KR101178946B1 - alpha 1,3-galactosyltransferase gene targeting vector - Google Patents

Abstract

The present invention can remove porcine alpha 1,3-galactosyltransferase gene by homologous recombination method, and alpha 1,3-galactosyltransferase gene that can be easily identified whether or not targeting including LacZ gene To a targeting vector.

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

Description

Alpha 1,3-galactosyltransferase gene targeting vector {alpha 1,3-galactosyltransferase gene targeting vector}

The present invention can remove porcine alpha 1,3-galactosyltransferase gene by homologous recombination method, and alpha 1,3-galactosyltransferase gene that can be easily identified whether or not targeting including LacZ gene To a targeting vector.

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

Microinjection has been widely used to produce transgenic animals by the classical method of inserting foreign DNA into the embryonic 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 rate of production of transformed eggs derived from fertilized eggs with foreign DNA inserted by microinjection was very low (Clark et al., Transgenic Res., 9; 263-275, 2000). The insertion position of the gene and removal of certain internal genes are impossible.

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

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

In order to produce genetically targeted animals, the use of embryonic stem cells has been considered an essential factor. However, although embryonic stem cell-like cell lines have been reported in livestock, including pigs and cattle, the use of embryonic stem cells in livestock has been limited to date (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 general somatic cells as nuclear donor cells could 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).

Meanwhile, alpha 1,3-galactosyltransferase gene is a gene that causes a rapid immune rejection reaction between xenotransplantation. If this gene is removed, it is possible to develop a disease model animal for xenotransplantation which has been rejected. Do. In 2002, UK-based PPL produced the world's first somatic cloned pig, in which the hetero 1,3-galactosyltransferase gene was heterozygous removed (Yifan Dai et al., Nat Biotechnology, 20; 251). -255, 2002). In 2003, the company succeeded in replicating somatic cells in which the alpha 1,3-galactosyltransferase gene was homozygous removed, thereby producing a disease model animal for organ transplantation that can solve the current organ shortage. Has made breakthrough advances for this (Carol J. Phelps, Science, 299; 411-414, 2003). Later, a targeting method using a promoter trap principle, which can more efficiently remove alpha 1,3-galactosyltransferase gene, was introduced (Sharon Harrison et al., Cloning and stem). cells, 6 (4); 327-331, 2004). The use of gene targeting vectors using promoter-traps is limited to genes that are actively transcribed in somatic cells into which they are introduced. Alpha 1,3-galactosyltransferase genes are found in most porcine derived cells. It is available because it is expressed actively. The gene trap method is a method of capturing (traping) genes (expressed genes) that are transcribed and function in a specific cell, and gene trap methods known to date include enhancer traps and exon traps as well as promoter traps. .

In 2005, cloned pig-derived organs from which alpha 1,3-galactosyltransferase was removed were transplanted into monkeys, which resulted in delayed survival until 2-6 months after transplantation without acute immune rejection (Kenji Kuwaki). et al., Nature medicine, 11 (1); 29-31, 2005).

In order to select cells targeted by such a gene targeting method, it has been common to analyze nucleic acid sequences by PCR reaction. However, this identification method is time-consuming and cumbersome because it is possible to grow cells to at least 96 wells. In addition, passaging of somatic cells used as nuclear donor cells is limited, and since multiple passages cause aging and damage of cells, it is important to establish healthy targeted cell lines at the beginning of passaging.

Therefore, the inventors of the present invention, while removing the alpha 1,3-galactosyltransferase gene of the miniature pig using the principle of gene target (alpha) 1,3-galactosyltransferase Since the LacZ gene can be expressed under the promoter of the gene, a targeting vector for easy gene targeting identification was produced, and a method for efficiently targeting the cell line was developed using the vector.

Since alpha 1,3-galactosyltransferase gene is actively expressed in most porcine somatic cells, it can be expected to express LacZ, a foreign gene introduced therefrom. When the LacZ gene is expressed, X-gal is decomposed to show a blue color. Therefore, the expression can be easily checked with the naked eye. Therefore, the vector of the present invention can primarily select whether the alpha 1,3-galactosyltransferase gene is targeted by X-gal treatment, which is much more than the PCR method used as a conventional target cell line selection method. In addition to the simplicity, it is possible to secure a target cell line in a healthy state in the early passages in that targeting can be determined as soon as the cell line is formed. Such a vector cassette can be usefully used for the production of cloned pigs for xenotransplantation.

