WO2009069986A2 - Genetically-modified cell line for producing cloned miniature pigs for xenotransplantation and method for preparing the same. - Google Patents

Abstract

The present invention relates to a targeting vector specific for a xenoantigen determining antigen synthesizing gene capable of knock-outing the xenoantigen determining antigen synthesizing gene using homologous recombination technologies and integrating human complement inhibiting gene and/or human CD39 gene at the loci of the xenoantigen determining antigen synthesizing gene.

Description

[DESCRIPTION]

[invention Title]

GENETICALLY-MODIFIED CELL LINE FOR PRODUCING CLONED MINIATURE PIGS FOR XENOTRANSPLANTATION AND METHOD FOR PREPARING THE SAME

[Priority]

The present application claims benefit of Korean Patent Application No. 2007-123823 filed on November 30, 2007, the disclosure of which is hereby incorporated by reference into this application.

[Technical Field]

The present invention relates to a genetically modified cell line for producing cloned miniature pigs for xenotransplantation and a method for preparing the same.

[Background Art]

According to the report by Korean Network for Organ Sharing, the number of patients who are waiting for transplantation reaches approximately 20,000 every year in

Korea. However, donation cannot even reach 10% by the demand. In the USA, patients waiting for organ transplantation increase by one patient every 16 minutes but 11 of those in the waiting list die every day without getting chance of transplantation. Accordingly, the advance of biotechnology paves the way to development of techniques for xenotransplantation.

Constant development of animal genetics facilitates disclosure of functions of each gene and production of commercially useful transgenic animals by inserting or deleting a gene of interest. As for the method for producing transgenic animals, random gene integration using microinjection or viral infection, etc, and gene targeting which targets a particular gene of interest using embryonic stem cells or somatic cells have been used.

Microinjection is a classic method of inserting a heterologous DNA into the pronucleus of a fertilized egg and has been widely used for the production of transgenic animals (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, transgenic offsprings originated from fertilized eggs introduced with a heterologous DNA take only 2-3%, indicating a very low efficiency (Clark et al., Transgenic Res., 9: 263-275, 2000), and it is impossible to regulate the location of the heterologous gene insertion and to eliminate a particular endogenous gene. Viral infection is also a method widely used for animal gene manipulation (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 a particular heterologous gene to be incorporated is introduced into an animal gene by using a viral vector, but still has a problem of inability of locating the heterologous gene to a specific site and eliminating a particular endogenous gene. In addition, the maximum size of a heterologous gene to be inserted is limited to 7 kb and a virally encoded protein is also a major interference (Wei et al., Annu. Rev. Pharmacol. Toxicol., 37: 119-141, 1997; Yanez et al., Gene Ther. , 5 (2) : 149-159, 1998) .

To overcome the above problems, gene targeting techniques which enable deletion or insertion of a gene of interest can be used. This techniques were first used in the study of gene function using mouse embryonic stem cells. Genetically-modified transgenic animals can be produced by inserting mouse embryonic stem cells in which a particular gene is targeted using homologous recombination into an embryo at the blastocyst stage. Numbers of gene-targeted mice have been generated by applying the gene targeting method (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. MoI. 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 the gene targeting techniques are applied in domestic animals, an animal bioreactor facilitating mass-production of a therapeutic protein or an animal model capable of being used for xenotransplantation, which is prepared by eliminating a gene related to immune rejection or by over-expressing a gene of interest can be prepared. Consequently, enormous industrial and economical profits may be expected.

To produce gene targeted animals, embryonic stem cells have been regarded to be essential factors. Although some cell lines similar to embryonic stem cell have been reported in domestic animals including pigs and cows, the utility of embryonic stem cells of the domestic animals has been restricted (Doetschman et al., Dev. Biol., 127(1) : 224-227, 1988; Stice et al., Biol. Reprod. , 54(1) : 100-110, 1996; Sukoyan et al., MoI. 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, general somatic cells have been proposed as nucleus donor cells for gene targeting, by which cloned large animals could be produced (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) .

Miniature pigs are considered to be the best organ donor for xenotransplantation because they have organs in similar sizes to human organs and physiological characteristics of the animals are also similar to those of human and mass-production of their organs can be possible.

Success of transplantation of pig organs depends on a series of immunorejections (hyperacute, acute vascular, cell-mediated, and chronic immunorejections) . It has been reported that hyperacute immunorejection occurring within a few minutes after transplantation can be overcome by eliminating the gene involved in synthesis of alpha-1,3- galactosyl antigenic determinant and by over-expressing a human complement regulatory gene.

Particularly, somatic cell-cloned pig in which alpha-

1, 3-galactosyltransferase (referred as ¹GT' hereinafter) was heterozygously deleted was generated for the first time by PPL Co., England in 2002 (Yifan Dai et al., Nat. Biotechnology, 20: 251-255, 2002) . GT is the gene causing hyperacute immunorejection right after xenotransplantation. Once this gene is knocked-out , animal disease model for xenotransplantation not causing in vivo hyperacute immunorejection can be developed. In 2003, said company succeeded in producing somatic cell-cloned animals in which GT gene was homozygously deleted, making a great progress in production of animal disease models for organ transplantation to solve the problem of organ shortage (Carol J. Phelps, Science, 299: 411-414, 2003) .

In 2005, an organ originated from GT knock-out cloned pig was transplanted in a monkey. As a result, the monkey survived 2-6 months without hyperacute immunorejection after transplantation (Kenji Kuwaki et al., Nature Medicine, 11(1) : 29-31, 2005) . However, although GT gene was deleted, human complement genes are activated by xenoantigen via other pathways, which can cause serious immunorejection after organ transplantation (Tanemura, M. et al., Biochem. Biophys. Res. Commun. , 235: 359-364, 1997; Komoda, H. et al., Xenotransplantation, 11: 237-246, 2004) . To overcome the above problem, a method has been established to produce cloned pigs over-expressing decay- accelerating factor (referred as 'DAF' hereinafter), membrane co-factor protein (referred as 'MCP' hereinafter) and human complements such as CD59 after deletion of GT gene (Yoichi Takahagi, Molecular Reproduction and Development 71: 331-338, 2005 Cozzi, Eb et al . , Transplant Proc, 26: 1402-1403, 1994; Fodor, W. L. et al., Proc. Natl. Acad. Sci. USA 91: 11153-11157, 1994; Adams, D. H. et al., Xenotransplantation, 8: 36-40, 2001) .

N-glycolylneuraminic acid (referred as 'Neu5Gc' hereinafter) antigenic determinant presented in every mammals except human can cause immunorejection after 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 (referred as 'Neu5Ac' hereinafter) by cytidine monophospho-N- acetylneuraminic acid hydroxylase (referred as ^ΛCMAH' hereinafter) .

Complement is a protein complex (C1-C9) composed of proteins involved in immune response. Once antigen- antibody complex is formed, a complement binds to cell membrane of bacteria to make a hole, which is called complement fixation or the complement binds to the antigen- antibody complex to promote phagocytosis, which is called opsonization. Regulator proteins capable of regulating the activity of complements have been identified. Thus, it has been tried to regulate the functions of complements by suppressing the activation of complements or by accelerating degradation of activated complements. DAF found in cell membrane of a host can interrupt the interaction between C2 and C4b. MCP promotes degradation of C4b to prevent the activation of complements in a host cell, and as a result destruction of a host cell can be prevented. CD59 existing on the surface of a host cell interrupts the binding of C7, C8 and C5b6 to prevent the formation of membrane attack complex.

