KR101763196B1 - Transgenic cloned piglets for xenotransplantation and producing method thereof - Google Patents
Transgenic cloned piglets for xenotransplantation and producing method thereof Download PDFInfo
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- KR101763196B1 KR101763196B1 KR1020150092205A KR20150092205A KR101763196B1 KR 101763196 B1 KR101763196 B1 KR 101763196B1 KR 1020150092205 A KR1020150092205 A KR 1020150092205A KR 20150092205 A KR20150092205 A KR 20150092205A KR 101763196 B1 KR101763196 B1 KR 101763196B1
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Abstract
The present invention relates to transgenic reproduction pigs in which alpha 1, 3-GT (Alpha 1,3-Galactosyltransferase) gene is knocked out and human CD39 gene is knocked out. The transgenic cloned pig according to the present invention can overcome the immunity rejection occurring in heterologous organ transplantation, and thus can be usefully used as donor for intergeneric organ and cell transplantation.
Description
The present invention relates to transgenic reproduction pigs in which
Xenotransplantation is one of several ways to replace human organs. Until recently, xenotransplantation has developed around organ transplantation. As a heterogeneous organs or source of cells, primates such as Baboons were widely used around 1963 when a heterotopic transplant was first attempted. In particular, Reemtsma, who is called the father of xenotransplantation, maintained chimpanzee kidneys transplanted into kidney failure patients for up to 9 months (Reemtsma K, et al. Ann Surg 1964; 160: 384-410) We have survived for 20 days by transplanting the baboon's heart to Baby Fae with congenital heart disease (Bailey LL, et al., JAMA 1985; 254: 3321-9). However, primates are not only endangered, they are difficult to breed, have a high social rejection to utilize them as heterogeneous organs, and have a high risk of infection. Therefore, pigs with the advantage that the size of organs other than primates are very similar to those of humans, the gestation period is as short as 127 days and breeding is easy, and a large number of pups (6 to 12) . However, there has been a problem that, when transplanting organs of pigs into the human body, xenograft rejection, which is much more severe than allograft, occurs.
On the other hand, alpha-1,3-galactosyltransferase is a gene that causes hyperacute rejection when pig organs are transplanted into humans. Since antibodies against galactose, which is present on the surface of all animal cells other than primates, are naturally present in the human body, an antibody-antigen reaction occurs, leading to a hyper-immune rejection reaction. The transferase synthesizes galactose. Therefore, a study on transgenic pigs inhibiting the function of the gene has been conducted, and in 2002, a cloned pig with alpha1,3-galactosyltransferase removed through the gene targeting method was produced for the first time in the world (Yifan Dai et al., Nat Biotechnology, 20: 251-255, 2002). In 2003, a homozygous cloned pig with both alpha-1,3-galactosyltransferase gene loci removed was prepared, and a platform for realizing a heterologous organ transplantation study as a heterotopic transplantation model was established (Carol J. Phelps, Science, 299: 411-414, 2003). In 2005, we transplanted cloned pig organs with
The present inventors have made efforts to overcome the xenotransplantation rejection and to develop a transgenic cloned pig which can maintain a stable transplantation function by preventing rapid heterologous organ damage at the early stage of transplantation. As a result, A knock-in vector capable of overexpressing the human CD39 gene was prepared by hunting the human CD39 gene, the thrombosis suppressor gene, to the? 1,3-GT gene so as to inhibit the reaction. In the transgenic cloned pig produced using this, The present invention has been completed.
It is an object of the present invention to provide a knock-in vector in which the human-derived CD39 gene is hits in a porcine-derived alpha-1,3-galactosyltransferase gene, And to provide a method for producing the same.
In order to achieve the above object, the present invention provides a method for producing a recombinant vector comprising the human-derived CD39 gene represented by SEQ. ID. NO: 1, which is loaded on a porcine-derived alpha-1,3-galactosyltransferase gene. Provide a knock in vector.