The object of the present invention is (1) a 2-4 kb long nucleic acid sequence homologous to the pig's alpha 1,3-galactosyltransferase nucleic acid sequence, and thus the porcine alpha 1,3-galactosyltransferase gene. A first region comprising a portion of intron 8; (2) internal ribosomal initiation site; (3) LacZ gene region; (4) positive screening marker regions; (5) a portion of exon 9 of the pig alpha 1,3-galactosyltransferase gene as a nucleic acid sequence 0.5 to 2 kb long that is homologous to the pig alpha 1,3-galactosyltransferase nucleic acid sequence. It is to provide an alpha 1,3-galactosyltransferase gene targeting vector, characterized in that it has a second region comprising, wherein the (1) to (5) regions are configured in order from the 5 'end.

Another object of the present invention is to provide a transformant transformed with the alpha 1,3-galactosyltransferase gene targeting vector.

In one embodiment, the present invention relates to a pig's alpha 1,3-galactosyltransfer as (1) a swine alpha 1,3-galactosyltransferase nucleic acid sequence homologous to the pig's alpha 1,3-galactosyltransferase nucleic acid sequence. A first region comprising a portion of intron 8 of an aze gene; (2) internal ribosomal initiation site; (3) LacZ gene region; (4) positive screening marker regions; (5) a portion of exon 9 of the pig alpha 1,3-galactosyltransferase gene as a nucleic acid sequence 0.5 to 2 kb long that is homologous to the pig alpha 1,3-galactosyltransferase nucleic acid sequence. It relates to an alpha 1,3-galactosyltransferase gene targeting vector, characterized in that it has a second region comprising, and the (1) to (5) regions are configured in order from the 5 'end.

As used herein, the term "gene targeting vector" refers to a vector capable of removing or inserting a gene of interest into a specific gene position of a genome, and to target a gene such that homologous recombination occurs. Contains homologous base sequences. The gene targeting vector of the present invention is an alpha 1,3-galactosyltransferase targeting vector in which the LacZ gene is inserted into the alpha 1,3-galactosyltransferase gene on the genome in a 5 '→ 3' arrangement. The terms "vector" and "vector cassette" are used in the same sense and may be of circular or linear form.

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

The "first region" of the present invention is characterized by having a nucleic acid sequence of 2 to 4 kb in length that is homologous to the nucleic acid sequence of the alpha 1,3-galactosyltransferase gene. The length of the first region is an important factor in determining the gene targeting efficiency, preferably 2 to 4 kb, and more preferably 2.6 kb. In addition, the first region preferably comprises part of intron 8 of the porcine alpha 1,3-galactosyltransferase gene. Herein, part of the restriction enzyme SalI recognition site at the 5 'end of Intron 8 to the BamHI recognition site at the 3' end is preferable.

“Second region” of the invention is characterized by having a nucleic acid sequence of 0.5 to 2 kb in length that is homologous to the alpha 1,3-galactosyltransferase gene nucleic acid sequence. Preferably it has a length of 1.0kb. In this case, the first region corresponds to the upstream of the 5 ′ → 3 ′ sequence of the nucleic acid sequence of the alpha 1,3-galactosyltransferase gene, and the second region corresponds to the downstream. In addition, the second region preferably comprises a portion of exon 9 of the porcine alpha 1,3-galactosyltransferase gene. As for a part, the restriction enzyme BamHI recognition site of the 5 'end of exon 9 to the 3' end of exon 9 is preferable. Porcine alpha 1,3-galactosyltransferase genes are known to have exon 1 to exon 9 and some introns, most of which are associated with exon 9 sites, so if these sites are removed by targeting, It is expected to be able to remove the activity.