In addition to said complement regulation genes, thrombosis developed after xenotransplantation can be suppressed by over-expression of a gene encoding human CD39 (US20080003212A; Karren, M. D., The Journal of Clinical Investigation, 113: 1440-1446, 2004) .

According to previous reports, ectopic expression of a foreign gene results in disorder of embryo development, which particularly is severe to neuronal system developed mainly in the late stage of embryo development and right after birth (Gao et al . , Neurochem Res., 24(9) : 1181-1188, 1999) . To overcome the above problems, the present inventors constructed targeting vectors for over-expressing human complement regulatory gene or thrombosis suppressor gene by deleting GT gene involved in alpha-1, 3-galactosyl antigenic determinant synthesis or CMAH gene involved in Neu5Gc antigenic determinant synthesis and inserting human complement regulatory gene or thrombosis suppressor gene capable of suppressing side effects of xenotransplantation such as immunorejection or thrombosis at the loci where the GT or the CMAH gene were removed. And further, the present inventors succeeded in the preparation of a somatic cell line in which the GT gene or the CMAH gene was knocked-out and instead a complement regulatory gene or a thrombosis suppressor gene were inserted at the loci where the GT or the CMAH gene were removed by using the above vector. The targeting vectors and the transgenic cell line prepared according to the method of the present invention facilitating the regulation of expressions of genes involved in immunorejection can be effectively used for the production of cloned pigs for xenotransplantation.

[Disclosure] [Technical Problem]

It is an object of the present invention to provide a gene targeting vector facilitating knock-out of xenoantigenic determinant synthetic gene and insertion of complement regulatory gene or thrombosis suppressor gene.

It is another object of the present invention to provide a transgenic cell line transfected with said gene targeting vector and over-expressing complement regulatory gene or thrombosis suppressor by knocking out xenoantigenic determinant synthetic gene and inserting complement regulatory gene or thrombosis suppressor gene.

It is also an object of the present invention to provide a non-human cloned animal prepared by nuclear transplantation using the transgenic somatic cell line.

It is further an object of the present invention to provide a preparation method of organs for xenotransplantation free from immunorejection containing the steps of raising the non-human cloned animal and extracting organs therefrom.

[Description of Drawings]

The application of the preferred embodiments of the present invention is best understood with reference to the accompanying drawings, wherein:

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

Figure 2 illustrates the nucleic acid sequence of polynucleotide (SEQ. ID. NO: 12) containing the entire sequence of intron 4 and a part of exon 5 (underlined) of miniature pig GT gene. Figure 3 is a diagram illustrating the targeting of GT gene by using pGTKOneoTK vector.

Figure 4 is a diagram illustrating the targeting of GT gene by using pGTKOIRESKITK vector.

Figure 5 is a diagram illustrating the construction process of pGTKOneoTK vector.

Figure 6 is a diagram illustrating the construction process of pGTKOIRESKITK vector.

Figure 7 is a diagram illustrating the construction process of pGTKOIRESCD59KITK vector. Figure 8 is a diagram illustrating the targeting of GT gene by using pGTKOIRESCD59KITK vector.

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

Figure 10 is a diagram illustrating the construction process of pCMAHKODT vector.

Figure 11 is a diagram illustrating the construction process of pCMAHKOCD39KIDT vector.

Figure 12 is a diagram illustrating the targeting of CMAH gene by using pCMAHKOCD39KIDT vector. Figure 13 is a photograph illustrating the pESF cell before the insertion of pCMAHKOCD39KIDT and the cell line resulted from the insertion of CMAH gene.

Figure 14 is a photograph illustrating the targeting of CMAH gene in said cell line, confirmed by PCR: V, targeting vector; WG, cell line in which gene is not inserted; #1 and #2, targeted cell lines;

#3-#7, cell lines in which gene is inserted randomly; and M, marker DNA.

[Best Mode] Definition of Terms

Hereinafter, the terms used in this invention are described.

In this invention, the term "gene targeting vector" indicates a vector capable of deleting or inserting a particular gene of interest from or into the loci where the particular gene exist in the genome, which can add homologous nucleotide sequence to the particular gene to induce homologous recombination thereon. The terms "vector" and "vector cassette" are regarded as same terms herein and they may be circular or linear form.

In this invention, the term "homologous" indicates homology between region 1 or region 2 and corresponding nucleic acid sequence, which shows at least 90% homology and more preferably 95% homology.

In this invention, the term "xenoantigenic determinant" indicates a region recognized as an antigen by immune system of a recipient of xenotransplantation, which is exemplified by galactose-αl, 3-galactose (referred as

'αGal' hereinafter) and N-glycolylneuraminic acid

(referred as 'NeuδGc' hereinafter) . The term

"xenoantigenic determinant synthetic gene" indicates a gene encoding an enzyme synthesizing xenoantigenic determinant. The most representative examples of said enzyme are alpha-1, 3-galactosyltransferase (referred as

¹GT' hereinafter) involved in αGal biosynthesis and CMP- acetylneuraminic acid hydroxylase (referred as 'CMAH' hereinafter) involved in NeuδGc biosynthesis.

In this invention, the term "selection marker" is a material used for selecting a transfected cell with the gene targeting vector, which can be markers making phenotypes distinguishable such as drug resistance, auxotrophy, cytotoxic agent resistance or surface protein expression, etc, and can include positive selection markers and negative selection markers.

The term "positive selection marker" indicates a marker facilitating positive selection of those cells expressing a particular marker after treated with a selective agent. And the term "positive selection marker gene" indicates the gene encoding said positive selection marker. For example, neomycin phosphotransferase (referred as 'neo' hereinafter) is used for the selection of stable transgenic cells from eukaryotic cells after culturing them in a medium supplemented with neomycin.

The term "negative selection marker gene" is a marker gene facilitating negative selection by eliminating those cells with random insertion. This marker plays a role in preventing transfection by random insertion by killing the marker specific cells selectively.

The term "internal ribosome entry site (referred as 'IRES' hereinafter)" indicates a nucleic acid sequence that allows for translation initiation in the middle of a messenger RNA (mRNA) sequence instead of 5' -cap structure during protein synthesis in eukaryotes. Multiple proteins having different functions can be produced from one mRNA by using the IRES.

The term "complement regulatory protein" indicates a protein preventing complements activated by a serial reaction of complement activation proteins which are critical barriers for xenotransplantation from being bound to membrane of a host cell to induce acute immunorejection.

The term "thrombosis suppressor protein" indicates a protein inhibits thrombosis. After allograft or xenograft, platelets of a recipient begin to interact with endothelial cells of a transplanted organ, followed by activation.

Then, the platelets are coagulated in endothelium of the transplanted organ to develop thrombosis making the transplantation to fail. ATP-diphosphohydrolase (referred as 'NTPDase' hereinafter) is the most representative thrombosis suppressor protein.

The term "transformation" indicates a process of making host DNA replicable as extrachromosomal factor or by integrating in chromosome. Transformation method includes any methods which insert nucleic acid molecules into an organism, a cell, a tissue or an organ and proper standard techniques are hired according to host cells by those in the art. To distinguish the transformation of eukaryotes by plasmid or non-plasmid naked DNA from the transformation as tumorigenesis of a cell, it is often called as ' transfection ' , but in this invention, both terms are used as same.