The present invention also provides a transformed cell line obtained by transforming the knock-in vector into somatic cells.
The present invention also provides a transgenic transgenic pig produced by nuclear transfer of the transformed cell line and a method for producing the same.
The transgenic cloned pig according to the present invention can suppress the activity of
1 is a schematic diagram showing a vector map of GTKO / hCD39-KI vector.
FIG. 2 is a graph showing the results of a left-arm PCR analysis on the transformed cell lines (G103 and G107).
FIG. 3 is a graph showing the results of light-arm PCR analysis of the transformed cell lines (G103 and G107).
FIG. 4 is a diagram showing the results of long PCR analysis in the transformed cell lines (G103 and G107).
FIG. 5 is a graph showing the results of left arm PCR analysis (SM: size marker, donor: donor cell used for nuclear transfer, WT: wild-type pig fibroblast, SG: surrogate mother) in transgenic cloned pigs (P1 and P2).
Fig. 6 shows the result of light-arm PCR analysis (SM: size marker, donor: donor cell used for nuclear transfer, WT: wild-type pig fibroblast, SG: surrogate moth) in transgenic cloned pigs (P1 and P2).
Fig. 7 is a diagram showing the result of a long PCR analysis (SM: size marker, donor: donor cell used for nuclear transfer, WT: wild-type pig fibroblast, SG: surrogate mother) in transgenic cloned pigs (P1 and P2).
Fig. 8 is a diagram showing the result of full PCR analysis (SM: size marker, donor: donor cell used for nuclear transfer, WT: wild-type pig fibroblast, SG: surrogate mother) in transgenic cloned pigs (P1 and P2).
FIG. 9 is a graph showing the results of real-time RT-PCR of GalT expression in transformed cell lines (G103 and G107).
FIG. 10 shows the results of RT-PCR analysis of the expression of hCD39 in the transformed cell lines (G103 and G107).
FIG. 11 shows the results of real-time RT-PCR analysis of expression of GalT in transgenic cloned pigs (P1 and P2).
FIG. 12 shows the results of RT-PCR analysis of hCD39 expression in transgenic cloned pigs (P1 and P2).
Fig. 13 shows the results of Western blot analysis of hCD39 expression in transgenic cloned pigs (P1 and P2).
FIG. 14 is a graph showing the results of FACS analysis of down-regulation of GalT on the cell surface of transgenic cloned pigs (P1 and P2).
15 is a diagram showing the results of FCDS analysis of expression of hCD39 on the cell surface of transgenic cloned pigs (P1 and P2).
16 is a graph showing the cell survival rate according to human serum treatment time in fibroblasts obtained from transgenic cloned pigs (P1 and P2).
Hereinafter, the present invention will be described in more detail.
The present invention relates to a method for screening a human-derived CD39 gene represented by SEQ. ID. NO. 1 knocked in a pig-derived alpha-1,3-galactosyltransferase (alpha1,3-GT) ) Vector.
The term " knock-in " in the present invention refers to the use of an exogenous nucleotide sequence which is not present in another species or originally living organism to a DNA sequence derived from the genome or the organism of the living organism .
In the present invention, the knock-in vector comprises a first region consisting of a left arm comprising a part of
The
The left arm and the right arm mean two regions where homologous recombination occurs.
The first region may be of a size of 4 kb in the second cycle, and preferably consists of the nucleotide sequence of SEQ ID NO: 2.
The second region may have a size of 5 kb in the third trimester, and preferably the nucleotide sequence of SEQ ID NO: 3.
If the sizes of the first and second regions are smaller than the specific range, homologous recombination does not occur. If the size of the first region and the second region is larger than the specific range, the vector size becomes unnecessarily large and unstable, .
The human-derived CD39 gene expression cassette may include a promoter and a selectable marker gene in addition to the human-derived CD39 gene.
