times and 72 ° C. for 10 minutes to cut the restriction enzymes Kpn I, Bam H I and connect two DNA fragments to the pDsRed2-C1 vector (Clontech, USA).

The CD39 gene linked to the pDsRed2-C1 vector is a forward primer of SEQ ID NO: 29 (5'-GACTCGAGCATGGAAGATACAAAGGAGTCTAACG-3 ') linked to the Xho I, Pac I position with a polyA sequence, and SEQ ID NO: 30 (5'-GCATTAATTAAGATTTAACAAAAATTTAACGCC-3). Taq DNA polymerase (Genotech, Korea) with a reverse primer of ') was prepared and preheated at 94 ° C for 2 minutes, 94 ° C for 20 seconds, 60 ° C for 45 seconds, and 72 ° C for 2 minutes 35 and 72 ° C for 10 minutes. PCR reaction under the condition of, and cut with restriction enzymes Kpn I, Bam H I, and then connected to the pCMAHKOneoDT vectors Xho I and Pac I position.

The linked CD39 gene is located behind the ATG at exon 4 of the CMAH gene, thereby being expressed by the promoter of the CMAH gene. As a result, a pCMAHKOCD39KIDT vector was completed that could simultaneously remove the CMAH gene and overexpress the CD39 gene by hitting it. The pCMAHKOCD39KIDT vector can be linearized by restriction enzyme Pml I and then introduced into somatic cells. When homologous recombination occurs, the DT gene is eliminated (FIG. 11). Targeting was performed by using a forward primer of SEQ ID NO: 31 (5'-ACCCCAGCTCACAATGAGCAACACCAGAT-3 ') and a reverse primer of SEQ ID NO: 32 (5'-AGAGGGACCCCAATGACAGTAACAGCAGA-3') using LA Taq polymerase (Takara, Japan) at 94 ° C. It was confirmed by PCR reaction under the conditions of preheating, 94 ° C., 20 seconds, 60 ° C., 45 seconds, and 72 ° C. for 2 minutes 35 times and 72 ° C. for 10 minutes (FIG. 12).

Example  3: gene Targeting  Cell line manufacture

3-1: CMAH  Gene targeting cell line production

The present inventors purified the pCMAHKOCD39KIDT plasmid prepared in Example 2 using a plasmid separation kit (QIAfilter Plasmid Midi kits, Qiagen, USA), linearized with restriction enzyme Pml I, and then obtained ear tissue fibroblasts derived from miniature pigs ( pESF). pESF cells consisted of DMEM (Gibco, Invitrogen corporation, USA), 10% FBS (Hyclone, USA), 0.001% gentamycin (Gibco, Invitrogen corporation, USA), 1% MEM non-essential amino acids (Gibco, Invitrogen corporation, USA) The culture solution was cultured at 5% CO ₂ , 37 ° C. conditions and the culture solution was changed every 2-3 days. 5 μg of linearized DNA was introduced into 2 × 10 ⁶ pESF cells of passage 2 or 3 using the Nucleofector (Amaxa Biosystems, USA) method, and then cultured in a culture solution containing 300 μg / ml G418 (Gibco, USA). After 12-14 days of culture, cell lines with a diameter of about 6-10 mm were observed (FIG. 13), and they were passaged into 96 well culture vessels. One half of the cell lines grown to 85-95% in the culture vessels were subcultured to 96 wells again, and half were used for targeting. Cells for targeting confirmation were lysed at 55 ° C. for 1 hour in lysis buffer consisting of 1M KCl, 1M Tris pH8.3, 1M MgCl ₂ , 0.45% NP40, 0.45% Tween 20, 10 μg / ml Proteinase K After processing at 100 to 5 minutes, PCR reaction was performed under the conditions described in Example 2-2 using the forward primer of SEQ ID NO: 31 and the reverse primer of SEQ ID NO: 32 (Fig. 14). Homologous recombination was attempted for a total of 132 cell lines, and as a result, two cell lines were hit. The CMAH gene targeting cell lines thus established were named CMAHKI # 1 and CMAHKI # 2. Among them, CMAHKI # 1 was deposited with the Korea Cell Line Bank under accession number KCTC 11433BP as of November 24, 2008.

The gene targeting vector of the present invention targets an endogenous antigenic determinant synthetic gene of an organ donor animal and simultaneously encodes a complement inhibitory protein and / or a thrombosis inhibitory protein to be expressed at the locus of the endogenous antigenic determinant synthetic gene. By knock-in can solve the problems such as gene silencing following the random insertion of the gene to be introduced, cancer-causing caused by the activation of the germination gene, it can be very effectively used to prepare donor animals for safer cross-organ transplantation have.

Figure 1 shows the nucleic acid sequence (SEQ ID NO: 5) of the polynucleotide containing the entire exon 4 (underscore) and part of the intron 2 of the GT gene of the miniature pig.

Figure 2 shows the nucleic acid sequence (SEQ ID NO: 12) of the polynucleotide containing the entire intron 4 and part of exon 5 (underlined) of the GT gene of the miniature pig.

Fig. 3 is a diagram illustrating the targeting of the GT gene using the pGTKOneoTK vector.

4 is a schematic diagram illustrating the GT gene hit using the pGTKOIRESKITK vector.

Figure 5 is a schematic diagram showing the manufacturing process of the pGTKOneoTK vector.

6 is a schematic diagram illustrating a manufacturing process of a pGTKOIRESKITK vector.

7 is a schematic diagram showing the manufacturing process of the pGTKOIRESCD59KITK vector of the present invention.

Fig. 8 is a schematic diagram illustrating the GT gene hit using the pGTKOIRESCD59KITK vector.

9 is a schematic diagram illustrating the targeting of the CMAH gene using the pCMAHKODT vector.

10 is a schematic diagram showing a manufacturing process of a pCMAHKODT vector.

11 is a schematic diagram showing a manufacturing process of a pCMAHKOCD39KIDT vector.

12 is a schematic diagram illustrating the targeting of the CMAH gene using the pCMAHKOCD39KIDT vector of the present invention.

Fig. 13 is a photograph of pESF cell state before the introduction of pCMAHKOCD39KIDT and the cell line formed after the CMAH gene introduction.

Figure 14 is a photograph showing the results of experiments confirmed by PCR reaction whether the actual CMAH gene hit in the cell line:

V, targeting vector;

WG, non-transgenic cell line;

# 1 and # 2, targeted cell lines;

# 3- # 7, cell line with randomly inserted gene; And

M, marker DNA.

