KR20060106590A - Transgenic pig expressing hla-g to inhibit activity of nk cell and the method of producing thereof - Google Patents

Abstract

The present invention relates to a transgenic cloned pig expressing the HLA-G gene and a method for producing the same, and more specifically, 1) separating somatic cells from a pig embryo; 2) preparing an expression vector comprising the HLA-G gene and introducing it into the somatic cells of step 1; 3) selecting and culturing the somatic cells into which the expression vector is introduced; 4) removing the nuclei of the eggs collected from the sows and fusing them with the somatic cells; And 5) transgenic cloned pigs expressing the HLA-G gene comprising the step of transplanting the fused cloned eggs into surrogate sows and giving birth to piglets, and a method of manufacturing the same. Transgenic cloned pigs expressing the HLA-G gene by the production method of the present invention can overcome problems such as post-translational modification or mass production of the existing method, natural killer cells (natural killer) It inhibits cell and NK cell activity and prevents lysis of target cells, thereby inhibiting graft rejection, which is useful for research on cell utilization and heterogeneous organ transplantation such as pig production expressing HLA-G gene through nuclear transplantation. Can be used.

HLA-G

Description

Transgenic pig expressing HLA-G to inhibit activity of NK cell and the method of producing etc.

Figure 1 a is an electrophoresis picture showing the PCR product of the HLA-G gene in JEG-3 cells, Figure 1 b is an electrophoresis picture showing the PCR product of the GAPDH gene.

2 is a schematic diagram showing a cleavage map of the pcDNA3.1 vector.

Figure 3 a is an electrophoresis picture showing the insertion of the HLA-G gene by cutting the pcDNA3.1-HLA-G vector with Bam H I and Hin d III restriction enzyme, Figure 3b is Sca Electrophoresis picture showing single-stranded pcDNA3.1-HLA-G vector digested with I restriction enzyme.

Figure 4 is a DNA electrophoresis picture showing the results of PCR analysis to identify the HLA-G gene and neomycin resistance gene (neo) in genomic DNA of the selected colony.

5 is a graph showing the results of FACS analysis for confirming the expression of HLA-G protein in HLA-G positive clone somatic cells.

Figure 6 is a photograph showing a Western blot analysis for confirming the expression of HLA-G protein in HLA-G positive clone somatic cells.

7 is a photograph showing immunohistochemical staining analysis to confirm the expression of HLA-G protein in HLA-G positive clone somatic cells.

Figure 8 is a DNA electrophoresis picture showing the results of PCR analysis for identifying the HLA-G gene and neomycin resistance gene (neo) in fetal cell genomic DNA.

9 is a graph showing the results of FACS analysis for confirming the expression of HLA-G protein in fetal cells.

10 is a photograph showing immunohistochemical staining analysis for confirming the expression of HLA-G protein in fetal cells # 1.

Figure 11 is a DNA electrophoresis picture showing the results of PCR analysis for identifying the HLA-G gene and neomycin resistance gene (neo) in genomic DNA of the cloned pigs.

12 is a photograph showing a transformed porcine litter expressing HLA-G protein.

The present invention relates to a transgenic cloned pig expressing the HLA-G gene and a method for manufacturing the same, and more particularly, inhibiting the activity of natural killer cells to prevent lysis of target cells to inhibit transplant rejection as well as nuclear transfer. It relates to a method for producing HLA-G gene expression cloned pigs.

Organs in pigs are much more physiologically similar to humans, and much effort has been made to transplant them. But the rejection of transplanting organs into humans was a major obstacle to xenotransplantation. This problem is due to the simultaneous elimination of pig genes involved in rejection (Lai et al., Science , 295: 1089-1092, 2002; Dai et al., Nat. Biotechnol ., 20: 251-255, 2002). By suppressing cells involved in rejection, xenografts are more feasible. If xenotransplantation is possible, porcine somatic cells may be transplanted.

Transplanting pig organs into the human body results in rejection by natural killer cells (NK cells) in addition to the superacute rejection caused by α-gal epitopes. When the organs of the pig enter the human body, the natural killer cells recognize the endothelial cells (EC) of the pigs non-magnetically and cause cell lysis, but the transgenic pigs are made of genes that inhibit the activity of the natural killer cells. If it is possible to prevent the cytotoxicity of natural killer cells, the gene that can be used to make such a transgenic pig is human leukocyte antigen-G (HLA-G) (Hitomi et al. , Journal of Immunology , 163: 6301-6305, 1999; Wang et al., Transplantation Proceeding , 36: 2473-2474, 2004).