Detailed Description of the Invention

Hereinafter, the present invention is described in detail .

In a preferred embodiment of the present invention, the present invention provides a gene targeting vector facilitating knock-out of internal xenoantigenic determinant synthetic gene and inserting a gene encoding complement regulatory protein or thrombosis suppressor protein comprising sequentially:

(1) region 1 containing 2-4 kb long nucleic acid sequence corresponding to xenoantigenic determinant synthetic gene;

(2) a positive selection marker gene;

(3) an internal ribosome entry site (referred as 1IRES' hereinafter); (4) a gene encoding complement regulatory protein or thrombosis suppressor protein; and

(5) region 2 containing 6-8 kb long nucleic acid sequence corresponding to the xenoantigenic determinant synthetic gene. In a preferred embodiment of the present invention regarding the gene targeting vector, the region 1 corresponds to left arm containing 5' -non-translation region and the region 2 corresponds to right arm containing exon including open reading frame (ORF) . The region 1 is preferably 2-4 kb long and more preferably 2.5-3.5 kb . The region 2 has the nucleic acid sequence of 5-8 kb long and more preferably has the nucleic acid sequence of 5.5-7.5 kb.

In another preferred embodiment of the present invention regarding the gene targeting vector, the xenoantigenic determinant synthetic gene is the gene encoding alpha-1, 3-galactosyltransferase (referred as 'GT' hereinafter) or CMP-acetylneuraminic acid hydroxylase (referred as ¹CMAH' hereinafter), but not always limited thereto . In the case that the xenoantigenic determinant synthetic gene is GT, the region 1 preferably contains a part of or the whole intron 2 and exon 4 of GT gene and more preferably contains 3.1 kb long nucleic acid sequence represented by SEQ. ID. NO: 1. The region 2 contains intron 4 and a part of or the whole exon 5 of GT gene and more preferably contains 6.9 kb long nucleic acid sequence represented by SEQ. ID. NO: 2. In the case that the xenoantigenic determinant synthetic gene is CMAH, the region 1 contains a part of intron 3 and a part of or the whole exon 4 of CMAH gene and more preferably contains 3.5 kb long nucleic acid sequence represented by SEQ. ID. NO: 3. The region 2 contains a part of exon 6 and a part of or the whole intron 6 of CMAH gene and more preferably contains 6 kb long nucleic acid sequence represented by SEQ. ID. NO: 4. The GT gene targeted by said vector is preferably mammal originated GT, for example GT originated from a cow, a sheep, a goat, a pig, a horse, a rabbit, a dog, and a monkey, more preferably pig originated GT gene and most preferably miniature pig originated GT gene, but not always limited thereto.

In addition, the CMAH gene targeted by said vector is preferably mammal originated CMAH gene, for example CMAH originated from a cow, a sheep, a goat, a pig, a horse, a rabbit, a dog, and a monkey, more preferably pig originated CMAH gene and most preferably miniature pig originated CMAH gene, but not always limited thereto.

The vector of the present invention includes a positive selection marker gene.

The positive selection marker gene of the present invention can be selected from the group consisting of neomycin phosphotransferase (neo) , hygromycin phosphotransferase (hyg) , histidinol dehydrogenase (hisD) , puromycin (puro) and guanine phosphosribosyltransferase

(gpt) , but not always limited thereto. The positive selection marker gene can be knocked-in to be translated using start codon of endogenous xonoantigenic determinant synthetic gene without a promoter by promoter trap method or can be operably linked to a constitutive promoter such as cytomegalovirus (CMV) promoter. The gene targeting vector of the present invention includes an internal ribosome entry site (IRES) . The IRES activates translation of downstream of 5' -end methylguanosine cap (CAP structure) independently and contains a sequence translating two cistrons (open reading frame) from a single transcript in animal cells. IRES is known to provide an independent ribosome entry site for the translation of ORF located on the downstream. The positive selection marker of the invention, neo gene, and human complement regulatory gene or thrombosis suppressor gene inserted by MCS can be expressed as a single mRNA, but can be expressed as two different proteins by IRES binding ribosome .

The vector of the present invention includes a gene encoding complement regulatory protein and/or thrombosis suppressor protein as a knock-in gene. The complement regulatory protein herein is exemplified by CD59, DAF, MCP or CD46, and preferably CD59 is used but not always limited thereto. DAF (decay accelerating factor) inhibits the activation of complement by interrupting the binding between C2 and C4b. MCP (membrane cofactor protein) inhibits the activation of complement in a host cell by accelerating degradation of C4b. CD59 (homologous restriction factor) inhibits the formation of membrane attack complex (MAC) by interrupting the binding of C7 to C8 and C5b6. In the meantime, the thrombosis suppressor protein is preferably ATP-diphosphohydrolase (referred as 'NTPDase' hereinafter) and more preferably CD39, but not always limited thereto. To generate animals for efficient organ transplantation, the vector of the present invention may be designed for simultaneous knock-in of both genes respectively encoding a complement regulatory protein and thrombosis suppressor protein. At this time, the complement regulatory protein and the thrombosis suppressor protein can be produced as a fusion protein or can be translated into two different proteins using IRES after being transcribed as one cistron.

The vector of the present invention can also include a negative selection marker. The negative selection marker can be selected from the group consisting of herpes simplex virus-thymidine kinase (HSV-tk) , hypoxanthine phosphoribosyl transferase (Hprt) , cytosine deaminase and diphtheria toxin (DT) , and particularly, thymidine kinase or diphtheria toxin is preferred, but not always limited thereto. The negative selection marker can be located in the 5' -end of region 1 or in the 3' -end of region 2.

Once the gene targeting vector of the present invention is targeted in a host cell, the endogenous xenoantigenic determinant synthetic gene in the host cell genome is homologously recombinated with the targeting vector, and as a result nucleotide sequence is replaced. The positive selection marker gene and the gene encoding complement regulatory protein or thrombosis suppressor protein of the vector is expressed by the promoter of endogenous xenoantigenic determinant synthetic gene or the promoter inserted in 5' -end of the positive selection marker gene in order to induce expression of the positive selection marker. The former is a kind of a promoter trap vector. Not only promoter trap but also enhancer trap and exon trap can be used to trap functional genes (genes to be express) transcribed in a cell. In another preferred embodiment of the present invention, this invention provides a gene targeting vector facilitating knock-out of internal xenoantigenic determinant synthetic gene and targeting a gene encoding complement regulatory protein or thrombosis suppressor protein, comprising sequentially:

(1) region 1 containing 2-4 kb long nucleic acid sequence corresponding to the xenoantigenic determinant synthetic gene;

(2) the gene encoding complement regulatory protein or thrombosis suppressor protein;

(3) a positive selection marker gene operably linked to a promoter; and (4) region 2 containing 5-8 kb long nucleic acid sequence corresponding to the xenoantigenic determinant synthetic gene.

In a preferred embodiment of the present invention regarding the gene targeting vector, the region 1 corresponds to left arm containing 5' -non-translation region and the region 2 corresponds to right arm containing exon. The region 1 is preferably 2-4 kb long and more preferably 2.5-3.5 kb. The region 2 has the nucleic acid sequence of 5-8 kb long and more preferably has the nucleic acid sequence of 5.5-7.5 kb. In another preferred embodiment of the present invention regarding the gene targeting vector, the xenoantigenic determinant synthetic gene is the gene encoding alpha-1, 3-galactosyltransferase (referred as 'GT' hereinafter) or CMP-acetylneuraminic acid hydroxylase (referred as 'CMAH' hereinafter), but not always limited thereto .