<110> Korea Research Institute of Bioscience and Biotechnology <120> Genetically-modified cell line for producing cloned miniature          pigs for xenotransplantation and method for preparing the same <130> 8P-11-47 <150> KR2007-123823 <151> 2007-11-30 <160> 32 <170> KopatentIn 1.71 <210> 1 <211> 2972 <212> DNA <213> Sus scrofa <400> 1 gaattcatga ttattatcct ccaagcctgt tcctcctcca gcccatctga gaaaatacta 60 caacccccct gcttaagcag aaatcttggg tcttccttgt ctcatctctg ataacaaaat 120 taccaaccac gtcctatcaa ttctctctcc aaagtatata tatatatatt tttttttaat 180 tttttcccgc tgtacagcat ggggatcaag ttattcttac atgtatattt tccccccacc 240 ctttgttccg ttgcaatatg agtatctaga catagttctc aatgctactc agcaggatct 300 ccttgtaaat ctaagttgta tctgataacc ccaagctccc gatccctccc actccctccc 360 tctcctgtcg ggcagccaca agtctattct ccaagtccat gattttcttt tctgtggaga 420 tggtcatttg tgctggatat tagattccag ttataagtga tatcatatgg tatttgtcaa 480 agtatatatt ttatttttct ttgtcttttt gtcttttgtc tttttttttt ttgttgttgt 540 tgttgttgtt gttgttgttg ctattacttg ggccgctccc gcggcatatg gaggttccca 600 ggctaggagt tgaatcggag ctgtagccac cggcctacgc cagagccaca gcaacgcggg 660 atccgagccg cgtctgcaac ctacaccaca gctcacggca acgctggatc cttaacccac 720 tgagcaaggg cagggaccga acccgcaacc tcatggttcc tagtcggatt cgttaaccac 780 tgcgccacga cgggaactcc caaagtatat tttgaatcaa gccacccttt gagccaggcc 840 acctcctctt tatggtcatg agaacggtct gcccttgtcc ttttctccat tctccacact 900 cagcacccag atgggtctct ctaggtgaag ttggatcagg ggattctcca gctttagatg 960 ctttttggga ttccccaccc tactttccat acctttccag gttctgactg cctctgcccc 1020 ccttctgact gcctagcacc agccactcaa gggggacagt gtcagtcact atttttttct 1080 tgtccaggtt ttttgctttt gtttttttca aacacgagca gctctttctc ttgtctgcct 1140 ggtatagatg ctgtttccaa aatattctca tcccttctca cggcccttgt catcctttcc 1200 catcctatct tcatcccttg ggaagctcta aagtcatctc cccaaattga agggtgacta 1260 aagagtttcc cagaaggaaa aactgagttt ccaactacta cactgacttg caagaaatgt 1320 ttgtgtcttc attaaatgaa aaagaaaaaa ctgtaacaag atatgagaaa atacagaaag 1380 gaaataataa gactagaaaa gtcaaatata tagtgaaggt gttgcatcaa acacttaaat 1440 aaactagtac agatgttaaa agactaaatt atatagttga aggatagctg tgaagatgta 1500 aactatgaca tctaaaacac aaaatgttgg cgttcccgtc acggcacagt ggaaacgaat 1560 ccgactagga accatgaggt tgcaggttca attcctgccc ttgctcagtg ggttaaggat 1620 ccggtgttgc cgtgagctgt ggtgtaggta gccaatgagg cttggatccc gcgttgctgt 1680 ggctctggtg taggccggtg gctacagctc cgattcgacc cctagcctgg gaacctccat 1740 atgccgcggg agcggcccta aaaagacaaa agaccaaaaa aaaaaaaaaa caaaaaaccc 1800 acaaaatgtt gggaatcagt cctctactag tattatgtta ttgtcaagtt ttccttttat 1860 gtctgttaat atttgcgttc tagatgtagg tgctctgata tcgtgtgcat atatgttaac 1920 caatgttatg tcttcctctg gtattgatcc ctttgttatt atgtaatgcc ctactttatc 1980 ttttgttaca ttctttgttt atgagtattg ctgatatgtg gctagctgcc acacttttct 2040 tgtcctttcc atttacaata aatatctttc tatctccacc caaattaaag tactccgcaa 2100 cctgttattc cacccagcat cccttccctc ttcaactaca atttcatgca gcgatcaaga 2160 aatacgaatg taccgactgt ttgccacttg tgtgggtgca ttggggaaaa gctgggtggg 2220 aagtggcaga gcctagatta taaaggacca gggtgagagt tcccattgtg gctcagctga 2280 aatgaatctg actagcatcc atgaggacga aggtttgatc cctggcctca atcagtgggt 2340 taaggatctg gcgttgctgt ccgtgagttg tggtgtagtt cgcagacaag gcgtggactt 2400 agtgtggctg tggctgtggc ataggctagt ggctacagct ctgattcgac ccctagcctg 2460 ggaatctcta tatgctgtga gtgtggccct aaaatttaaa tgaaattaaa taaaggacca 2520 gggtatattt ttctttgagg ataaggtaca tagtcagtat atcagggaca gtagacctag 2580 gaaacggatg cttcctctag tctgtgatgc gaggtggggc atctgagttg ggggcggctg 2640 gagcccttag ggaccattaa ctaaacccgt cactctccca catctcggtg gaccttggga 2700 tcagtcagga tgcttcccct ttgagcctca aaatggcctt agtatccttc ccaacccaga 2760 cggccctgtc agttcattga cttggctaat ttgccagtgt aggcctatgc aaattaaggt 2820 agaacgcact ccttagcgct cgttgactat tcatcaactt ttccttttag aaaagatatt 2880 ggtataagca cttcttaaaa aaccatattc cactctgggt gtatttaatc taattttccc 2940 ttctcctttt cttttcccag gagaaaataa tg 2972 <210> 2 <211> 6846 <212> DNA <213> Sus scrofa <400> 2 ctgtcaatgc tgcttgtctc aactgtaatg gttgtgtttt gggaatacat caacaggtag 60 ttatgaaaca tgatgaaatg atgttgatga aagtctcctc taatctccta gttatcagcc 120 aagtcaccag cttgcattaa aagtaggatt cactgacacc gtaaagaaag cattccagag 180 agttgccgtt gtggctcagg ggcagcaaac ccaattagga tccaagagga ggtgggtttg 240 atccctggcc ttgctctttg gcttaaggat ccggcattgc cgtgacctgt ggtgtaggtt 300 gcagatgcag ctcggatctg gcattgctgt ggctgtggcg taggctggtg gcttcagctc 360 cagtttgacc cctagcctgg gaacttccat atcccacact tgcggcccta aaaagcaaag 420 aaagaaagaa aatattctac ccttcctgta tccctgagcc cttaaatacc gtctttaaag 480 tcattagatc ttcaagtacc ttccagctaa ttaattatct tccttcctgc catgttgcca 540 ttgtcctgat ttttatacct ctgcagttct gggtagacta gagccagaaa taataaggtc 600 atgttaagac caagatataa tattaaatta tttatatgac cagatatgga agttaccttg 660 agaactttca gacaggaatt ccatgagaaa tacaccctga tttttgcaat cctaaaatat 720 ttgcagagtt taaaggaaca actcaagttg ttgacttttg ctgcaaaaca cactgagtcg 780 ctggtgattc atttgtgcct ggctaaactt ttgggtgttt tgtctctttt tttttaactc 840 tggaaagcaa aatgaattaa acatttctga gttttcaaat tcatcagtgg attcacccca 900 aatatttgag ctgcttcttt gcttttggaa actacgatgc cttggagatt ccagctggag 960 acgcttctga cagaaagaaa tgtctgcaag cagctacaaa aatgcatgat ggctttgact 1020 taagaggcat tgataccgct tggcctttct ttcaaaaagg ccaccttaca acttggcctg 1080 aaggcattcc cgtggtggtg cagcggaaaa tgaatctgac taggaacccc gaggttgtgg 1140 gttcaatccc tggccttgct cagtggctta aggatcgggt gttgaagtaa