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

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

In addition, the vector of the present invention comprises a positive selection marker region. 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 screening marker" refers to a marker that allows positive selection by allowing only cells expressing the selection marker to survive in an environment in which a selective agent is treated, such as neomycin (Neo), hygromycin (hygromycin: Hyg), histidinol dehydrogenase gene (hiD) and guanine phosphosribosyltransferase (Gpt), and the like, but are preferably neomycin markers, but are not limited thereto.

In addition, the positive selection marker of the present invention may include a separate promoter in the vector for transcription of the selection marker gene, or may be transcribed through a promoter on the host cell genome. In a specific embodiment of the present invention, the vector of the present invention has a neomycin marker as a positive selection marker and comprises a phosphoglycerate kinase (PGK) promoter as a promoter for transcription of the neomycin marker gene. do.

In addition, the vector of the present invention may further include a negative selection marker. The term “negative selection marker” refers to a marker that allows negative selection to select and remove cells that have undergone random insertion. Herpes simplex virus-thymidine kinase (HSV- TK), hypoxanthine phosphoribosyl transferase (Hprt), cytosine deaminase, and Diphtheria toxin, and preferably the Herpes simplex virus, HSV) Cymidine kinase (TK) with a promoter, but is not limited thereto. The negative selection marker may be located at the 5 'end of the first region or at the 3' end of the second region.

In addition, the alpha 1,3-galactosyltransferase gene targeting vector of the present invention includes an internal ribosome entry site (IRES). IRES is the site of formation or entry of ribosomal complexes to initiate protein synthesis. The IRES element independently comprises a sequence that functionally activates the translational initiation of the 5'-terminal methylguanosnium cap (CAP structure) upstream of the gene and reads two cystrons (open reading frame) from a single transcript in animal cells. do. IRES elements are known to provide independent ribosomal entry sites for the reading of open reading frames located immediately downstream. The alpha 1,3-galactosyltransferase gene region and the inserted LacZ gene region of the present invention are expressed as one mRNA, but the LacZ gene can be expressed as an independent protein by ribosomes bound to IRES.

In addition, the alpha 1,3-galactosyltransferase gene targeting vector of the present invention may further include a polyA sequence at the 3 'end. polyA sequence means a DNA sequence that directs both transcription termination and polyadenylation of the initial RNA transcript. Efficient polyadenylation of recombinant transcripts is desirable because transcripts without polyA tails are unstable and rapidly degrade.

Meanwhile, the alpha 1,3-galactosyltransferase gene targeting vector of the present invention comprises a LacZ gene region. In the present invention, the term "LacZ gene region" is a gene region encoding beta-galactosidase. Beta-galactosidase protein is known as an enzyme that breaks down lactose, and it breaks down the blue color by decomposing lactose analog X-gal (5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside). Make it When the LacZ gene is expressed in the host cell, the X-gal is decomposed to have a blue color when the X-gal is treated, and thus the expression of the LacZ gene can be easily visually identified.

 When the alpha 1,3-galactosyltransferase targeting vector of the present invention is targeted into a host cell, homology is made between the endogenous alpha 1,3-galactosyltransferase gene and the targeting vector on the host cell genome. As recombination occurs, the base sequence is replaced. The LacZ gene on the vector is expressed by the promoter of the endogenous alpha 1,3-galactosyltransferase gene, and the beta-galactosidase protein produced by the LacZ gene expression breaks down X-gal to become blue. do. That is, the vector of the present invention has the advantage that it is easy to visually check whether targeting using LacZ and X-gal system.

In addition, since the LacZ gene on the vector is targeted and then expressed by a promoter of an endogenous alpha 1,3-galactosyltransferase gene, the targeting vector is a promoter using the principle of promoter-trap. It can be called a trap vector. In addition to a promoter trap, there are enhancer traps and exon traps as a method of capturing (traping) genes (expressed genes) that are transcribed and function in cells.

In a specific embodiment of the present invention, the present inventors prepared pGTKOLacZneoTK vector as an alpha 1,3-galactosyltransferase gene targeting vector, which facilitates identification of targeting, and produced pBstTK and pBstTKGT.