In the case that the xenoantigenic determinant synthetic gene is GT, the region 1 preferably contains a part of or the whole intron 2 and exon 4 of GT gene and more preferably contains 3.1 kb long nucleic acid sequence represented by SEQ. ID. NO: 1. The region 2 contains intron 4 and a part of or the whole exon 5 of GT gene and more preferably contains 6.9 kb long nucleic acid sequence represented by SEQ. ID. NO: 2. In the case that the xenoantigenic determinant synthetic gene is CMAH, the region 1 contains a part of intron 3 and a part of or the whole exon 4 of CMAH gene and more preferably contains 3.5 kb long nucleic acid sequence represented by SEQ. ID. NO: 3. The region 2 contains a part of exon 6 and a part of or the whole intron 6 of CMAH gene and more preferably contains 6 kb long nucleic acid sequence represented by SEQ. ID. NO: 4.

The GT gene targeted by said vector is preferably mammal originated GT, for example GT originated from a cow, a sheep, a goat, a pig, a horse, a rabbit, a dog, and a monkey, more preferably pig originated GT gene and most preferably miniature pig originated GT gene, but not always limited thereto.

In addition, the CMAH gene targeted by said vector is preferably mammal originated CMAH gene, for example CMAH originated from a cow, a sheep, a goat, a pig, a horse, a rabbit, a dog, and a monkey, more preferably pig originated CMAH gene and most preferably miniature pig originated CMAH gene, but not always limited thereto. The vector of the present invention includes the gene encoding complement regulatory protein and/or thrombosis suppressor protein as a knock-in gene. The complement regulatory protein herein is exemplified by CD59, DAF, MCP or CD46, and preferably CD59 is used but not always limited thereto. DAF (decay accelerating factor) inhibits the activation of complement by interrupting the binding of C2 and C4b. MCP (membrane cofactor protein) inhibits the activation of complement in a host cell by accelerating degradation of C4b. CD59 (homologous restriction factor) inhibits the formation of membrane attack complex (MAC) by interrupting the binding of Cl, C8 and C5b6. In the meantime, the thrombosis suppressor protein is preferably ATP-diphosphohydrolase (referred as 'NTPDase' hereinafter) and more preferably CD39, but not always limited thereto. To generate animals for efficient organ transplantation, the vector of the present invention was designed for simultaneous knock-in of both genes respectively encoding complement regulatory protein and thrombosis suppressor protein. At this time, the complement regulatory protein and the thrombosis suppressor protein can be produced as a fusion protein and can be translated later into two different proteins using IRES after being transcribed as one cistron.

A promoter can be additionally included in 5' -end of the gene encoding complement regulatory protein and/or thrombosis suppressor protein. And at this time, the promoter can be general eukaryotic promoters such as CMV promoter, EFIa promoter, and SV40 early promoter, and SV40 early promoter is more preferred. A promoter is not necessarily included, though. And if a promoter is not included, transcription is performed by the promoter of endogenous xenoantigenic determinant synthetic gene.

The vector of the present invention also includes said positive selection marker gene. The vector of the present invention additionally includes said negative selection marker.

In a preferred embodiment of the present invention, pGTKOIRESCD59KITK vector capable of eliminating pig alpha-

1, 3-galactosyltransferase gene and at the same time targeting human complement regulatory gene was constructed as a targeting vector of alpha-1, 3-galactosyltransferase gene. In the process, pGTKOneoTK and pGTKOIRESKITK vectors capable of targeting alpha-1, 3-galactoxyltransferase gene were also constructed. First, to obtain homologous region with nucleic acid sequence of alpha-1, 3-galactosyltransferase included in the pGTKOIRESCD59KITK vector, a part of nucleotide sequence of miniature pig alpha-1, 3-galactosyltransferase gene targeted in miniature pig alpha-1, 3-galactosyltransferase gene was identified.

To determine the region of alpha-1, 3- galactosyltransferase gene of a miniature pig defined as region 1 and region 2, primers were constructed based on pig nucleotide sequences already reported (Kihiro Koike et al., Transplantation, 70: 1275-1283, 2000), followed by PCR using Taq polymerase capable of proofreading.

The pGTKOneoTK vector contains region 1 of 3.1 kb in size comprising a part of intron 2 and the whole nucleotide sequence of exon 4 of alpha-1, 3-galactosyltransferase gene. It also includes region 2 of 6.9 kb in size comprising the entire exon 4 and a part of exon 5 nucleotide sequence. The vector also contains neo gene as a positive selection marker, poly A gene region and TK gene region as a negative selection marker. The pGTKOIRESKITK vector contains IRES gene region and multi-cloning site (MCS) in addition to the same composition as the pGTKOneoTK vector. A gene encoding a target protein is cloned into MCS of the pGTKOIRESKITK vector, which is then expressed in a host cell. In this invention, a gene encoding complement regulatory protein is inserted.

The pGTKOIRESCD59KITK vector is prepared by inserting CD59 gene that is a kind of genes encoding complement regulatory gene into MCS of the pGTKOIRESKITK vector. The pGTKOIRESCD59KITK is the vector capable of knocking out pig alpha-1, 3-galactosyltransferase gene and at the same time capable of targeting human complement regulatory gene, and therefore it is the most appropriate vector for the present invention. Particularly, the vector contains 3.1 kb long region 1 comprising a part of intron 2 and the entire nucleotide sequence of exon 4 of pig alpha-1, 3- galactosyltransferase gene in 5' -end; neo gene operably linked to SV40 early promoter as a positive selection marker; multi-cloning site having IRES, Sac I and Not I restriction enzyme sites; and 6.9 kb long region 2 comprising poly A, the entire nucleotide sequence of intron 4 and a part of exon 5. The vector additionally contains a gene encoding complement regulatory protein in the restriction enzyme site (Figure 8) . Construction of said vector can be performed by the conventional gene recombination technique well-known to those in the art, and particularly site-specific DNA cleavage and ligation can be performed with a general enzyme well-known and accepted in this field.

In another preferred embodiment of the present invention, the present inventors constructed pCMAHKOCD39KIDT vector capable of knocking out pig CMAH gene and at the same time capable of targeting a gene encoding human thrombosis suppressor protein as a gene targeting vector of CMAH. In the process, pCMAHKOneoDT vector capable of targeting CMAH gene was also constructed.

First, to obtain homologous region with nucleic acid sequence of CMAH included in the pCMAHKOCD39KIDT vector, a part of nucleotide sequence of miniature pig CMAH gene targeted in miniature pig CMAH gene was identified.

To determine the region of CMAH gene of a miniature pig defined as region 1 and region 2, primers were constructed based on pig nucleotide sequences already reported (Kihiro Koike et al . , Transplantation, 70: 1275- 1283, 2000), followed by PCR using Taq polymerase capable of proofreading.