gctgtggtgt 1200 agattgcaga cgcagcttgg atctggtgtt gctgtggctt tggtgtaggc cggcagctac 1260 agctccactt ggacccctag tctgggaacc tccatatgcc acaggtgtgg ccctaaaagg 1320 aaaaaagaca acaaacaaac aaaaaaccaa aaaacaactt ggcctggaga gctatgtcat 1380 caccattgat attttgatgg gtagtgtttt agtagcccct caagttcagg atgatggcct 1440 ggattaacgt tagaatgtct cttaaattct aagacttgat gagccagcag gaccattttg 1500 gccacttaga aaggaactgc atcttcaggt ccatcagtag aaggaggatt ctctagggag 1560 ttctctctta gctcagcggg ttcaagaatt cagtcttgtc cctacagcag ctcaggtgac 1620 tgctatggct tggctttgat ccctggccca ggaatttctg catgctgcag gtgcagccaa 1680 aaaaaaaaaa aaaaaggagg aggtggattc cctagaataa gaagctgtca ttcctttgga 1740 tgcttcatag atctaaccac ttctggaaca gttattccct ctcattctga agaactcatt 1800 ttaagaaaaa caagacgagc tagagagtga acaaatgtct acaaaccaac cttttcgaat 1860 tgaggaaact gtggtacttc ctctgaagaa aagatgacag cgttggatgc agagaccctg 1920 gggctccctt aggtacttga ggactgagga gatattctca gtggaggctg gagctaggct 1980 gcctggggtt ggtcctgtgc caccacttcc ctcctctgtg actttgggca agtttcccta 2040 tctttaaaaa tggggatgat agtagtacct gcttcatagg gttgttggat aaaataagtt 2100 gtgaataaag cactaagggc aacgtactta gtaagcgctg gctgccatca ccaccaccac 2160 tatcaccatc tgtccggagg gcagcatagg acaggagatt ttggcaaata gaaggaagag 2220 ttctaggagt tcccgttgtg gtgcagggga aatgaatcca actaggaact aggaggtttc 2280 gggttcaatc ccgcgcctcg ctcagtgggt taaggatcca gtgttgccat gagctgtggt 2340 gtagattgca gacatggcta ggatctggag ttgctatggc tgtggtgtaa gctggcagct 2400 gtagctcgga ttctacccct agcctgggaa tttccgtatg ccacaggttt ggccctacaa 2460 agaaaaaaga aaaagaaaaa gaaaaaattc taggggctga aagaatctaa cagaagagca 2520 agttccccat ggggttcctg acctgagttg agatgcttgt gtaggcaacc ttcaagctct 2580 gaactcttga ttgttttgaa ttgcagccag agttatactt ccatattttg ggtacttcac 2640 aaaattaaaa cacagaagcc aaaggcccag aagtgcatat tggtgctggc ctcccataaa 2700 gagggttgtt ttgcagtgct gggcacactc tctcttcaca gtaactggag cagattctgg 2760 ctgctcttca gggccgtagt ctggcaccca gactgcagcc acatcattct tcaatgtgag 2820 gaatctattt gaacatctgc aaggggttta aaaggcagga gattctttgc caccttgtga 2880 attggtctga ggtgagctga gggcactaac cttagacagg tgggtagcac tgtagctaaa 2940 gaggattaca ggagttcctg ttgtggctta gtggtaacaa atccaactag tatccatgag 3000 gattcaggtt cgatccctgg cctcgctcag tgggtcaggt atccggtgtt gctgtggctg 3060 tggtgtaggc tggcagcttc agctctgatt tgacccctag cctgggaact tccatgtgct 3120 gtaggtaagg cccttgaaaa aaaaaaaaaa gagatttaca aaataactcc atcaaacaca 3180 tacagctgtt taagaatgtc atccaggaca gcatttggtt aaaggctaga tgaaaaaaaa 3240 aaaaaaaatc ttagaatttt atttatttat tttttctttt tagggccaga cctgtggcct 3300 atggaaatgc ctgggctagg ggtggaatca gagctgccta caccacagcc atagccacgc 3360 cagatccaag ccccgtctgt gacctacacc acagctcatg gcaaacactg gatccttaat 3420 ccactgagtg aggccaggaa ttgaacccac attctcatgg atgctagttg ggttcttaag 3480 ccactgagcc acaagcttag aattttagag gtggaagaaa ctttaagagc tataataaag 3540 taatgatggt gatggtgatt ttgatgttag cggctactag ttattgagtg tttgcttgtg 3600 ccaggaactc cactgttcat tccctcctgt ttttaaaaca gccctggaag gtcagtgtta 3660 gtccacattt ctagatgagg aatactgagt ttccacaata ttaaatgtga acgttcaagg 3720 tcacattttt aggaagattt aggtccaggg ctgtctgact tgggtaacct gggtaaccct 3780 tcctttagtc aaggtttcca ttgttcaggc gatggacaag taggtgaaat gccttaacag 3840 tgaacttatg tctaacttct aattagaact cagatcttct gattcatcat ctggggctcc 3900 ttctggagct ggttgttcat gccaaatgct gcgaggggta cagtgtgcgc caaggagaat 3960 ccctaccctc aaggggttat gctgtagatg gagcaggcag aggtacccat gaaagcccaa 4020 caacacaggc tagaaggagg atgtcagaga gagagagcaa aggaacgtga gagttcaggg 4080 agggcaagat tatgtttggc ttggagatgg atctatgttt tgcatttatt tttttggggg 4140 gggggtcttt ttgctacttc ttgggctgct cccgaggcat atggaggttc ccaggctagg 4200 ggtctaattg gagccgcagc caccagccta tgccagagcc acagcaacgc aggatctgag 4260 ccgcgtctgc aaccttcacc acagctcacg gcaacgccag atcgttaacc cactgagcaa 4320 gggcagggac cgaacctgca acctcatggt tcctagtcag attcgttaag cactgcgcca 4380 cgacgggaac tccctcattt agaaatattt attgagcacc tactgtatgc caggcattgt 4440 gctaggttca taccaaagaa ggctcagaag agatggcatc cgagctgtgc cttgaaggat 4500 gaatatgtgt taaatgccgt acacttcagg gtggttgttg ctgtgacctg aggtgttgaa 4560 ggcttctggg aaaggagggt gagatgagga agagggaggg gttactaaaa agatgggacg 4620 aggtggcaaa tccaaatcta taaattgatg ccctgagtgc ctcggcagga gggtggggct 4680 cctgagtgct gggtggcacg ggccctcccc ctcctcttgc ccctttccct tccccctctt 4740 gtaggatctg aagtcagatt ccccaggttc aaatactgtt tcttccctta gcagtatgac 4800 cttgggcaaa ataatttatt gcctctgtcc ctctgaggag gaatagaacc tccttcattg 4860 actgttatta gaatttaatg agctaataca tgtcagttgc ttagaaaggt ccccagccaa 4920 ctattagcta ttatgaatat tatcagatca atagacagat ttagaaacaa gggactttag 4980 agctgggtcc atgggtactg agcttagagg ggaaaccata ggtggtagga aggcatgtat 5040 ttcattccta ccaggagatg tggactccca gctggggcag aaggcagagg gaggagatcg 5100 gggctttggc agaatctcaa acaaatatta gtggttagtg gttttttgtt tctgttttaa 5160 gagatgaggg caggcgtttc cgatgtggcg cagtggaaac gaatctgact agtatccatg 5220 aggatgcagg ttccatccct ggactcactc agtgagttaa ggatccggca ttgccgtgag 5280 ttgtggtgta ggtcacagac acagctcaga tctggcattg ctgtggctgt ggtgtaggct 5340 agcagctgta gctccaattc aacccctagc ctgggaactt ccacatgccg caggtgcaac 5400 cccaaaagat aaatgaataa ataaataaat atgcgacctt cctttcttgg ggcctttgca 5460 tgtttttctc tctgttaggc acactcttgc taatccctct tcactgggcc tcctatgtat 5520 ccttcagaac tcagctaaaa catcatcccc tcccctgggg agccttcgag gtcttcctgt 5580 taagtgctcc tatgctttct tggagttttg aagtcctata atgatgtgtt tatcaaaata 5640 