First, in order to obtain alpha 1,3-galactosyltransferase intron 8 and exon 9 sites of a miniature pig comprising a gene portion defined as a first region and a second region on the vector, bases of pigs already reported Primers were prepared based on the sequence (Kihiro Koike et al., Transplantation, 70; 1275-1283, 2000) and subjected to PCR reaction with a tag polymerase with proofreading function.

The pBstTK vector is the pBluescriptIISK (+) plasmid backbone and contains the HSV-TK gene region, a negative selection marker, and the pBstTKGT vector inserts the intron 8 and exon 9 sites of the alpha 1,3-galactosyltransferase gene into pBstTK. Form.

pGTKOLacZneoTK vector is a form in which IRES, LacZ, PGK-neo gene cassette is inserted into the pBstTKGT vector. pGTKOIRESCD59KITK vector is a vector that can remove the pig alpha 1,3-galactosyltransferase gene, and can be easily identified through the screening method through X-gal treatment, which best meets the purpose of the present invention. It can be called a vector. Specifically, from the 5 'end, the gene is composed of the HSV-TK gene region as a negative screening marker, and a part of the base sequence of the intron 8 of the pig alpha 1,3-galactosyltransferase gene. 1 region, 3 IRES, 4 LacZ gene region, 5 positive selection marker, and a second region of about 1.0 kb in size consisting of neo gene region and 6 base sequence of exon 9.

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.

On the other hand, after targeting the alpha 1,3-galactosyltransferase gene targeting vector of the present invention to a cell, the cell can be confirmed by targeting by X-gal treatment. Targeted cell line expresses LacZ gene by promoter of alpha 1,3-galactosyltransferase gene on water cell genome, and X-gal treated by expression of LacZ is degraded and becomes blue. . Therefore, the blue cell line is primarily selected for targeting. Targeting confirmation using LacZ and X-gal systems in mouse-derived cells has been reported several times (Takafumi shintani, Neuroscience letters, 247 (2-3); 135-138: Jonathan E. Dodge, Molecular and cellular biology, 24 (6); 2478-2486, 2004).

In another embodiment, the present invention relates to a transformant into which the alpha 1,3-galactosyltransferase targeting vector is introduced.

The term "transformation" of the present invention means that DNA is introduced into a host so 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. 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 acetic acid Lithium-DMSO method and the like, but are not limited thereto.

Among the targeting vectors produced in the specific embodiment of the present invention, pGTKOLacZneoTK was selected from Escherichia coli. coli) were introduced a deposit of arcs deposited in KCTC (Korean Collection for Type Cultures, Korea Daejeon Science and 111 Bungee Korea Research Institute of Bioscience and Biotechnology in) on December 05, 2007 No. KCTC 11246BP.

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

Example  One : miniature  Pig-derived genomic DNA From alpha 1,3- Galactosyl Transferase  Gene Intron  Sequence corresponding to 8 and 9 exons Cloning

The alpha 1,3-galactosyltransferase gene of pigs consists of nine exons, and some nucleotide sequences of exons 1 to exon 9 and introns have been reported (Kihiro Koike et al., Transplantation, 70; 1283, 2000). Since most of the catalytic activity of the alpha 1,3-galactosyltransferase gene is present in exon 9 (Yifan Dai, Nature, 20; 251-255, 2002), the present invention can remove the exon 9 site. We created a targeting vector. First, we cloned the base sequences of the alpha 1,3-galactosyltransferase genes intron 8 and exon 9 of miniature pigs that were hit to target the alpha 1,3-galactosyltransferase gene of the miniature pig. It was. Primers were prepared at the positions of Intron 8 and Exon 9 and PCR were performed using polymerase (Intron biotechnology) having a proofreading function. The base sequence and reaction conditions of the primer used in the PCR reaction are as follows.

 SEQ ID NO: 1 Forward primer: 5'- aggtcgtgaccataaccagatggaaggctc-3 '

SEQ ID NO: 2 Reverse primer: 5'- cttgcaccatgaagtctctgcactccagaa-3 '

1 cylcle 94 ℃ 3 minutes 30 cycles 94 ℃ 40 sec 68 ℃ 40 sec 72 ℃ 10 minutes 1 cycle 72 ℃ 10 minutes

The synthesized PCR product was cloned into a T-easy vector (promega), and the restriction enzyme position was confirmed by sequencing (FIG. 1).