Natural killer cells play an important role in innate immunity. In particular, the most important role is to selectively attack cells in the body where the function of Major Histocompatibility Complex (MHC class I) is reduced by infection of tumor cells, bacteria invading the body and viruses. will be. Natural killer cells also cause rejection when transplanting bone marrow. The activity and inactivation of spontaneous killer cells is achieved by the binding of specific inhibitory or activating receptors to the ligands of the target cell. That is, cytotoxicity of natural killer cells is regulated by activating signals such as MIC and ULBP and inhibitory signals of MHC class I and non-MHC class I. Specific inhibitory receptors that inactivate killer cells include the killer cell Ig-like receptor (KIR) and LIR1 / ILT2, CD94 / NKG2A. These receptors bind non-MHC class I and MHC class I molecules to inactivate natural killer cells (Lewis et al., Annu . Rev. Immunol. , 16: 359-93, 1998).

Natural killer cells also play an important role in xenotransplant rejection. During xenotransplantation between humans and pigs, natural killer cells rapidly produce xenogeneic cytotoxicity to pig cells. This is because the deficiency of MHC class I and swine leukocyte antigen (SLA class I) are delivered as a positive signal to natural killer cell inhibitory receptors. As a result, natural killer cells attack target cells, secrete pre-inflammatory cytokine, and lose their function. Therefore, inhibiting cytotoxic cell activity and heterologous cytotoxicity is an excellent approach to reduce rejection of xenotransplantation.

HLA-G is a non-classical MHC class I molecule, located at the end of HLA-A, located on human chromosome 6, and quite consistent with the nucleotide sequence of HLA-A2. HLA-G is also similar to HLA-B and C and contains eight exons. Exon 1 is a peptide signal, exon 2 is α1-domain, exon 3 is α2-domain, exon 4 is α3-domain, exon 5 is transmembrane site, exon 6-7 is cytoplasmic tail The tail region, and exon 8, encodes an untranslated region. HLA-G is an isoform of G1, G2, G3, G4 and two soluble forms G5, G6 by messenger RNA splicing, where the largest HLA-G1 is exon All of them are located on 1-8 (Juan et al., Journal of Immunology , 158: 5736-5743, 1997). Among these isoforms of HLA-G, it is known that HLA-G1 binds to inhibitors of natural killer cells, KIR, CD94 / NKG2A, and LIR1 / ILT2, to inactivate killer cells (Schneider et al., Scand . Immunol ., 54: 70-75, 2001; Nathalie e t al ., Proc . Natl . Acad . sci . USA , 94; 11520-11525, 1997).

On the other hand, HLA-C can be used to inactivate natural killer cells in addition to HLA-G. However, HLA-C is a polymorphic allele and expression of HLA-C during vascularizd xenograft. There is a report of cytotoxicity by T cells. In contrast, HLA-G is a minimally polymorphic molecule that is specifically expressed in the placenta and amnion during pregnancy, and the expressed HLA-G protects the fetus from natural killer cells. It is not known to cause an immune response. Therefore, HLA-G has been selected as a good gene to suppress the immune rejection response by natural killer cells during xenotransplantation and many studies have been reported (Sasaki et al., Trasnplantation , 67: 31-37, 1999; Miyagawa et al. Transpl Immunol ., 11: 147-53, 2003).

To date, most of the long-term biodevelopment has focused on superacute rejection. However, in organ transplantation, there may be a treatment method using heterologous organs and heterologous cells, and in each case, the rejection reaction to be centrally handled may have to be appropriately studied in this situation.

In order to solve the above problems of xenotransplantation, the present invention provides a transgenic cloned pig that expresses the HLA-G gene capable of suppressing cytotoxicity of natural killer cells generated during organ transplantation. Was completed.

It is an object of the present invention to provide a method for producing a pig clone somatic cell line expressing the HLA-G gene and a pig clone somatic cell line produced by the method.