gggtccaccc tccctgccag cttctctaca ccacagacac atggtgtctg tttcagtcaa 5700 cactgtatgt ctggcacttg acatgtaacg catgctcagc aggtatttgt tgaatgaatg 5760 gaggcggtct gctagagtcg tcatatattt actgatcccg tcttgtagga tggtctcact 5820 gcttttgtta gcttaagaag tacctttttt tttttttttt aatggccaca cccatggcat 5880 atagaaattc cacgaaggaa ggaagaaaga aagaaagaaa gaaggaaatt cctgggtcag 5940 ggattgaatc caagccacag gtgcaacctg agctgcagtt gcggcaacac cacatctttt 6000 aacccactgt gctgggccag ggatcatacc tgtgcatcta cagcgaccca agccacggca 6060 gtcagattct ttttctgcct ttctttcttt cttttctttt tttttttttt ttttttttgt 6120 ctttttgcct tttctaggtg cggcatatgg aggttcccag gctaggtgtc gaatcagagc 6180 tgtagacgcc ggcctaaacc acagccacag caacacagga tccaagcctt gtctgtgacc 6240 tacaccacag ctcacggcaa cgctggatcc ttaacccgct gagcgaggcc agggattgaa 6300 cccgcaacct catggttctt agttggattc gttaaccact gagccatgat gggaactcct 6360 gcagtcagat tcttaaccca ctatgccaca gcaggaactc ctagaagtgc cctttgaggc 6420 tactctgtag acagctttga gccagcgagg caagacctgt ttttctggag gaagataaat 6480 cctgggtgag ggatgggtgg gctgtggtct tcctgggacc catctctgga gcctctctcc 6540 ctcagcaaag ccaccttgga caataagagc tgccatctat tttttttttc tttaaactaa 6600 gatttgatat tttccagaga cctccctccc accgtttgat ctgagtaatt ctgaaatgac 6660 gagagtcccg tgatatcatt ttttcgatct cgaaggtgga aacctgggag tagccacaac 6720 ccaggctctc agctcagcct agggtttcaa tgataatgat tgcaaaatag cttttctctg 6780 cattccaagt aacatgatat gtttttattt ccatttgctt ttagcccaga aggttctttg 6840 ttctgg 6846 <210> 3 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Forward for GT partial intron 2 and exon 4 <400> 3 tccatgaaca acttcgattg catg 24 <210> 4 <211> 27 <212> DNA <213> Artificial Sequence <220> Reverse for GT partial intron 2 and exon 4 <400> 4 tggctgataa ctaggagatt agaggag 27 <210> 5 <211> 3242 <212> DNA <213> Sus scrofa <400> 5 tccatgaaca acttcgattg catggtctgc ctacatctct ctgatgaact ttagacttga 60 atgtccactt gtctccctgt ccccttttag gtattcgcac actccccgac attcacacgt 120 ccaaaaggga attcatgatt attatcctcc aagcctgttc ctcctccagc ccatctgaga 180 aaatactaca acccccctgc ttaagcagaa atcttgggtc ttccttgtct catctctgat 240 aacaaaatta ccaaccacgt cctatcaatt ctctctccaa agtatatata tatatatttt 300 tttttaattt tttcccgctg tacagcatgg ggatcaagtt attcttacat gtatattttc 360 cccccaccct ttgttccgtt gcaatatgag tatctagaca tagttctcaa tgctactcag 420 caggatctcc ttgtaaatct aagttgtatc tgataacccc aagctcccga tccctcccac 480 tccctccctc tcctgtcggg cagccacaag tctattctcc aagtccatga ttttcttttc 540 tgtggagatg gtcatttgtg ctggatatta gattccagtt ataagtgata tcatatggta 600 tttgtcaaag tatatatttt atttttcttt gtctttttgt cttttgtctt tttttttttt 660 gttgttgttg ttgttgttgt tgttgttgct attacttggg ccgctcccgc ggcatatgga 720 ggttcccagg ctaggagttg aatcggagct gtagccaccg gcctacgcca gagccacagc 780 aacgcgggat ccgagccgcg tctgcaacct acaccacagc tcacggcaac gctggatcct 840 taacccactg agcaagggca gggaccgaac ccgcaacctc atggttccta gtcggattcg 900 ttaaccactg cgccacgacg ggaactccca aagtatattt tgaatcaagc caccctttga 960 gccaggccac ctcctcttta tggtcatgag aacggtctgc ccttgtcctt ttctccattc 1020 tccacactca gcacccagat gggtctctct aggtgaagtt ggatcagggg attctccagc 1080 tttagatgct ttttgggatt ccccacccta ctttccatac ctttccaggt tctgactgcc 1140 tctgcccccc ttctgactgc ctagcaccag ccactcaagg gggacagtgt cagtcactat 1200 ttttttcttg tccaggtttt ttgcttttgt ttttttcaaa cacgagcagc tctttctctt 1260 gtctgcctgg tatagatgct gtttccaaaa tattctcatc ccttctcacg gcccttgtca 1320 tcctttccca tcctatcttc atcccttggg aagctctaaa gtcatctccc caaattgaag 1380 ggtgactaaa gagtttccca gaaggaaaaa ctgagtttcc aactactaca ctgacttgca 1440 agaaatgttt gtgtcttcat taaatgaaaa agaaaaaact gtaacaagat atgagaaaat 1500 acagaaagga aataataaga ctagaaaagt caaatatata gtgaaggtgt tgcatcaaac 1560 acttaaataa actagtacag atgttaaaag actaaattat atagttgaag gatagctgtg 1620 aagatgtaaa ctatgacatc taaaacacaa aatgttggcg ttcccgtcac ggcacagtgg 1680 aaacgaatcc gactaggaac catgaggttg caggttcaat tcctgccctt gctcagtggg 1740 ttaaggatcc ggtgttgccg tgagctgtgg tgtaggtagc caatgaggct tggatcccgc 1800 gttgctgtgg ctctggtgta ggccggtggc tacagctccg attcgacccc tagcctggga 1860 acctccatat gccgcgggag cggccctaaa aagacaaaag accaaaaaaa aaaaaaaaca 1920 aaaaacccac aaaatgttgg gaatcagtcc tctactagta ttatgttatt gtcaagtttt 1980 ccttttatgt ctgttaatat ttgcgttcta gatgtaggtg ctctgatatc gtgtgcatat 2040 atgttaacca atgttatgtc ttcctctggt attgatccct ttgttattat gtaatgccct 2100 actttatctt ttgttacatt ctttgtttat gagtattgct gatatgtggc tagctgccac 2160 acttttcttg tcctttccat ttacaataaa tatctttcta tctccaccca aattaaagta 2220 ctccgcaacc tgttattcca cccagcatcc cttccctctt caactacaat ttcatgcagc 2280 gatcaagaaa tacgaatgta ccgactgttt gccacttgtg tgggtgcatt ggggaaaagc 2340 tgggtgggaa gtggcagagc ctagattata aaggaccagg gtgagagttc ccattgtggc 2400 tcagctgaaa tgaatctgac tagcatccat gaggacgaag gtttgatccc tggcctcaat 2460 cagtgggtta aggatctggc gttgctgtcc gtgagttgtg gtgtagttcg cagacaaggc 2520 gtggacttag tgtggctgtg gctgtggcat aggctagtgg ctacagctct gattcgaccc 2580 ctagcctggg aatctctata tgctgtgagt gtggccctaa aatttaaatg aaattaaata 2640 aaggaccagg gtatattttt ctttgaggat aaggtacata gtcagtatat cagggacagt 2700 agacctagga aacggatgct tcctctagtc tgtgatgcga ggtggggcat ctgagttggg 2760 ggcggctgga gcccttaggg accattaact aaacccgtca ctctcccaca tctcggtgga 2820 ccttgggatc agtcaggatg cttccccttt gagcctcaaa atggccttag tatccttccc 2880 aacccagacg gccctgtcag ttcattgact tggctaattt gccagtgtag gcctatgcaa 2940 attaaggtag aacgcactcc ttagcgctcg ttgactattc atcaactttt