Example  2 : pGTKOLacZneoTK  The making of the vector

2-1. pBstTK  The making of the vector

For the construction of the pGTKOLacZneoTK vector, the HSV-TK gene, TK (thymidine kinase) gene, which is a negative selection marker to which the HSV promoter is linked to the Xho I and Hind III restriction sites of pBluescript II (+) (Stratagene), was first restricted to Xho I and Hind III. PBstTK vector was prepared by cutting and inserting with enzyme (FIG. 2). 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 may occur to remove the TK gene of the targeting vector (FIG. 6), but the cell line may be grown in a medium containing a cyclocyclovir. However, when the targeting vector is inserted into an arbitrary position of a chromosome by random insertion, the TK gene is present together. As it is expressed and enters, it fails to grow in a medium containing liver cyclovir and dies.

2-2. pBstTKGT  The making of the vector

The alpha 1,3-galactosyltransferase gene intron 8 and exon 9 sites cloned in Example 1 were cut into Sal I and Not I, and then inserted into Sal I and Not I positions of the prepared pBstTK vector. Was completed (FIG. 3).

2-3. pGTKOLacZneoTK  The making of the vector

Of pBstTKGT vector prepared in Example 2-2 After cutting the BamHI restriction enzyme positions at the positions of intron 8 and exon 9 of the alpha 1,3-galactosyltransferase gene, IRES, derived from the p1049 vector (Hehls M, Science, 272; 886-889, 1996), The pGTKOLacZneoTK vector was completed by cutting the LacZ and PGK-neo gene cassettes with BamHI and connecting them to each other (FIG. 4).

The introduced LacZ gene is linked to only polyA without a promoter, and Neo is expressed as an independent transcript since both promoter and polyA are linked. By introducing the IRES sequence, alpha 1,3-galactosyltransferase and LacZ gene are expressed as one mRNA, but can be expressed as LacZ gene-independent proteins by ribosomes bound to IRES (Louis). Marie Houdebine et al., Transgenic research, 8; 157-177, 1999).

The completed pGTKOLacZneoTK vector was linearized by NotI restriction enzyme and then It can be introduced into somatic cells (FIG. 5). Endogenous alpha 1,3-galactosyltransferra as a result of the double crossing of the left and right arm portions, the same sequence between the pGTKOLacZneoTK targeting vector and the endogenous alpha 1,3-galactosyltransferase gene By replacing the nucleotide sequence of the aze gene with the nucleotide sequence of the targeting vector, expression of the alpha 1,3-galactosyltransferase gene is eliminated, and the LacZ gene is under the promoter of the alpha 1,3-galactosyltransferase gene. It is expressed in the same mRNA as the alpha 1,3-galactosyltransferase gene but is expressed as an independent protein due to the characteristics of the IRES sequence. In addition, the neogene is expressed as an independent transcript because a separate PGK promoter and polyA are linked. HSV-TK gene, a negative selection marker, was eliminated as a result of homologous recombination (FIG. 6).

Pig alpha 1,3-galactosyltransferase gene targeting vector of the present invention can be easily identified through a screening method through X-gal treatment, whether targeting can be determined as soon as the cell line is formed. Healthy target cell lines in early passages are possible. Such a vector can be usefully used for the production of cloned pigs for xenotransplantation.

Figure 1 shows the results of cloning the pGEM-T vector (promega) after PCR amplification from the genomic DNA derived from miniature pigs from the alpha 1,3-galactosyltransferase gene exon 8 to exon 9 amplification. The SalI and BamHI restriction enzyme positions are in the alpha 1,3-galactosyltransferase gene and the NotI position is in the pGEM-T easy vector (Promega).

Fig. 2 shows the insertion of the HSV-TK gene, a TK (thymidine kinase) gene, which is a negative selection marker to which the HSV promoter is linked to the Xho I and HindIII restriction enzyme positions of the pBluescript II (+) (Stratagene) vector, with Xho I and HindIII restriction enzymes. This is the result of completing the pBstTK vector. In addition, the pBstTK vector has restriction enzyme positions such as Kpn1, SalI, NotI, and SacI.