The pCMAHKOneoDT vector contains region 1 of 3.5 kb in size comprising a part of intron 3 and a part of exon 4 of CMAH gene. It also includes region 2 of 6.0 kb in size comprising a part of exon 6 and a part of intron 6. The vector also contains neo gene region as a positive selection marker, poly A gene region and DT gene region as a negative selection marker. Next, the gene encoding CD39, the thrombosis suppressor protein, was inserted in front of neo gene of the pCMAHKOneoDT vector, resulting in the construction of pCMAHKOCD39KIDT vector. The gene encoding CD39 was overlapped with the start codon of CMAH gene, in order for the vector to be designed to be capable of knocking out pig CMAH gene and to be capable of targeting human CD39 gene at the same time.

The pCMAHKOCD39KIDT is the vector capable of knocking out pig CMAH gene and at the same time capable of targeting human CD39 gene, and therefore it is the most appropriate vector for the present invention. Particularly, the vector contains 3.5 kb long region 1 comprising a part of intron 3 and a part of exon 4 of pig CMAH gene; the gene encoding CD39 protein linked to the frame of the start codon of CMAH gene; neo gene region operably linked to SV40 early promoter; 6.0 kb long region 2 comprising poly A, a part of exon 6 and a part of intron 6; and DT gene as a negative selection marker.

Construction of said vector can be performed by the conventional gene recombination technique well-known to those in the art, and particularly site-specific DNA cleavage and ligation can be performed with a general enzyme well-known and accepted in this field.

In another preferred embodiment of the present invention, the present invention provides a transformant transfected with said gene targeting vector.

The transfection can be performed by inserting nucleic acid molecules into an organism, a cell, a tissue or an organ, and as informed to those in the art, appropriate standard technique can be selected according to host cells. For example, transfection can be performed by the method selected from the group consisting of electroporation, CaPO₄ precipitation, CaCl₂ precipitation, microinjection, PEG method, DEAE-dextran method, cationic liposome method, and lithium acetate-DMSO method, but not always limited thereto.

The targeting vector constructed in a preferred embodiment of the present invention, pCMAHKOCD39KIDT, was introduced in miniature pig ear tissue originated fibroblasts (pESF) to construct two gene targeted cell lines (CMAHKI#1, CMAHKI#2) . The CMAHKI#1 cell line was deposited at KCTC (Korean Collection for Type Cultures, 111 Gwahangno, Yuseong-gu, Daejeon, Korea, Korea Research Institute of Bioscience and Biotechnology) on November 24, 2008 under Accession No: KCTC 11433BP. The depository authority above is appointed according to Budapest Treaty regarding on deposit of microorganisms.

Therefore, the present inventors guarantee said depository authority meets the rules and conditions set by Budapest Treaty.

The present invention also provides a non-human cloned animal prepared by nuclear transplantation using the transgenic somatic cell line. The non-human cloned animal herein is exemplified by sheep, goat, pig, dog or any other mammals in similar sizes to human, and a pig is more preferred and a miniature pig is most preferred.

Nuclear transplantation used for the generation of cloned animals can be performed by one of the methods well known to those in the art and preferably one of the methods described in USβ, 781, 030B, USβ, 603, 059B, US6, 235, 969B, US7, 355, 094B, US7 , 071, 372B, KR862298B, KR500412B, KR807644B, JP4153878B, USβ, 700, 037B, US7, 291, 764B, USβ, 258, 998B, USβ, 548, 741B, WO03/089632A, and US7 , 371, 922B, and if a pig is targeted, one of the methods described in KR500412B, KR807644, JP4153878B, USβ, 700, 037B, US7,291,764B, USβ, 258 , 998B, USβ, 548, 741B, WO03/089632A, and US7,371,922B is preferred. These patent documents are all incorporated by references entirely in this document. In addition, the present invention provides a preparation method of organs for xenotransplantation containing the steps of raising the non-human cloned animals and extracting necessary organs therefrom. The organs herein can be extracted by surgical operation after raising cloned donor animals under regulated feed conditions considering gender, age, weight, height, etc. The organs can be transplanted in a recipient right after the extraction or quickly stored in a refrigerator.

[Mode for Invention]

Practical and presently preferred embodiments of the present invention are illustrative as shown in the following Examples.

However, it will be appreciated that those skilled in the art, on consideration of this disclosure, may make modifications and improvements within the spirit and scope of the present invention.

Example 1: Construction of GT gene targeting vector <!-!> Identification of the entire sequence of exon 4 and a part of intron 2 of miniature pig alpha-1,3- galactosyltransferase gene Alpha-1, 3-galactosyltransferase gene of a pig comprises 9 exon and the entire exon nucleotide sequences and a part of intron nucleotide sequences have been reported (Kihiro Koike et al., Transplantation, 70: 1275- 1283, 2000) . In this invention, a part of alpha-1,3- galactosyltransferase gene nucleotide sequence was identified to use for the targeting of alpha-1,3- galactosyltransferase of miniature pig. To determine a part of intron 2 and the entire exon 4 nucleotide sequence of miniature pig alpha-1, 3-galactosyltransferase gene, the forward primer represented by SEQ. ID. NO: 3 (5 ' -TCCATGAAC- AACTTCGATTGCATG-3 ' ) and the reverse primer represented by SEQ. ID. NO: 4 (5'-TGGCTGATAACTAGGAGATTAGAGGAG-3') were constructed based on the whole pig nucleotide sequences reported so far (Kihiro Koike et al., Transplantation, 70: 1275-1283, 2000), followed by PCR using Taq DNA polymerase

(Taq polymerase, Intron Biotechnology, Korea) capable of proofreading as follows: predenaturation at 94 °C for 2 minutes, denaturation at 94°C for 20 seconds, annealing at 60°C for 45 seconds, polymerization at 68°C for 4 minutes, 35 cycles from denaturation to polymerization, and final extension at 68 °C for 10 minutes.

The PCR product was cloned into T-easy vector (Promega, USA), followed by sequencing with 4 clones. As a result, a common nucleotide sequence was identified (SEQ. ID. NO: 5, Figure 1) . Intron 2 region of miniature pig alpha-1, 3-galactosyltransferase was represented by SEQ. ID.

NO: 6 and intron 4 region was represented by SEQ. ID. NO: 7.

The left arm region used for the targeting vector was prepared by PCR with the forward primer represented by SEQ. ID. NO: 8 (5'-GAATTCATGATTATTATCCTCCCAAGC-3' ) ligated to

EcoR I restriction enzyme site and the reverse primer represented by SEQ. ID. NO: 9 (5'-CATGAATTCCATTATTTTCTCCT-

GGG-3') using Taq DNA polymerase (Intron Biotechnology,

Korea) as follows; predenaturation at 94°C for 2 minutes, denaturation at 94°C for 20 seconds, annealing at 60°C for 45 seconds, polymerization at 68 °C for 3 minutes 30 seconds, 35 cycles from denaturation to polymerization, and final extension at 68°C for 10 minutes.

The obtained clone was digested with EcoR I, which was used as the left arm composing region 1 (SEQ. ID. NO: 1) of the targeting vector (Figures 5 and 6) .