ccttttagaa 3000 aagatattgg tataagcact tcttaaaaaa ccatattcca ctctgggtgt atttaatcta 3060 attttccctt ctccttttct tttcccagga gaaaataatg aatgtcaaag gaagagtggt 3120 tctgtcaatg ctgcttgtct caactgtaat ggttgtgttt tgggaataca tcaacaggta 3180 attatgaaac atgatgaaat gatgttgatg aaagtctcct ctaatctcct agttatcagc 3240 ca 3242 <210> 6 <211> 3088 <212> DNA <213> Sus scrofa <400> 6 tccatgaaca acttcgattg catggtctgc ctacatctct ctgatgaact ttagacttga 60 atgtccactt gtctccctgt ccccttttag gtattcgcac actccccgac attcacacgt 120 ccaaaaggga attcatgatt attatcctcc aagcctgttc ctcctccagc ccatctgaga 180 aaatactaca acccccctgc ttaagcagaa atcttgggtc ttccttgtct catctctgat 240 aacaaaatta ccaaccacgt cctatcaatt ctctctccaa agtatatata tatatatttt 300 tttttaattt tttcccgctg tacagcatgg ggatcaagtt attcttacat gtatattttc 360 cccccaccct ttgttccgtt gcaatatgag tatctagaca tagttctcaa tgctactcag 420 caggatctcc ttgtaaatct aagttgtatc tgataacccc aagctcccga tccctcccac 480 tccctccctc tcctgtcggg cagccacaag tctattctcc aagtccatga ttttcttttc 540 tgtggagatg gtcatttgtg ctggatatta gattccagtt ataagtgata tcatatggta 600 tttgtcaaag tatatatttt atttttcttt gtctttttgt cttttgtctt tttttttttt 660 gttgttgttg ttgttgttgt tgttgttgct attacttggg ccgctcccgc ggcatatgga 720 ggttcccagg ctaggagttg aatcggagct gtagccaccg gcctacgcca gagccacagc 780 aacgcgggat ccgagccgcg tctgcaacct acaccacagc tcacggcaac gctggatcct 840 taacccactg agcaagggca gggaccgaac ccgcaacctc atggttccta gtcggattcg 900 ttaaccactg cgccacgacg ggaactccca aagtatattt tgaatcaagc caccctttga 960 gccaggccac ctcctcttta tggtcatgag aacggtctgc ccttgtcctt ttctccattc 1020 tccacactca gcacccagat gggtctctct aggtgaagtt ggatcagggg attctccagc 1080 tttagatgct ttttgggatt ccccacccta ctttccatac ctttccaggt tctgactgcc 1140 tctgcccccc ttctgactgc ctagcaccag ccactcaagg gggacagtgt cagtcactat 1200 ttttttcttg tccaggtttt ttgcttttgt ttttttcaaa cacgagcagc tctttctctt 1260 gtctgcctgg tatagatgct gtttccaaaa tattctcatc ccttctcacg gcccttgtca 1320 tcctttccca tcctatcttc atcccttggg aagctctaaa gtcatctccc caaattgaag 1380 ggtgactaaa gagtttccca gaaggaaaaa ctgagtttcc aactactaca ctgacttgca 1440 agaaatgttt gtgtcttcat taaatgaaaa agaaaaaact gtaacaagat atgagaaaat 1500 acagaaagga aataataaga ctagaaaagt caaatatata gtgaaggtgt tgcatcaaac 1560 acttaaataa actagtacag atgttaaaag actaaattat atagttgaag gatagctgtg 1620 aagatgtaaa ctatgacatc taaaacacaa aatgttggcg ttcccgtcac ggcacagtgg 1680 aaacgaatcc gactaggaac catgaggttg caggttcaat tcctgccctt gctcagtggg 1740 ttaaggatcc ggtgttgccg tgagctgtgg tgtaggtagc caatgaggct tggatcccgc 1800 gttgctgtgg ctctggtgta ggccggtggc tacagctccg attcgacccc tagcctggga 1860 acctccatat gccgcgggag cggccctaaa aagacaaaag accaaaaaaa aaaaaaaaca 1920 aaaaacccac aaaatgttgg gaatcagtcc tctactagta ttatgttatt gtcaagtttt 1980 ccttttatgt ctgttaatat ttgcgttcta gatgtaggtg ctctgatatc gtgtgcatat 2040 atgttaacca atgttatgtc ttcctctggt attgatccct ttgttattat gtaatgccct 2100 actttatctt ttgttacatt ctttgtttat gagtattgct gatatgtggc tagctgccac 2160 acttttcttg tcctttccat ttacaataaa tatctttcta tctccaccca aattaaagta 2220 ctccgcaacc tgttattcca cccagcatcc cttccctctt caactacaat ttcatgcagc 2280 gatcaagaaa tacgaatgta ccgactgttt gccacttgtg tgggtgcatt ggggaaaagc 2340 tgggtgggaa gtggcagagc ctagattata aaggaccagg gtgagagttc ccattgtggc 2400 tcagctgaaa tgaatctgac tagcatccat gaggacgaag gtttgatccc tggcctcaat 2460 cagtgggtta aggatctggc gttgctgtcc gtgagttgtg gtgtagttcg cagacaaggc 2520 gtggacttag tgtggctgtg gctgtggcat aggctagtgg ctacagctct gattcgaccc 2580 ctagcctggg aatctctata tgctgtgagt gtggccctaa aatttaaatg aaattaaata 2640 aaggaccagg gtatattttt ctttgaggat aaggtacata gtcagtatat cagggacagt 2700 agacctagga aacggatgct tcctctagtc tgtgatgcga ggtggggcat ctgagttggg 2760 ggcggctgga gcccttaggg accattaact aaacccgtca ctctcccaca tctcggtgga 2820 ccttgggatc agtcaggatg cttccccttt gagcctcaaa atggccttag tatccttccc 2880 aacccagacg gccctgtcag ttcattgact tggctaattt gccagtgtag gcctatgcaa 2940 attaaggtag aacgcactcc ttagcgctcg ttgactattc atcaactttt ccttttagaa 3000 aagatattgg tataagcact tcttaaaaaa ccatattcca ctctgggtgt atttaatcta 3060 attttccctt ctccttttct tttcccag 3088 <210> 7 <211> 89 <212> DNA <213> Sus scrofa <400> 7 gagaaaataa tgaatgtcaa aggaagagtg gttctgtcaa tgctgcttgt ctcaactgta 60 atggttgtgt tttgggaata catcaacag 89 <210> 8 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Forward for left arm <400> 8 gaattcatga ttattatcct cccaagc 27 <210> 9 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Reverse for left arm <400> 9 catgaattcc attattttct cctggg 26 <210> 10 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Forward for GT intron 4 and partial exon 5 <400> 10 gtcgacctgt caatgctgct tg 22 <210> 11 <211> 24 <212> DNA <213> Artificial Sequence <220> Reverse for GT intron 4 and partial exon 5 <400> 11 ctcgagccag aacaaagaac cttc 24 <210> 12 <211> 6858 <212> DNA <213> Sus scrofa <400> 12 gtcgacctgt caatgctgct tgtctcaact gtaatggttg tgttttggga atacatcaac 60 aggtagttat gaaacatgat gaaatgatgt tgatgaaagt ctcctctaat ctcctagtta 120 tcagccaagt caccagcttg cattaaaagt aggattcact gacaccgtaa agaaagcatt 180 ccagagagtt gccgttgtgg ctcaggggca gcaaacccaa ttaggatcca agaggaggtg 240 ggtttgatcc ctggccttgc tctttggctt aaggatccgg cattgccgtg acctgtggtg 300 taggttgcag atgcagctcg gatctggcat tgctgtggct gtggcgtagg ctggtggctt 360 cagctccagt ttgaccccta gcctgggaac ttccatatcc cacacttgcg gccctaaaaa 420 gcaaagaaag aaagaaaata ttctaccctt cctgtatccc