The term 'cassette' in the present invention means a specific region of a vector on which an exogenous base sequence desired to be inserted can be freely inserted using a gene recombination technique, and an exogenous gene amplified by PCR or the like can be recombined .
The term "promoter" in the present invention is intended to induce overexpression of a target gene. The promoter may further include not only a basal element necessary for transcription but also an enhancer which can be used for promoting and regulating expression have.
The term "selection marker" in the present invention is intended to select cells transfected with a knock-in vector and includes a selectable phenotype such as drug resistance, resistance to nutritional requirements, tolerance to cytotoxic agents, And includes positive selection markers and negative selection markers.
The "positive selection marker" means a marker capable of allowing positive selection by allowing only cells expressing the specific marker to survive in a treatment environment of a selective agent. For example, neomycin phosphotransferase neomycin phosphotransferase, hygromycin phosphotransferase, puromycin, histidinol dehydrogenase, guanine phosphotransferae, zeocin, But are not limited thereto. The positive selectable marker gene can be cloned such that it can be translated by the promoter trap method, i.e., without using a promoter, using the start codon of the endogenous antigenic determinant synthetic gene, or the cytomegalovirus, CMV Lt; RTI ID = 0.0 > promoter. ≪ / RTI >
The "negative selection marker" is a marker gene that enables negative selection to selectively remove the cells that have undergone random insertion. By selectively killing only the cells expressing the gene, (Eg, Herpes simplex virus-thymidine kinase, Hypoxanthine phosphoribosyltransferase, Cytosine dehydrogenase, and the like) deaminase, Diphtheria toxin-A, and the like.
The human-derived CD39 gene expression cassette may preferably be one comprising the nucleotide sequence of SEQ ID NO: 4.
In one embodiment of the present invention, a GTKO / hCD39-KI vector having a human CD39 gene inserted into a porcine α1,3-GT locus was prepared, and its vector map is shown in FIG.
The present invention also provides a transformed cell line obtained by transforming the knock-in vector into somatic cells.
The somatic cells are preferably fibroblasts, but are not limited thereto.
The term "transformed" in the present invention means that DNA is introduced into the host and the DNA becomes replicable as an extrachromosomal factor or by chromosome integration completion. Such transformation includes any method of introducing the nucleic acid molecule into an organism, cell, tissue or organ, and may be carried out by selecting a suitable standard technique depending on the host cell, as is known in the art, A method such as electroporation, calcium phosphate (CaPO 4 ) precipitation, calcium chloride (CaCl 2 ) precipitation, microinjection, polyethylene glycol (PEG) method, DEAE-dextran method, cationic liposome method, and lithium acetate- But are not limited thereto. On the other hand, in order to distinguish transformation of eukaryotic cells by plasmid or non-plasmid naked DNA from transformation as a meaning of tumorigenization of cells, it is also called 'transfection', but in the present invention, It is used as a meaning.
When the knock-in vector of the present invention is transformed into a host cell, homologous recombination occurs between the endogeneous α1,3-GT gene on the host cell genome and the target knock-in vector, and the nucleotide sequence is changed.
In addition, the present invention provides a transgenic transgenic pig produced by nuclear transfer of the transformed cell line.
(A) preparing the transformed cell line; (b) transplanting the transformed cell line of step (a) into an enucleated oocyte and then fusing to produce a nuclear transfer embryo; And (c) transplanting the nuclear transfer embryo of step (b) into the tubal duct of the surrogate mother to produce an embryo, and a method for producing the transgenic cloned embryo.
The term 'nuclear transfer' in the present invention refers to a genetic engineering technique that artificially binds nucleus-free cells to nucleus-free cells of other cells to have the same traits. It is possible to use any method known in the art without limitation.
The term " nuclear transfer embryo " in the present invention refers to an oocyte into which a donor nuclear cell has been introduced or fused.
The term " enucleated oocyte " in the present invention means that the nucleus of the oocyte is removed.