<l-2> Identification of the entire sequence of intron 4 and a part of exon 5 of miniature pig alpha-1, 3- galactosyltransferase gene

To determine the entire intron 4 nucleotide sequence and a part of exon 5 of miniature pig alpha-1, 3- galactosyltransferase gene, the forward primer represented by SEQ. ID. NO: 10 (5'-GTCGACCTGTCAATGCTGCTTG-3') and the reverse primer represented by SEQ. ID. NO: 11 (5 ' -CTCGAGCC- AGAACAAAGAACCTTC-3 ') were constructed based on the whole pig nucleotide sequences reported so far (Kihiro Koike et al., Transplantation, 70: 1275-1283, 2000), followed by PCR using Taq DNA polymerase (Taq polymerase, Intron Biotechnology, Korea) capable of proofreading as follows: predenaturation at 94 °C for 2 minutes, denaturation at 94°C for 20 seconds, annealing at 60°C for 45 seconds, polymerization at 68°C for 7 minutes, 35 cycles from denaturation to polymerization, and final extension at 68 °C for 10 minutes.

The PCR product was cloned into T-easy vector

(Promega, USA), followed by sequencing with 4 clones. As a result, a common nucleotide sequence was identified (SEQ.

ID. NO: 14, Figure 2) . The obtained clone was digested with Sal I and Xho I, which was used as the right arm composing region 2 (SEQ. ID. NO: 2) of the targeting vector

(Figures 5 and 6) .

<l-3> Construction of pGTKOneoTK vector The left arm having EcoR I restriction enzyme site and the right arm having Sal I and Xho I restriction enzyme sites prepared in Examples <1-1> and <l-2> were inserted respectively into EcoR I and Sal I sites of pBst-TK vector containing the negative selection marker TK (thymidine kinase) gene (Figure 5) . TK gene degrades gancyclovir, once it is included in a medium, to produce a cytotoxic enzyme product. The cell line in which TK gene is deleted by homologous recombination (Figures 3, 4 and 8) can grow in a medium containing gancyclovir, but if the targeting vector is inserted by random insertion, TK gene might be co-expressed, indicating that the cell line cannot grow in the medium containing gancyclovir. The positive selection marker neo gene proceeded to PCR along with poly A region and pcDNA3.1 vector (Invitrogen, USA) with the forward primer represented by SEQ. ID. 13 (5'-CTAGGAATTCCTTCGCGA- TGTACGGGCC-3 ' ) ligated to EcoR I restriction enzyme site and the reverse primer represented by SEQ. ID. NO: 14 (5'- CAGTGATATCCTTATCGCTATCGATTCACAC-3 ' ) ligated to EcoR V restriction enzyme site using Taq DNA polymerase (Intron Biotechnology, Korea) capable of proofreading as follows: predenaturation at 94 °C for 2 minutes, denaturation at 94°C for 20 seconds, annealing at 60°C for 45 seconds, polymerization at 68°C for 1 minute 30 seconds, 35 cycles from denaturation to polymerization, and final extension at 68 °C for 10 minutes.

The PCR product was inserted into EcoR I and EcoR V sites of pBst-TK vector (Figure 5) . As a result, pGTKOneoTK vector was constructed. After linearization with the restriction enzyme Not I or Xho I, it can be introduced in vivo. TK gene was deleted by homologous recombination. Targeting was confirmed by PCR with the primer set composed of the forward primer represented by SEQ. ID. NO: 15 (5 ' -GGTCTGCCTACATCTCTCTGATGAAC-3' ) and the reverse primer represented by SEQ. ID. NO: 16 (5 ' -GGCATCAG- AGCAGCCGATTG-3' ) and the other primer set composed of the forward primer represented by SEQ. ID. NO: 17 (5 ' -CCTTCTAT- CGCCTTCTTGACGAG-3' ) and the reverse primer represented by SEQ. ID. NO: 18 ( 5 ' -CTAGAGATATTTGGAATTCAAAGCACTTAC-3 ' ) using Taq DNA polymerase (Takara, Japan) . PCR using the primer set each represented by SEQ. ID. NO: 15 and NO: 16 was performed as follows: predenaturation at 94 °C for 2 minutes, denaturation at 94°C for 20 seconds, annealing at 60°C for 45 seconds, polymerization at 68°C for 3 minutes, 35 cycles from denaturation to polymerization, and final extension at 68°C for 10 minutes. PCR using the primer set each represented by SEQ. ID. NO: 17 and NO: 18 was performed as follows: predenaturation at 94 °C for 2 minutes, denaturation at 94°C for 20 seconds, annealing at 60°C for 45 seconds, polymerization at 68 °C for 8 minutes, 35 cycles from denaturation to polymerization, and final extension at 68°C for 10 minutes (Figure 3) .

Internal ribosome entry site (IRES) , multicloning site (MCS, Sal I, Not I), and poly A region proceeded to

PCR with the forward primer represented by SEQ. ID. NO: 19 (5'-GATATCCGCGTCGAGCATGC-3') ligated to EcoR V restriction enzyme site of pIRES (Clontech, USA) vector and the reverse primer represented by SEQ. ID. NO: 20 (5'-CTCGAGTCGCCATTCA- GGC-3 ' ) ligated to Xho I restriction enzyme site using Taq DNA polymerase (Intron Biotechnology, Korea) capable of proofreading as follows: predenaturation at 94 °C for 2 minutes, denaturation at 94°C for 20 seconds, annealing at 60°C for 45 seconds, polymerization at 68°C for 1 minute 30 seconds, 35 cycles from denaturation to polymerization, and final extension at 68°C for 10 minutes. The positive selection marker neo gene and the human complement regulatory gene to be inserted in MCS can be expressed as one mRNA by IRES but can be expressed as two different proteins by IRES binding ribosome (Louis Marie Houdebine et al., Transgenic research, 8: 157-177, 1999) .

The PCR product was cloned into pBst-TK vector digested with Hind III, treated with Klenow enzyme and digested with Sal I again (Figure 6) . As a result, pGTKOIRESKITK vector cassette having MCS (Sal I, Not I) where human complement regulatory gene could be inserted was constructed. After linearization with the restriction enzyme Xho I, it could be introduced into somatic cells. TK gene was deleted by homologous recombination (Figure 4) .

<l-4> Construction of pGTKOIRESCD59KITK vector cassette CD59, the human complement regulatory gene was inserted into MCS {Sal I, Not I) site of pGTKOIRESKITK vector cassette (Figure 7) . To clone CD 59 gene, PCR was performed with the forward primer represented by SEQ. ID. NO: 21 (5 ' -CTGAGTCGACATGGGAATCCAAGGAGGGTCTG-3') ligated to Sal I restriction enzyme site and the reverse primer represented by SEQ. ID. NO: 22 (5'-CTGAGCGGCCGCTTAGGGATG- AAGGCTCCAGG-3' ) ligated to Not I restriction enzyme site using Taq DNA polymerase (Intron Biotechnology, Korea) capable of proofreading as follows: predenaturation at 94 °C for 2 minutes, denaturation at 94 °C for 20 seconds, annealing at 60°C for 45 seconds, polymerization at 68 °C for 40 seconds, 35 cycles from denaturation to polymerization, and final extension at 68°C for 10 minutes .

The PCR product was cloned into MCS site of pGTKOIRESKITK vector cassette digested with Sal I and Not I (Figure 7) . As a result, pGTKOIRESCD59KITK vector capable of deleting alpha-1, 3-galactosyltransferase gene and targeting CD59 gene was constructed. After linearization with the restriction enzyme Xho I, the pGTKOIRESCD59KITK vector could be introduced into somatic cells. TK gene was deleted by homologous recombination (Figure 8) . Targeting was confirmed by PCR with the primer set each represented by SEQ. ID. NO: 15 and NO: 16 and the other primer set each represented by SEQ. ID. NO: 17 and NO: 18 as show in Example <l-3> using Taq DNA polymerase (Takara, Japan) (Figure 8) . PCR conditions were also the same as described in Example <l-3>.