tgagccctta aataccgtct 480 ttaaagtcat tagatcttca agtaccttcc agctaattaa ttatcttcct tcctgccatg 540 ttgccattgt cctgattttt atacctctgc agttctgggt agactagagc cagaaataat 600 aaggtcatgt taagaccaag atataatatt aaattattta tatgaccaga tatggaagtt 660 accttgagaa ctttcagaca ggaattccat gagaaataca ccctgatttt tgcaatccta 720 aaatatttgc agagtttaaa ggaacaactc aagttgttga cttttgctgc aaaacacact 780 gagtcgctgg tgattcattt gtgcctggct aaacttttgg gtgttttgtc tctttttttt 840 taactctgga aagcaaaatg aattaaacat ttctgagttt tcaaattcat cagtggattc 900 accccaaata tttgagctgc ttctttgctt ttggaaacta cgatgccttg gagattccag 960 ctggagacgc ttctgacaga aagaaatgtc tgcaagcagc tacaaaaatg catgatggct 1020 ttgacttaag aggcattgat accgcttggc ctttctttca aaaaggccac cttacaactt 1080 ggcctgaagg cattcccgtg gtggtgcagc ggaaaatgaa tctgactagg aaccccgagg 1140 ttgtgggttc aatccctggc cttgctcagt ggcttaagga tcgggtgttg aagtaagctg 1200 tggtgtagat tgcagacgca gcttggatct ggtgttgctg tggctttggt gtaggccggc 1260 agctacagct ccacttggac ccctagtctg ggaacctcca tatgccacag gtgtggccct 1320 aaaaggaaaa aagacaacaa acaaacaaaa aaccaaaaaa caacttggcc tggagagcta 1380 tgtcatcacc attgatattt tgatgggtag tgttttagta gcccctcaag ttcaggatga 1440 tggcctggat taacgttaga atgtctctta aattctaaga cttgatgagc cagcaggacc 1500 attttggcca cttagaaagg aactgcatct tcaggtccat cagtagaagg aggattctct 1560 agggagttct ctcttagctc agcgggttca agaattcagt cttgtcccta cagcagctca 1620 ggtgactgct atggcttggc tttgatccct ggcccaggaa tttctgcatg ctgcaggtgc 1680 agccaaaaaa aaaaaaaaaa aggaggaggt ggattcccta gaataagaag ctgtcattcc 1740 tttggatgct tcatagatct aaccacttct ggaacagtta ttccctctca ttctgaagaa 1800 ctcattttaa gaaaaacaag acgagctaga gagtgaacaa atgtctacaa accaaccttt 1860 tcgaattgag gaaactgtgg tacttcctct gaagaaaaga tgacagcgtt ggatgcagag 1920 accctggggc tcccttaggt acttgaggac tgaggagata ttctcagtgg aggctggagc 1980 taggctgcct ggggttggtc ctgtgccacc acttccctcc tctgtgactt tgggcaagtt 2040 tccctatctt taaaaatggg gatgatagta gtacctgctt catagggttg ttggataaaa 2100 taagttgtga ataaagcact aagggcaacg tacttagtaa gcgctggctg ccatcaccac 2160 caccactatc accatctgtc cggagggcag cataggacag gagattttgg caaatagaag 2220 gaagagttct aggagttccc gttgtggtgc aggggaaatg aatccaacta ggaactagga 2280 ggtttcgggt tcaatcccgc gcctcgctca gtgggttaag gatccagtgt tgccatgagc 2340 tgtggtgtag attgcagaca tggctaggat ctggagttgc tatggctgtg gtgtaagctg 2400 gcagctgtag ctcggattct acccctagcc tgggaatttc cgtatgccac aggtttggcc 2460 ctacaaagaa aaaagaaaaa gaaaaagaaa aaattctagg ggctgaaaga atctaacaga 2520 agagcaagtt ccccatgggg ttcctgacct gagttgagat gcttgtgtag gcaaccttca 2580 agctctgaac tcttgattgt tttgaattgc agccagagtt atacttccat attttgggta 2640 cttcacaaaa ttaaaacaca gaagccaaag gcccagaagt gcatattggt gctggcctcc 2700 cataaagagg gttgttttgc agtgctgggc acactctctc ttcacagtaa ctggagcaga 2760 ttctggctgc tcttcagggc cgtagtctgg cacccagact gcagccacat cattcttcaa 2820 tgtgaggaat ctatttgaac atctgcaagg ggtttaaaag gcaggagatt ctttgccacc 2880 ttgtgaattg gtctgaggtg agctgagggc actaacctta gacaggtggg tagcactgta 2940 gctaaagagg attacaggag ttcctgttgt ggcttagtgg taacaaatcc aactagtatc 3000 catgaggatt caggttcgat ccctggcctc gctcagtggg tcaggtatcc ggtgttgctg 3060 tggctgtggt gtaggctggc agcttcagct ctgatttgac ccctagcctg ggaacttcca 3120 tgtgctgtag gtaaggccct tgaaaaaaaa aaaaaagaga tttacaaaat aactccatca 3180 aacacataca gctgtttaag aatgtcatcc aggacagcat ttggttaaag gctagatgaa 3240 aaaaaaaaaa aaaatcttag aattttattt atttattttt tctttttagg gccagacctg 3300 tggcctatgg aaatgcctgg gctaggggtg gaatcagagc tgcctacacc acagccatag 3360 ccacgccaga tccaagcccc gtctgtgacc tacaccacag ctcatggcaa acactggatc 3420 cttaatccac tgagtgaggc caggaattga acccacattc tcatggatgc tagttgggtt 3480 cttaagccac tgagccacaa gcttagaatt ttagaggtgg aagaaacttt aagagctata 3540 ataaagtaat gatggtgatg gtgattttga tgttagcggc tactagttat tgagtgtttg 3600 cttgtgccag gaactccact gttcattccc tcctgttttt aaaacagccc tggaaggtca 3660 gtgttagtcc acatttctag atgaggaata ctgagtttcc acaatattaa atgtgaacgt 3720 tcaaggtcac atttttagga agatttaggt ccagggctgt ctgacttggg taacctgggt 3780 aacccttcct ttagtcaagg tttccattgt tcaggcgatg gacaagtagg tgaaatgcct 3840 taacagtgaa cttatgtcta acttctaatt agaactcaga tcttctgatt catcatctgg 3900 ggctccttct ggagctggtt gttcatgcca aatgctgcga ggggtacagt gtgcgccaag 3960 gagaatccct accctcaagg ggttatgctg tagatggagc aggcagaggt acccatgaaa 4020 gcccaacaac acaggctaga aggaggatgt cagagagaga gagcaaagga acgtgagagt 4080 tcagggaggg caagattatg tttggcttgg agatggatct atgttttgca tttatttttt 4140 tggggggggg gtctttttgc tacttcttgg gctgctcccg aggcatatgg aggttcccag 4200 gctaggggtc taattggagc cgcagccacc agcctatgcc agagccacag caacgcagga 4260 tctgagccgc gtctgcaacc ttcaccacag ctcacggcaa cgccagatcg ttaacccact 4320 gagcaagggc agggaccgaa cctgcaacct catggttcct agtcagattc gttaagcact 4380 gcgccacgac gggaactccc tcatttagaa atatttattg agcacctact gtatgccagg 4440 cattgtgcta ggttcatacc aaagaaggct cagaagagat ggcatccgag ctgtgccttg 4500 aaggatgaat atgtgttaaa tgccgtacac ttcagggtgg ttgttgctgt gacctgaggt 4560 gttgaaggct tctgggaaag gagggtgaga tgaggaagag ggaggggtta ctaaaaagat 4620 gggacgaggt ggcaaatcca aatctataaa ttgatgccct gagtgcctcg gcaggagggt 4680 ggggctcctg agtgctgggt ggcacgggcc ctccccctcc tcttgcccct ttcccttccc 4740 cctcttgtag gatctgaagt cagattcccc aggttcaaat actgtttctt cccttagcag 4800 tatgaccttg ggcaaaataa tttattgcct ctgtccctct gaggaggaat agaacctcct 4860 tcattgactg ttattagaat ttaatgagct aatacatgtc agttgcttag aaaggtcccc 4920 