3 is a result of completing the pBstTKGT vector by cutting the alpha 1,3-galactosyltransferase gene intron 8 and exon 9 into Sal I and Not I from the vector of FIG. 1 and inserting them into Sal I and Not I positions of the pBstTK vector. .

4 shows the pBstTKGT vector. BamHI restriction enzymes at the positions of intron 8 and exon 9 of the alpha 1,3-galactosyltransferase gene were removed to remove a portion of the intron 8 site and exon 9 site, and the p1049 vector (Hehls M, Science, 272). ; 886-889, 1996), IRES-LacZ / PGK-neo gene cassette was cloned to complete the pGTKOLacZneoTK vector.

FIG. 5 shows the 1.85 kb HSV-TK gene, 2.6 kb left arm, 4.8 kb IRES-LacZ, neo gene, 1 kb light arm constituting the pGTKOLacZneoTK vector. pGTKOLacZneoTK can be cut and linearized with restriction enzyme NotI and then transfected into cells.

FIG. 6 shows the original sequence by homologous chromosomal recombination in the left and right arm regions, which are identical sequences between the pGTKOLacZneoTK targeting vector and the endogeneous alpha 1,3-galactosyltransferase gene. By replacing the nucleotide sequence of the alpha 1,3-galactosyltransferase gene with the nucleotide sequence of the targeting vector, the inherent function of the alpha 1,3-galactosyltransferase gene is removed, and the LacZ gene is alpha 1,3. It is expressed as the same mRNA by the promoter of the galactosyltransferase gene but expresses the beta-galactosidase protein independently due to the presence of the IRES sequence. Neo genes are also expressed as independent transcripts due to the presence of a separate PGK promoter and polyA. The HSV-TK gene, a negative selection marker, is eliminated as a result of homologous recombination.

<110> Korea Research Institute of Bioscience and Biotechnology <120> alpha 1,3-galactosyltransferase gene targeting vector <130> PA070768 / KR <160> 2 <170> Kopatentin 1.71 <210> 1 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> forward primer for amplifying alpha 1,3-galactosyltransferase          gene <400> 1 aggtcgtgac cataaccaga tggaaggctc 30 <210> 2 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for amplifying alpha 1,3-galactosyltransferase          gene <400> 2 cttgcaccat gaagtctctg cactccagaa 30  

Claims (9)

(1) Alpha 1,3-galactosyltransferase gene nucleic acid sequence of a pig 3 from the restriction enzyme SalI of the 5 'end of intron 8 of the alpha 1,3-galactosyltransferase gene of pigs A first region of 2 to 4 kb in length comprising a BamHI recognition site on the terminal side; (2) internal ribosomal initiation site; (3) LacZ gene region; (4) positive screening marker regions; (5) Exon from the restriction enzyme BamHI recognition site of the 5 'end of the exon 9 of the alpha 1,3-galactosyltransferase gene of pigs as the nucleic acid sequence of the pig alpha 1,3-galactosyltransferase gene Alpha 1,3-galactosyl having a second region of 0.5 to 2 kb length including the 3 'end of 9, wherein the (1) to (5) regions are configured in order from the 5' end Transferase Gene Targeting Vector. The vector of claim 1, wherein the pig is a miniature pig. The vector of claim 1, wherein the first region has a length of 2.6 kb. The vector of claim 1, wherein the second region has a length of 1.0 kb. The method of claim 1, wherein the positive selection markers are neomycin (Neo), hygromycin (Hyg), histidinol dehydrogenase gene (hiD) and guanine phosphoribosyltransferase (guanine). phosphosribosyltransferase (Gpt). The vector of claim 1, further comprising a negative selection marker. The vector of claim 1, wherein the vector is pGTKOLacZneoTK disclosed in FIG. 5. The transformant to which the vector of Claim 1 was introduced. The transformant of claim 8, which is Accession No. KCTC 11246BP.

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