In the transgenic cloned pig according to the present invention, the activity of alpha1,3-galactosyltransferase is inhibited, and overexpression of human CD39 can overcome the immune rejection reaction occurring in heterologous organ transplantation.
Therefore, the transgenic cloned pig according to the present invention can be usefully used as a donor for intergeneric organ and cell transplantation.
Hereinafter, the present invention will be described in detail with reference to Examples. The following examples are for illustrative purposes only and are not intended to limit the scope of the present invention.
Example 1. α1,3- GT Locus Human for insertion CD39 Production of gene expression cassettes
A GTKO / hCD39-KI vector capable of knocking out α1,3-GT and inserting hCD39 was prepared by inserting human CD39 gene into the locus of pig α1,3-GT, which is an antigenic determinant gene. First, 2.5 kb of the left arm of α-
Left arm: 5'-GCGGCCGCAATTCATGATTATTATCCTCCAAGC-3 'and 5'-CTCGAGTATTTTCTCCTGGGAAAAGAAAAGG-3'
Right arms: 5'-GTCGACCTGTCAATGCTGCTTGTCTCAACTG -3 'and 5'-GGCGCGCCTCTGCCTGCTCCATCTACAGCATAA-3'
hCD39: 5'-CTCGAGCATGGAAGATACAAAGGAGTCTAAC-3 'and 5'-TTAATTAAGATGGAACAAAAATTTAACGCGAAT-3'
The genes amplified by PCR were inserted into T vectors (Promega Inc, USA) and restriction enzyme was used to confirm insertion of the gene (Not I and Xho I for left arm, Asc I and Sal I for right arm, , hCD39 uses Xho I and Pac I as restriction enzymes). The 5 'and 3' DNA fragments of a-gal were then inserted into the modified PGKneolox2DTA.2 (Addgene plasmid 13449) vector, respectively, and the hCD39pA gene inserted between the neomycin sites of the left arm and mPGKneo.
The vector map of the GTKO / hCD39-KI vector prepared by the above procedure and the positions of the respective genes are shown in Fig.
Example 2. Transfection with pig primary fibroblasts
Fibroblasts were isolated from the ear of a 2 month old miniature pig. More specifically, after removing the cartilage of the ear, the ear tissue was cut into small pieces and transferred to a culture dish, and then the ear-derived fibroblasts were kept in the culture medium until they became about 90% confluence of the plate . Cells were resuspended in DMEM (Dulbecco's modified Eagle's medium (Invitrogen) containing 15% fetal bovine serum (Invitrogen, CA, USA), 1% nonessential amino acid (Invitrogen), 0.1 mM mercaptoethanol (Invitrogen) and 1% antibiotics / ) ≪ / RTI > (Invitrogen) medium. The transfection was carried out using the Amaxa Basic Nucleofector Kit according to the manufacturer's method. After 48 hours, the cells were inoculated into 10 cm dishes and cultured in the presence of 400 μg / mL of G418 (Invitrogen) to generate an antibiotic-resistant clone Respectively.
Example 3. Human CD39 Production of transgenic pigs expressing genes
3-1. Oocytes ( oocyte ) Preparation
Ovaries of immature female pigs were obtained and transferred to a laboratory in a 0.9% NaCl solution at 35 ° C. Cumulus-oocyte complexes (COCs) were inhaled from 2-6 mm diameter antral follicles using an 18-gauge needle fixed to a 10 mL disposable syringe. The above COCs were dissolved in a solution containing 0.1% polyvinyl alcohol, 3.05 mM D-glucose, 0.91 mM sodium pyruvate, 0.57 mM cysteine, 0.5 μg / mL LH (L-5269, Sigma-Aldrich Corp., St. Louis, (Sigma-Aldrich Corp.), 75 μg / mL penicillin G, and 50 μg / mL streptomycin, as well as 10 μg / mL FSH (F-2293, Sigma-Aldrich Corp.) And washed three times with TCM 199 (31100-035, Gibco Grand Island, NY, USA). Approximately 50-60 COCs were transferred to a 4-well multi-dish (Nunc, Roskilde, Denmark) covered with mineral oil, then 500 mL of the same medium was added and incubated at 5% CO 2 and 39 ° C.