agccaactat tagctattat gaatattatc agatcaatag acagatttag aaacaaggga 4980 ctttagagct gggtccatgg gtactgagct tagaggggaa accataggtg gtaggaaggc 5040 atgtatttca ttcctaccag gagatgtgga ctcccagctg gggcagaagg cagagggagg 5100 agatcggggc tttggcagaa tctcaaacaa atattagtgg ttagtggttt tttgtttctg 5160 ttttaagaga tgagggcagg cgtttccgat gtggcgcagt ggaaacgaat ctgactagta 5220 tccatgagga tgcaggttcc atccctggac tcactcagtg agttaaggat ccggcattgc 5280 cgtgagttgt ggtgtaggtc acagacacag ctcagatctg gcattgctgt ggctgtggtg 5340 taggctagca gctgtagctc caattcaacc cctagcctgg gaacttccac atgccgcagg 5400 tgcaacccca aaagataaat gaataaataa ataaatatgc gaccttcctt tcttggggcc 5460 tttgcatgtt tttctctctg ttaggcacac tcttgctaat ccctcttcac tgggcctcct 5520 atgtatcctt cagaactcag ctaaaacatc atcccctccc ctggggagcc ttcgaggtct 5580 tcctgttaag tgctcctatg ctttcttgga gttttgaagt cctataatga tgtgtttatc 5640 aaaatagggt ccaccctccc tgccagcttc tctacaccac agacacatgg tgtctgtttc 5700 agtcaacact gtatgtctgg cacttgacat gtaacgcatg ctcagcaggt atttgttgaa 5760 tgaatggagg cggtctgcta gagtcgtcat atatttactg atcccgtctt gtaggatggt 5820 ctcactgctt ttgttagctt aagaagtacc tttttttttt ttttttaatg gccacaccca 5880 tggcatatag aaattccacg aaggaaggaa gaaagaaaga aagaaagaag gaaattcctg 5940 ggtcagggat tgaatccaag ccacaggtgc aacctgagct gcagttgcgg caacaccaca 6000 tcttttaacc cactgtgctg ggccagggat catacctgtg catctacagc gacccaagcc 6060 acggcagtca gattcttttt ctgcctttct ttctttcttt tctttttttt tttttttttt 6120 ttttgtcttt ttgccttttc taggtgcggc atatggaggt tcccaggcta ggtgtcgaat 6180 cagagctgta gacgccggcc taaaccacag ccacagcaac acaggatcca agccttgtct 6240 gtgacctaca ccacagctca cggcaacgct ggatccttaa cccgctgagc gaggccaggg 6300 attgaacccg caacctcatg gttcttagtt ggattcgtta accactgagc catgatggga 6360 actcctgcag tcagattctt aacccactat gccacagcag gaactcctag aagtgccctt 6420 tgaggctact ctgtagacag ctttgagcca gcgaggcaag acctgttttt ctggaggaag 6480 ataaatcctg ggtgagggat gggtgggctg tggtcttcct gggacccatc tctggagcct 6540 ctctccctca gcaaagccac cttggacaat aagagctgcc atctattttt tttttcttta 6600 aactaagatt tgatattttc cagagacctc cctcccaccg tttgatctga gtaattctga 6660 aatgacgaga gtcccgtgat atcatttttt cgatctcgaa ggtggaaacc tgggagtagc 6720 cacaacccag gctctcagct cagcctaggg tttcaatgat aatgattgca aaatagcttt 6780 tctctgcatt ccaagtaaca tgatatgttt ttatttccat ttgcttttag cccagaaggt 6840 tctttgttct ggctcgag 6858 <210> 13 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Forward for cloning neo <400> 13 ctaggaattc cttcgcgatg tacgggcc 28 <210> 14 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Reverse for cloning neo <400> 14 cagtgatatc cttatcgcta tcgattcaca c 31 <210> 15 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Forward for validation of homologous recombination of GT left arm <400> 15 ggtctgccta catctctctg atgaac 26 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse for validation of homologous recombination of GT left arm <400> 16 ggcatcagag cagccgattg 20 <210> 17 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Forward for validation of homologous recombination of GT right          arm <400> 17 ccttctatcg ccttcttgac gag 23 <210> 18 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Reverse for validation of homologous recombination of GT right          arm <400> 18 ctagagatat ttggaattca aagcacttac 30 <210> 19 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward for cloning IRES <400> 19 gatatccgcg tcgagcatgc 20 <210> 20 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Reverse for cloning IRES <400> 20 ctcgagtcgc cattcaggc 19 <210> 21 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Forward for cloning CD59 <400> 21 ctgagtcgac atgggaatcc aaggagggtc tg 32 <210> 22 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Reverse for cloning CD59 <400> 22 ctgagcggcc gcttagggat gaaggctcca gg 32 <210> 23 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Forward for CMAH partial intron 3 and partial exon 4 <400> 23 gagctccatg acaggaactc ctgagatgaa tatc 34 <210> 24 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Reverse for CMAH partial intron 3 and partial exon 4 <400> 24 gcggccgctc gttgcctctc tccaggtatt aag 33 <210> 25 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Forward for CMAH partial exon 6 and partial intron 6 <400> 25 gatatcacca tcaatactga tcaatgtttt ctg 33 <210> 26 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Reverse for CMAH partial exon 6 and partial intron 6 <400> 26 gtcgacagca ggaaaaactg agtctgcagt tc 32 <210> 27 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Forward for cloning CD39 <400> 27 tgcaatttcg cctcttggc 19 <210> 28 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> Reverse for cloning CD39 <400> 28 gaggatccct ataccatatc tttccagaaa tatgaagg 38 <210> 29 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Forward for cloning CD39 + polyA <400> 29 gactcgagca tggaagatac aaaggagtct aacg 34 <210> 30 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Reverse for cloning CD39 + polyA <400> 30 gcattaatta agatttaaca aaaatttaac gcg 33 <210> 31 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Forward for validation of homologous recombination of CMAH <400> 31 accccagctc acaatgagca acaccagat 29 <210> 32 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Reverse for validation of homologous recombination of CMAH <400> 32 agagggaccc caatgacagt aacagcaga 29  