In order to achieve the above object, the present invention

1) separating the somatic cells from the pig embryo;

2) preparing an expression vector comprising the HLA-G gene and introducing it into the somatic cells of step 1; And

3) selecting and culturing the somatic cells into which the expression vector is introduced;

4) removing the nucleus of the oocytes taken from the sows and fusing them with the clonal somatic cells; And

5) It provides a method for producing a transgenic cloned pig expressing the HLA-G gene comprising the step of transplanting the fused cloned eggs into surrogate sows and give birth to piglets.

The present invention also provides a clonal cell line for producing a transgenic cloned pig by the above method.

In addition, the present invention provides a transgenic cloned pig prepared by the above method.

In the present invention, " clone cell line " refers to a cell line having the same position of a vector introduced into the genome.

Also, " gastric cavity " refers to the space between the zona pellucida of the egg and the egg cytoplasm.

In addition, " cloning " refers to the stage in which the ova and somatic cells are fused by electrical stimulation.

Hereinafter, the present invention will be described in detail.

The present invention

1) separating the somatic cells from the pig embryo;

2) preparing an expression vector comprising the HLA-G gene and introducing it into the somatic cells of step 1; And

3) selecting and culturing the somatic cells into which the expression vector is introduced

4) removing the nucleus of the oocytes taken from the sows and fusing them with the clonal somatic cells; And

5) It provides a method of producing a transgenic pig expressing the HLA-G gene comprising the step of transplanting the fused cloned eggs into surrogate sows and giving birth to piglets.

In step 1 , the pig fetus is preferably using a fetus of 20 to 50 days of gestation, more preferably using a fetus of 30 to 40 days of age. In a preferred embodiment of the present invention, pig somatic cells were isolated using a fetus of 35 days of gestation. As a method for isolating somatic cells, a general method used in the related art may be applied to the present invention in the same manner. In a preferred embodiment of the present invention, the somatic cells may be removed by chopping the pig fetus using a razor blade and then passaged with trypsin. Separated.

In step 2 , the HLA-G gene is preferably described by SEQ ID NO: 1 , but is not necessarily limited thereto. In addition, the expression vector including the HLA-G gene can be used without limitation any known expression vector that can be expressed in porcine cells. In a preferred embodiment of the present invention, the pcDNA3.1-HLA-G vector was used as the expression vector including the HLA-G gene.

In step 3 , the somatic cells into which the expression vector of step 2 is introduced can be easily selected by using the expression vector into which the selection marker is introduced. Antimicrobial resistance genes can be used as screening markers. The antibiotic resistance genes include, but are such as neo ^r, pac ^{r, r} bsr, hph ^r may be used, and is not necessarily limited to: In a preferred embodiment of the present invention, somatic cells were selected by introducing an expression vector containing the HLA-G gene by treating G418 with the cell culture using neo ^r .

In the present invention, the HLA-G gene is efficiently injected into porcine somatic cells to select a transgenic pig clone somatic cell line expressing the HLA-G protein, which is then transplanted into a porcine oocyte from which the nucleus has been removed. Transgenic pigs expressing the genes were prepared.

Therefore, the transgenic cloned pigs prepared by the above method according to the present invention are expressed in the HLA-G gene, thereby inhibiting the activity of natural killer cells, preventing lysis of the target cells and inhibiting the transplant rejection. It can be usefully used for research in the field and field of xenotransplantation.

The present invention also provides a clonal cell line for producing a transgenic cloned pig by the above method. The present invention also provides a transgenic cloned pig prepared by the above method.

The clone somatic cell line is characterized by the same position of the expression vector injected into the genome. If a cloned somatic cell line with an expression vector is not produced, the gene is introduced but the insertion site of the gene is different for each somatic cell. Since the expression patterns of the cells may differ slightly depending on which part of the chromosome the gene is inserted into, the expression pattern of the introduced gene may appear slightly different for each individual when the pigs are produced using these cells. , KW et al., Anim . Biotech. , 2001, 12: 173-181).

In the present invention to solve the problems of the method as described above to produce a clonal somatic cell line. This technique is expected to increase the production efficiency of transgenic animals using somatic cloning. In other words, after the useful protein gene is inserted into the genome of the somatic cell, several different clone cell lines can be made according to the region where the gene is inserted. Since the expression rate may vary depending on which part of the genome is inserted into the genome, a cloned animal may be used to produce a cloned animal, and then an individual having a good expression rate may be selected by examining a protein expression pattern.