Example 2: Construction of CMAH gene targeting vector <2-l> Construction of pCMAHKOneoDT vector

To use a part of intron 3 and a part of exon 4 of miniature pig CMAH gene nucleotide sequence

(www.ncbi.nlm.nih.gov, NW_001886419.1) as the left arm of the targeting vector, PCR was performed with the forward primer represented by SEQ. ID. NO: 23 (5'-GAGCTCCATGACA-

GGAACTCCTGAGATGAATATC-3' ) ligated to Sal I restriction enzyme site and the reverse primer represented by SEQ. ID. NO: 24 (5'-GCGGCCGCTCGTTGCCTCTCTCCAGGTATTAAG-3^? ) ligated to Not I restriction enzyme site using Taq DNA polymerase (ExTaq polymerase, Takara, Japan) capable of proofreading as follows: predenaturation at 94 °C for 2 minutes, denaturation at 94 °C for 20 seconds, annealing at 60 °C for 45 seconds, polymerization at 72°C for 4 minutes, 35 cycles from denaturation to polymerization, and final extension at 72 °C for 10 minutes.

The PCR product was digested with Sac I and Not I, followed by insertion into Sac I and Not I sites of pBCKIDT (AU 2005256120 B2 ) vector (Figure 10) .

Particularly, to use as the right arm of the targeting vector, a part of exon 6 and a part of intron 6 of CMAH gene nucleotide sequence proceeded to PCR with the forward primer represented by SEQ. ID. NO: 25 (5 ' -GATATCAC- CATCAATACTGATCAATGTTTTCTG-3' ) ligated to EcoR V restriction enzyme site and the reverse primer represented by SEQ. ID. NO: 26 (5'-GTCGACAGCAGGAAAAACTGAGTCTGCAGTTC- 3 ' ) ligated to Sal I restriction enzyme site using Taq DNA polymerase (ExTaq polymerase, Takara, Japan) capable of proofreading as follows: predenaturation at 94 °C for 2 minutes, denaturation at 94°C for 20 seconds, annealing at 60^°C for 45 seconds, polymerization at 72°C for 6 minutes, 35 cycles from denaturation to polymerization, and final extension at 72 °C for 10 minutes. The PCR product was digested with EcoR V and Sal I, followed by ligation to EcoR V and Sal I sites of pBCKIDT vector. As a result, pCMAHneoDT vector was constructed (Figure 10) .

<2-2> Construction of pCMAHKOCD39KIDT vector

Human CD39 gene was ligated to Xho I and Pac I sites of pCMAHKOneoDT vector (Figure 11) . CD39 gene in human cDNA library (Openbiosystems, USA) was digested with Sal I and Kpn I, which proceeded to PCR with the forward primer represented by SEQ. ID. NO: 27 (5'-ATGCAATTTCGCCTCTTGGC- 3 ' ) ligated to Kpn I restriction enzyme site and the reverse primer represented by SEQ. ID. NO: 28 (5 ' -GAGGATCC- CTATACCATATCTTTCCAGAAATATGAAGG-3 ' ) ligated to BamR I restriction enzyme site using Taq DNA polymerase (Genotech, Korea) capable of proofreading as follows: predenaturation at 94°C for 4 minutes, denaturation at 94°C for 20 seconds, annealing at 57°C for 45 seconds, polymerization at 72 °C for 1 minute, 35 cycles from denaturation to polymerization, and final extension at 72 °C for 10 minutes. The PCR product was digested with Kpn I and BamΑ I, followed by ligation to pDsRed2-Cl vector (Clontech, USA) .

The CD39 gene ligated to pDsRed2-Cl vector proceeded, along with poly A, to PCR with the forward primer represented by SEQ. ID. NO: 29 (5'-GACTCGAGCATGGAAGATACAA- AGGAGTCTAACG-3 ' ) ligated to Xho I restriction enzyme site and the reverse primer represented by SEQ. ID. NO: 30 (5'- GCATTAATTAAGATTTAACAAAAATTTAACGCG-3') ligated to Pac I restriction enzyme site using Taq DNA polymerase (Genotech, Korea) capable of proofreading as follows: predenaturation at 94°C for 2 minutes, denaturation at 94°C for 20 seconds, annealing at 60°C for 45 seconds, polymerization at 72°C for 2 minutes, 35 cycles from denaturation to polymerization, and final extension at 72°C for 10 minutes. The PCR product was digested with Kpn I and BamR I, followed by ligation to Xho I and Pac I sites of pCMAHKOneoDT vector.

The ligated CD39 gene was located behind ATG of exon 4 of CMAH gene, so it could be expressed by CMAH gene promoter. As a result, pCMAHKOCD39KIDT vector capable of deleting CMAH gene over-expressing CD39 gene by targeting was constructed. After linearization with the restriction enzyme PmI I, the pCMAHKOCD39KIDT vector could be inserted into somatic cells. DT gene was deleted by homologous recombination (Figure 11). Targeting was confirmed by PCR with the forward primer represented by SEQ. ID. NO: 31 (5'- ACCCCAGCTCACAATGAGCAACACCAGAT-3' ) and the reverse primer represented by SEQ. ID. NO: 32 (5'-AGAGGGACCCCAATGACAGTAA- CAGCAGA-3') using LA Tag polymerase (Takara, Japan) as follows: pre-denaturation at 94 °C for 2 minutes, denaturation at 94 ^°C for 20 seconds, annealing at 60 °C for 45 seconds, polymerization at 72°C for 2 minutes, 35 cycles from denaturation to polymerization, and final extension at 72 °C for 10 minutes (Figure 12) .

Example 3: Generation of gene targeted cell line

<3-l> Generation of CMAH gene targeted cell line

The present inventors purified the pCMAHKOCD39KIDT plasmid constructed in Example 2 by using plasmid separation kits (QIAfilter Plasmid Midi kits, Qiagen, USA), followed by linearization with the restriction enzyme PmI I, Then the plasmid was introduced in ear tissue fibroblasts (pESF) originated from a miniature pig. The pESF cells were cultured in DMEM (Gibco, Invitrogen Corporation, USA) supplemented with 10% FBS (Hyclone, USA), 0.001% gentamycin (Gibco, Invitrogen Corporation, USA) and 1% MEM nonessential amino acid (Gibco, Invitrogen Corporation, USA) in a 37 °C 5% CO₂ incubator. The medium was replaced every 2-3 days. 5 g of the linearized DNA was introduced in 2X10⁶ pESF cells (subculture 2 or 3) by Nucleofector (Amaxa Biosystems, USA), followed by culture in the medium containing 300 g/ml of G418 (Gibco, USA) . After 12-14 days of culture, cell lines of 6 - 10 mm in diameter were observed (Figure 13) . Those cell lines were sub-cultured in a 96-well culture plate. When the cell lines grew 85- 95% in the culture plate, half of them were transferred to a 96-well plate for sub-culture. The other half was used for confirmation of targeting. The cells used for confirmation of targeting were lysed in lysis buffer comprising IM KCl, IM Tris pH 8.3, IM MgCl₂, 0.45% NP40, 0.45% Tween 20, and 10 g/ml Proteinase K at 55°C for 1 hour, followed by heat treatment at 100 °C for 5 minutes. PCR was performed using the forward primer represented by SEQ. ID. NO: 31 and the reverse primer represented by SEQ. ID. NO: 32 under the same conditions as described in Example <2-2> (Figure 14) . Homologous recombination was induced with 132 cell lines. As a result, 2 cell lines were confirmed to be targeted. The confirmed CMAH gene targeted cell lines were named CMAHKI#1 and CMAHKI#2, among which CMAHKI#1 was deposited at KCTC (Korean Collection for Type Cultures) on November 24, 2008 under Accession No: KCTC 11433BP.