Claims (31)

(1) a first region having a nucleic acid sequence of 2 to 4 kb in length homologous to the antigenic determinant synthetic gene; (2) positive selection marker genes; (3) internal ribosome entry site (abbreviated as 'IRES'); (4) genes encoding complement inhibitory proteins or thrombosis inhibitory proteins; And (5) encoding a complement inhibitory protein or a thrombosis inhibitory protein while simultaneously removing a heterologous antigenic decider synthetic gene in the cell which includes a second region having a nucleic acid sequence of 6 to 8 kb in length homologous to the antigenic determinant synthetic gene. Gene targeting vectors that can target genes. The gene targeting vector of claim 1, wherein the antigenic determinant synthetic gene is a gene encoding alpha 1,3-galactosyltransferase (hereinafter, abbreviated as GT). The gene targeting vector of claim 1, wherein the antigenic determinant synthetic gene encodes CMP-acetylneuraminic acid hydroxylase (hereinafter, abbreviated as 'CMAH'). The gene targeting vector of claim 1, wherein the antigenic determinant gene is a gene derived from a mammal. The genetic targeting vector of claim 4, wherein the mammal is a cow, a sheep, a goat, a pig, a horse, a rabbit, a dog, or a monkey. The gene targeting vector of claim 5, wherein the mammal is a pig. The gene targeting vector of claim 6, wherein the pig is a miniature pig. The gene targeting vector of claim 1, wherein the first region is a left arm including a 5 ′ untranslated region. The gene targeting vector of claim 1, wherein the first region has a size of 2 to 4 kb. The gene targeting vector of claim 1, wherein the second region is a right arm including an open reading frame of the antigenic determinant synthetic gene. The gene targeting vector of claim 1, wherein the size of the second region is 5 to 8 kb. The method of claim 1, wherein the positive selection marker is neomycin phosphotransferase (Neo), hygromycin phosphotransferase (Hyg), histidinol dehydrogenase (histidinol dehydrogenase) , hisD), puromycin (Puro) and guanine phosphosribosyltransferase (Gpt) is selected from the group consisting of gene targeting vectors. The gene targeting vector of claim 1, wherein the complement inhibitory protein is CD59, DAF, MCP or CD46. The gene targeting vector of claim 1, wherein the thrombosis inhibiting protein is ATP-diphosphohydrolase (hereinafter abbreviated as 'NTPDase'). The gene targeting vector of claim 14, wherein the NTPDase is CD39. The gene targeting vector of claim 1, further comprising a negative selection marker gene at the 3 ′ end. The method of claim 16, wherein the negative selection marker gene is Herpes simplex virus-thymidine kinase (HSV-tk), hypoxanthine phosphoribosyl transferase (Hprt), cy A gene targeting vector, characterized by encoding a protein selected from the group consisting of cytosine deaminase and diphtheria toxin. (1) a first region having a nucleic acid sequence of 2 to 4 kb in length homologous to the antigenic determinant synthetic gene; (2) genes encoding complement inhibitory proteins or thrombosis inhibitory proteins; (3) a positive selection marker gene operably linked to a promoter; And (4) encoding a complement inhibitory protein or a thrombosis inhibitory protein while simultaneously removing the heterologous antigenic receptor synthetic gene in the cell which includes a second region having a nucleic acid sequence of 5 to 8 kb in length homologous to the antigenic determinant synthetic gene. Gene targeting vectors that can target genes. 19. The gene targeting vector of claim 18, wherein the promoter is selected from the group consisting of a CMV promoter, an EF1a promoter and an SV40 early promoter. The gene targeting vector according to claim 18, wherein the antigenic determinant synthetic gene is a gene encoding alpha 1,3-galactosyltransferase (hereinafter, abbreviated as 'GT'). The gene targeting vector of claim 18, wherein the antigenic determinant synthetic gene encodes CMP-acetylneuraminic acid hydroxylase (hereinafter abbreviated as 'CMAH'). 19. The gene targeting vector of claim 18, wherein the antigenic determinant gene is a gene derived from miniature pig. The method of claim 18, wherein the positive selection marker is neomycin phosphotransferase (Neo), hygromycin phosphotransferase (Hyg), histidinol dehydrogenase (histidinol dehydrogenase) , hisD), puromycin (Puro) and guanine phosphosribosyltransferase (Gpt) is selected from the group consisting of gene targeting vectors. 19. The gene targeting vector of claim 18, wherein the complement inhibitory protein is CD59, DAF, MCP or CD46. 19. The gene targeting vector of claim 18, wherein the thrombosis inhibiting protein is ATP-diphosphohydrolase (hereinafter, abbreviated as 'NTPDase'). The gene targeting vector of claim 25, wherein the NTPDase is CD39. A cell line transformed with the gene targeting vector of any one of claims 1 to 26. The cell line according to claim 27, which is CMAHKI # 1 deposited with accession number KCTC 11433BP. A non-human clone produced by nuclear transfer of the transformed cell line of claim 27. 30. The non-human clone of claim 29, which is a miniature pig. 30. A method for producing a heterologous organ for transplantation, wherein the immune rejection reaction is removed after breeding the non-human clone animal according to claim 29.

Images