The present inventors produced a transgenic cloned pig expressing the HLA-G gene by injecting the HLA-G gene that inhibits the activity of natural killer cells in porcine somatic cells. Several studies have shown that HLA-G genes can be useful for xenotransplantation in many ways, according to the research that can suppress not only rejection by natural killer cells but also immune rejection by T cells. Although there are many difficulties in reducing the rejection of xenotransplantation, the transgenic cloned pigs produced using the cell line of the present invention can be usefully used in the field of cell use therapy and the suppression of rejection during xenotransplantation. The present inventors deposited a transgenic pig clone somatic cell line in which the HLA-G gene was introduced into the Korea Cell Line Research Foundation on February 17, 2005 (Accession No .: KCLRF-BP-00110).

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited by the following examples.

< Example  1> Isolation of Porcine Somatic Cells

After 35 days of gestation, the pig fetus was chopped with a razor blade, and the cells were incubated for 5 minutes in DMEM medium (BioWhittaker, USA) to which trypsin-EDTA (Gibco, USA) was added. The supernatant was centrifuged (1,000 rpm, 5 minutes), incubated in DMEM medium containing 10% FBS (Fetal bovine serum, Hyclone, USA), and treated with trypsin when the cells reached 90% saturation. Subcultures were used, some of which were used for nuclear transfer and others were cryopreserved.

< Example  2> HLA -G Gene Expression Vector Preparation

Human chorionic carcinoma cell line JEG-3 (KCLB, KOREA) was incubated in a 37 ° C., 5% CO ₂ cell incubator and RNA was isolated using Trizol reagent (Invitrogen, USA). CDNA was prepared using AMV reverse transcriptase (Reverse Transcriptase, Invitrogen, USA), and the HLA-G gene described in SEQ ID NO: 1 was obtained by PCR using primers to which Bam H I / Hin d III recognition sites were added. The primers used in the PCR reaction to obtain the HLA-G gene is HLA-G forward primer (5'-ATGGATCCGGATGGTGGTCATGGCGCCC-3 ') and SEQ ID NO in the reverse primer (5'-ACTAAGCTTAGCCTGAGAGTAGCTCCCTCCTT-3 described 3 represented by SEQ. ID. NO: 2 ') Was used. PCR conditions were premodified at 95 ° C. for 4 minutes, then repeated 30 times at 95 ° C. for 1 minute, 60 ° C. for 30 seconds, and 72 ° C. for 30 seconds, and finally reacted at 72 ° C. for 10 minutes ( FIG. 1 ) . Figure 1 a is an electrophoresis picture showing the PCR product of the HLA-G gene of JEG-3 cells, Figure 1 b is an electrophoresis picture showing the PCR product of the GAPDH gene, as shown, HLA in JEG-3 cells -G gene could be identified.

As a vector containing the HLA-G gene, pcDNA 3.1 (Invitrogen, USA) was used ( FIG. 2 ). The pcDNA 3.1 vector was digested with Bam H I and Hin d III restriction enzymes and then conjugated to the HLA-G gene using T4 DNA ligase (Roche, USA) to express the HLA-G gene. -G vector was prepared. Then, the pcDNA3.1-HLA-G vector was digested with Bam H I and Hin d III restriction enzymes to confirm the insertion of the HLA-G gene by electrophoresis ( FIG. 3 a ), Sca Single stranded pcDNA3.1-HLA-G vector digested with I restriction enzyme was confirmed by electrophoresis ( b of FIG. 3 ).

< Example  3> HLA Preparation of Clonal Cell Lines Expressing -G Gene

Sca I site was linearized by cleaving Sca I site to the prepared pcDNA3.1-HLA-G vector ( Fig . 3b ), and DNA was isolated by phenol extraction. Lipid mediated method (Lipofectamine ^™ 2000, Invitrogen, USA) was performed to inject the prepared DNA into porcine somatic cells isolated in Example 1 . After incubating the cells for 48 hours, transformed somatic cells were selected by adding G418 (Geneticin, Gibco., USA), where the concentration of treated G418 was determined in various ways to determine the optimal concentration at 300 ㎍ / ml. It was. Clonal cell lines were treated with G418 at a concentration of 300 μg / ml for the transfected cells to obtain colonies formed.