[industrial Applicability] The gene targeting vector of the present invention can be effectively used for the construction of safer donor animals for xenotransplantation because the vector not only targets endogenous xenoantigenic determinant synthetic gene of an organ donor animal but also is capable of knocking in the gene encoding complement regulatory protein and/or thrombosis suppressor protein in the location of the targeted endogenous xenoantigenic determinant synthetic gene to express the gene, suggesting that the vector can overcome the problems of gene silence according to random insertion of a target gene and cancer development induced by the activation of oncogene.

Those skilled in the art will appreciate that the conceptions and specific embodiments disclosed in the foregoing description may be readily utilized as a basis for modifying or designing other embodiments for carrying out the same purposes of the present invention. Those skilled in the art will also appreciate that such equivalent embodiments do not depart from the spirit and scope of the invention as set forth in the appended claims.

Claims

[CLAIMS]

[Claim l]

A gene targeting vector capable of deleting endogenous xenoantigenic determinant synthetic gene and targeting a gene encoding complement regulation protein or thrombosis suppressor protein, the gene targeting vector comprises sequentially

(1) region 1 containing 2-4 kb long nucleic acid sequence corresponding to the xenoantigenic determinant synthetic gene;

(2) a positive selection marker gene;

(3) an internal ribosome entry site (referred as 'IRES' hereinafter);

(4) a gene encoding complement regulation protein or thrombosis suppressor protein; and

(5) region 2 containing 6-8 kb long nucleic acid sequence corresponding to the xenoantigenic determinant synthetic gene.

[Claim 2]

The gene targeting vector according to claim 1, wherein the xenoantigenic determinant synthetic gene is the gene encoding alpha 1, 3-galactosyltransferase (referred as 'GT' hereinafter) . [Claim 3]

The gene targeting vector according to claim 1, wherein the xenoantigenic determinant synthetic gene is the gene encoding CMP-acetylneuraminic acid hydroxylase (referred as 'CMAH' hereinafter) .

[Claim 4]

The gene targeting vector according to claim 1, wherein the xenoantigenic determinant synthetic gene is originated from a mammal.

[Claim 5]

The gene targeting vector according to claim 4, wherein the mammal is a cow, a sheep, a goat, a pig, a horse, a rabbit, a dog or a monkey.

[Claim 6]

The gene targeting vector according to claim 5, wherein the mammal is a pig.

[Claim 7]

The gene targeting vector according to claim 6, wherein the mammal is a miniature pig.

[Claim 8] The gene targeting vector according to claim 1, wherein the region 1 is the left arm containing 5' non- translation region.

[Claim 9]

The gene targeting vector according to claim 1, wherein the size of the region 1 is 2-4 kb.

[Claim 10] The gene targeting vector according to claim 1, wherein the region 2 is the right arm containing open reading frame of the xenoantigenic determinant synthetic gene.

[Claim 11]

The gene targeting vector according to claim 1, wherein the size of the region 2 is 5-8 kb.

[Claim 12] The gene targeting vector according to claim 1, wherein the positive selection marker is selected from the group consisting of neomycin phosphotransferase (neo) , hygromycin phosphotransferase {hyg) , histidinol dehydrogenase {hisD) and guanine phosphosribosyltransferase (gpt) . [Claim 13]

The gene targeting vector according to claim 1, wherein the complement regulatory protein is selected from the group consisting of CD59, DAF, MCP and CD46.

[Claim 14]

The gene targeting vector according to claim 1, wherein the thrombosis suppressor protein is ATP- diphosphohydrolase (referred as 'NTPDase' hereinafter) .

[Claim 15]

The gene targeting vector according to claim 14, wherein the NTPDase is CD39.

[Claim lβ]

The gene targeting vector according to claim 1, wherein the negative selection marker gene is additionally included at 3' -end.

[Claim 17]

The gene targeting vector according to claim 16, wherein the negative selection marker is selected from the group consisting of herpes simplex virus-thymidine kinase (HSV-tk) , hypoxanthine phosphoribosyl transferase (Hprt) , cytosine deaminase and diphtheria toxin (DT) .

[Claim 18]

A gene targeting vector capable of deleting endogenous xenoantigenic determinant synthetic gene and targeting a gene encoding complement regulation protein or thrombosis suppressor protein, the gene targeting vector comprise sequentially:

(1) region 1 containing 2-4 kb long nucleic acid sequence corresponding to the xenoantigenic determinant synthetic gene;

(2) a gene encoding complement regulation protein or thrombosis suppressor protein;

(3) a positive selection marker gene operably linked to promoter; and

(4) region 2 containing 5-8 kb long nucleic acid sequence corresponding to the xenoantigenic determinant synthetic gene.

[Claim 19]

The gene targeting vector according to claim 18, wherein the promoter is selected from the group consisting of CMV promoter, EFIa promoter, and SV40 early promoter.

[Claim 20] The gene targeting vector according to claim 18, wherein the xenoantigenic determinant synthetic gene is the gene encoding alpha 1, 3-galactosyltransferase (referred as 'GT' hereinafter) .

[Claim 21]

The gene targeting vector according to claim 18, wherein the xenoantigenic determinant synthetic gene is the gene encoding CMP-acetylneuraminic acid hydroxylase (referred as ¹CMAH¹ hereinafter) .

[Claim 22]

The gene targeting vector according to claim 18, wherein the xenoantigenic determinant synthetic gene is originated from a miniature pig.

[Claim 23]

The gene targeting vector according to claim 18, wherein the positive selection marker is selected from the group consisting of neomycin phosphotransferase (neo) , hygromycin phosphotransferase [hyg) , histidinol dehydrogenase {hisD) and guanine phosphosribosyltransferase {gpt) .

[Claim 24] The gene targeting vector according to claim 18, wherein the complement regulatory protein is selected from the group consisting of CD59, DAF, MCP and CD46.

[Claim 25]

The gene targeting vector according to claim 18, wherein the thrombosis suppressor protein is ATP- diphosphohydrolase (referred as 'NTPDase' hereinafter) .

[Claim 26]

The gene targeting vector according to claim 25, wherein the NTPDase is CD39.

[Claim 27] A cell line transformed with any one of the gene targeting vectors of claims 1 - 26.

[Claim 28]

The cell line according to claim 27, wherein the cell line is CMAHKI#1 deposited under Accession number KCTC 11433BP.

[Claim 29]

A non-human cloned animal generated by nuclear transplantation using the transgenic cell line of claim 27. [Claim 30]

The non-human cloned animal according to claim 29, wherein the animal is a miniature pig.

[Claim 31]

A preparation method of organs for xenotransplantation free from immunorejection comprising the steps of raising the non-human cloned animal of claim 29 and extracting organs therefrom.