< Example  4> clone In somatic cells HLA The introduction of -G gene

A portion of the porcine somatic colonies selected in Example 2 was boiled in distilled water, and positive cells into which the expression cells were introduced were selected by PCR analysis. Primers used in the PCR reaction to identify the HLA-G gene are primers described in SEQ ID NO: 2 and SEQ ID NO: 3 as in Example 2 . Further, the neomycin resistance gene (neomycin resistance gene, neo) neo described in SEQ ID NO: 4 to make the forward primer (5'-GGATTGCACGCAGGTTCTCCG-3 ') and SEQ ID NO: neo reverse primer (5'-ATTCGGCAAGCAGGCATCGCC- described as 5 3 ') was used. PCR conditions were premodified at 95 ° C. for 4 minutes, then repeated 1 time at 95 ° C., 30 seconds at 60 ° C., 30 seconds at 72 ° C., and finally reacted at 72 ° C. for 10 minutes ( FIG. 4 ). . Then, as a result of the electrophoresis of the PCR product, the cloned cell lines 8-1, 8-4, 8-6, and 8-8 transformed with the HLA-G gene and neomycin resistance gene were identified. The clone somatic cell line 8-4 clone cell line was deposited on February 17, 2005 to the Korea Cell Line Research Foundation ( accession number: KCLRF -BP-00110 ).

< Example  5> clone In somatic cells HLA Expression of G protein

<5-1> FACS  By analysis HLA -G protein expression confirmation

Cells 1-1 and 8-4 cells positively selected by PCR analysis in Example 4 were obtained by culturing in a 60 mm cell culture dish, followed by FACS (fluorescence activated cell sorting) as follows. The analysis was performed. Stained cells were fixed in fixed solution (phosphate buffer solution containing 1% formaldehyde), and then the primary antibody, anti-HLA-Gab7758 (MEM / G-9, abcam, UK) antibody or control antibody. After staining with IgG1-ab9404 (abcam, UK) antibody, stained with FITC conjugated anti-IgG.ab5874 (abcam, UK) antibody and analyzed by FACS ( FIG. 5 ). As a result, it was confirmed that HLA-G positive clone cell lines 8-1 and 8-4 markedly increased the expression of HLA-G protein compared to the mini pig control cells.

<5-2> West Blot  By analysis HLA -G protein expression confirmation

In Example 4 , Western blot analysis was performed to confirm the expression of HLA-G protein in cells positively selected by PCR analysis. After lysing the cells and quantifying the protein, electrophoresis was performed, and the electrophoresis gel was transferred to PVDF membrane to treat the antibody. Anti-HLA-G.4H84 (Santa Cruz, USA) antibody, a primary antibody diluted 1: 2000 in blocking solution, was incubated at room temperature for 2 hours, and then a hose diluted 1: 10000 in TBST solution. Goat anti-mouse IgG (H + L) secondary antibody (ZYMED, USA) conjugated with horse radish peroxidase (HRP) was incubated at room temperature for 1 hour and reacted with ECL solution. It was confirmed ( FIG. 6 ). As a result, the expression of 39 kDa HLA-G protein was confirmed in HLA-G positive clone cell lines 8-1 and 8-4.

<5-3> by immunohistochemical staining HLA -G protein expression confirmation

In order to confirm the expression of HLA-G in the cellular state, the cloned somatic cell line positively selected by PCR analysis was cultured on a chamber slide for 5 days to JEG-3 and negative HLA-G positive control cells. Immunohistochemistry using an anti-HLA-G.4H84 (Santa Cruz, USA) antibody diluted at 1: 100 after immobilization in a fixed solution (95% ethanol) with a control cell line, Mini pig somatic cell line (PWG). Staining was performed ( FIG. 7 ). As a result, expression of HLA-G protein was confirmed in HLA-G positive clone cell line 8-4.

< Example  6> Using somatic cloning HLA Production of transgenic cloned pigs with -G

<6-1> culture

Mature cultures were prepared in TCM199 (31100035; Gibco, Grand Island, NY, USA) in 0.1% polyvinylalcohol, 3.05 mM D-glucose (Sigma Chemical Co, St. Louis, MO, USA), 0.91 mM sodium pyruvate (sodium pyruvate, Sigma), 0.57 mM cysteine, 0.5 g / ml LH (Sigma), 0.5 g / ml FSH (Sigma), 10 ng / ml epidermal growth factor (Sigma), 75 g / ml penicillin G (Sigma) and 50 g / ml streptomycin (Sigma) were added.

The micromanipulation culture medium was added 0.3% BSA and 7.5 g / ml cytochalasin (cytochalasin B, CB, Sigma) to TCM199. The activated culture medium was added 1.0 mM CaCl ₂ H ₂ O, 0.1 mM MgCl ₂ 6H ₂ O, 0.5 mM HEPES to 0.3 M mannitol (Sigma). The embryonated culture of cloned eggs was added 0.4% BSA to NCSU (North Carolina State University) -23 medium.

<6-2> collection and fine manipulation of eggs ( Micromanipulation )

The ovaries of uncultivated pigs were collected from slaughterhouses and transported to 35-39 ° C and 0.9% physiological saline to the laboratory. Oocytes were collected by inhaling follicular fluid from a 2-6 mm diameter follicle by connecting an 18-gauge needle to a disposable 10-ml syringe. The eggs were washed in mature culture medium and 50-60 were placed in 4-well plates containing 500 μl culture and incubated for 42-44 hours.

After culturing the oocytes in a microcoagulant culture medium for 5-10 minutes, somatic cells were added to the culture medium, and the first polar body of the egg and its cytoplasm were removed with a 30 μm diameter micro glass tube, and the somatic cells were removed using the glass tube. The egg was injected into the gastric cavity.

<6-3> Cell fusion and activation

After micromanipulation, the nuclear transfer embryos were placed between platinum electrical wires 1 mm apart. Cell fusion and activation were induced by feeding two DC pulses 1.0-1.2 kV / cm and 30 μsec to a fusion device (BTX Electro-Cell Manipulation 2001), and then examined the fusion rate after 0.5-1 hour.

<6-4> Cloning  culture

Only fused cloned eggs were selected and 20-30 cloned eggs were incubated for 6 days in a 4-well plate containing 500 μl of culture medium. After incubation, staining with 5μg / ml bisbenzimide (Hoechst 33342) was examined for the nucleus of the cloned eggs under a fluorescence microscope.

<6-5> Transgenic Pig Production by Cloning Embryos

The fused cloned eggs examined above were incubated for 1 or 2 days in a 4-well plate containing 500ul of culture solution, and then put the cloned eggs in a freezing tube containing 2ml of the same culture solution and heated to 39 ° C. Embryo transfer kit (Minitube, USA) was delivered to the fertilized egg transplant site. Surrogate mothers for cloned embryo transplantation were prepared by selecting individuals whose estrus began, and after washing the surrogate mothers, 0.5 g of pentotal sodium was administered to the vein for temporary anesthesia. The anesthetized surrogate mother was fixed to the operating table and then inhaled anesthesia by supplying 5% isoflurane. The anesthetized surrogate mother was prepared by incision about 5cm along the abdominal line and exposed the uterus and ovary in vitro. At this time, the cloned eggs were inhaled with a catheter (Tom cat catheter, Monoject, USA) and pushed to the buccal region of the fallopian tube to transplant the cloned eggs. A total of 1243 cloned eggs were transplanted into 6 surrogate mothers for the production of HLA-G transgenic cloned pigs. Surrogate mothers who had cloned embryos completed their pregnancy test using 30-day ultrasound, and those with confirmed pregnancy induced delivery after 114 days. Three surrogate mothers were pregnant and one of them was excised from the uterus 35 days after pregnancy to secure four fetuses and the fetal cell line was made by the method of Example 1 . In order to confirm the expression of the HLA-G gene of the fetal cell line using the method of Example 4 , Example 5-1 and Example 5-3 , all four of the transformants were confirmed ( Fig. 8 , Fig. 9 and FIG. 10 ). The remaining two gave birth normally and produced 9 transgenic cloned pigs.

Born cloned pigs were treated with protein and K and treated with proteinase K (Gibco. Inc) to decompose the protein and then denature the protein with phenol to isolate genomic DNA. The isolated genomic DNA was transformed using primers described in SEQ ID NO: 2 and SEQ ID NO: 3 for HLA-G, and SEQ ID NO: 4 and SEQ ID NO: 5 for Neo, and all nine live transformants were transformed. This was confirmed ( FIG. 11 ).

As described above, transgenic cloned pigs expressing the HLA-G gene according to the production method of the present invention inhibit the activity of natural killer cells to prevent lysis of the target cells, thereby inhibiting the transplant rejection reaction, through nuclear transplantation. Pigs expressing the HLA-G gene can be produced and used for the field of cell-use treatment and xenotransplantation.

<110> MGenbio Inc.          PARK, KWANG-WOOK <120> Transgenic pig expressing HLA-G to inhibit activity of NK cell          and the method of producing <130> 5p-08-40 <160> 5 <170> KopatentIn 1.71 <210> 1 <211> 1017 <212> DNA <213> Homo sapiens HLA-G <400> 1 atggtggtca tggcgccccg aaccctcttc ctgctgctct cgggggccct gaccctgacc 60 gagacctggg cgggctccca ctccatgagg tatttcagcg ccgccgtgtc ccggcccggc 120 cgcggggagc cccgcttcat cgccatgggc tacgtggacg acacgcagtt cgtgcggttc 180 gacagcgact cggcgtgtcc gaggatggag ccgcgggcgc cgtgggtgga gcaggagggg 240 ccggagtatt gggaagagga gacacggaac accaaggccc acgcacagac tgacagaatg 300 aacctgcaga ccctgcgcgg ctactacaac cagagcgagg ccagttctca caccctccag 360 tggatgattg gctgcgacct ggggtccgac ggacgcctcc tccgcgggta tgaacagtat 420 gcctacgatg gcaaggatta cctcgccctg aacgaggacc tgcgctcctg gaccgcagcg 480 gacactgcgg ctcagatctc caagcgcaag tgtgaggcgg ccaatgtggc tgaacaaagg 540 agagcctacc tggagggcac gtgcgtggag tggctccaca gatacctgga gaacgggaag 600 gagatgctgc agcgcgcgga cccccccaag acacacgtga cccaccaccc tgtctttgac 660 tatgaggcca ccctgaggtg ctgggccctg ggcttctacc ctgcggagat catactgacc 720 tggcagcggg atggggagga ccagacccag gacgtggagc tcgtggagac caggcctgca 780 ggggatggaa ccttccagaa gtgggcagct gtggtggtgc cttctggaga ggagcagaga 840 tacacgtgcc atgtgcagca tgaggggctg ccggagcccc tcatgctgag atggaagcag 900 tcttccctgc ccaccatccc catcatgggt atcgttgctg gcctggttgt ccttgcagct 960 gtagtcactg gagctgcggt cgctgctgtg ctgtggagaa agaagagctc agattga 1017 <210> 2 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> HLA-G forward primer <400> 2 atggatccgg atggtggtca tggcgccc 28 <210> 3 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> HLA-G backward primer <400> 3 actaagctta gcctgagagt agctccctcc tt 32 <210> 4 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> neo forward primer <400> 4 ggattgcacg caggttctcc g 21 <210> 5 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> neo backward primer <400> 5 attcggcaag caggcatcgc c 21

Claims (8)

1) separating the somatic cells from the pig embryo; 2) preparing an expression vector comprising the HLA-G gene and introducing it into the somatic cells of step 1; And 3) selecting and culturing the somatic cells into which the expression vector is introduced; 4) removing the nucleus of the oocytes taken from the sows and fusing them with the clonal somatic cells; And 5) A method of producing a transgenic cloned pig expressing the HLA-G gene, comprising the step of transplanting the fused cloned eggs into surrogate sows and giving birth to piglets. The method of claim 1, Pig embryo of step 1 characterized in that the fetus of 30 to 40 days of gestation. The method of claim 2, Porcine fetus is characterized in that the fetus of 35 days of gestation. The method of claim 1, The HLA-G gene of step 2 is characterized in that SEQ ID NO: 1. The method of claim 1, The expression vector of step 2 is characterized in that the pcDNA3.1-HLA-G vector. A transgenic pig clone somatic cell line expressing the HLA-G gene produced by the method of claim 1. The method of claim 6, Transgenic pig clone somatic cell line deposited with accession number KCLRF-BP-00110. A transgenic pig expressing the HLA-G gene produced by the method of claim 1.

Images