3-2. Nuclear transfer
Nuclear transfer was performed with minor modification of the method of Park et al. (Biol. Reprod. 2002; 66: 1001-1005). More specifically, after 42-44 hours of culture, oocytes were separated from cumulus cells by strongly vortexing in TL-HEPES containing 0.1% PVA and 0.2% hyaluronidase for 4 minutes. In TCM 199 containing 0.3% BSA (Sigma-Aldrich Corp., A-8022) and 7.5 μg / mL cytochalasin B, the first polar body and adjacent cytoplasm were inhaled using a micropipette pipette, The nucleus was removed. For serum starvation prior to SCNT, the donor cells prepared in Example 2 were cultured in DMEM containing 0.5% FBS for 3 days. Single donor cells were placed in the perivitelline space of the oocyte in contact with the oocyte membrane. Inoculation of I was disposed between a 0.3 M mannitol, 1.0 mM CaCl 2 · H 2 O, 0.1 mM MgCl 2 · 6H 2 O , and in a culture medium consisting of HEPES 0.5 mM to 1 mm spacing 0.2 mm Two platinum electrodes having a diameter of oocytes. Fusion / activation was induced by applying DC pulse of 1.1 kV / cm 2 continuously for 30 μs (BTX, USA). 20 to 30 reconstructed embryos were then transferred to a 4-well multi-dish covered with mineral oil and NCSU (North Carolina State University) -23 medium supplemented with 500 mL of 0.4% BSA was added. After 1 or 2 days of incubation, the NT embryos were surgically implanted into the oviduct of the female pig, the first day of the standing estrus. The pregnancy status was confirmed by an ultrasonic scanner (Mysono 201, Medison Co., Ltd., Seoul, Korea).
Example 4. Screening of transformed cell lines and transformed pigs
4-1. Screening of transformed cell lines
A
As shown in FIG. 2 and FIG. 3, it was confirmed that target cells of 4.0 kb and 2.5 kb appeared in two transformed cell lines of G103 and G107, respectively, as a result of left arm and right arm PCR.
In addition, as shown in Fig. 4, it was verified that G103 and G107 transgenic cell lines were heterozygously targeted through long PCR.
G103 and G107 transformed cell lines selected through the above procedure were used as donor cells for nuclear transfer in Example 3.
4-2. Screening of transgenic pigs
In order to identify the transformed pig prepared in Example 3, the target clone was analyzed by performing left arm PCR, right arm PCR and long PCR in the same manner as in Example 4-1. Wild-type pig fibroblasts (WT) and surrogate moth (SG) were used as controls. The results are shown in Figs. 5 to 7. Fig.
As shown in Figs. 5 to 7, it was confirmed that two piglets (P1 and P2) were successfully transformed as a result of left arm, right arm and long PCR.
Next, full PCR was performed on the same transgenic piglets using primers located outside the homology region. The primer used was 207F (5'-CCT CCTCTATCCTACCTCTAA AGC-3 ', located at
As shown in FIG. 8, amplification of the target band (10.5 kb) and the endogenous band (7.5 kb) confirmed that the target vector was normally inserted into the GalT locus of the transgenic pig.
Example 5. Verification of transformed cell lines and transgenic pigs
5-1. In the transformed cell line, GT And hCD39 Gene expression analysis
Real-time RT-PCR was performed to confirm whether α1,3-GT was down-regulated by heterologous knockout in the transformed cell lines (G103 and G107) selected in Example 4 above. More specifically, total RNA was isolated from transfected cells using the easy-spinTM total RNA extraction kit (Intron, Korea) and cDNA was synthesized using the iScriptTM cDNA synthesis kit (Biorad, CA, USA). cDNA was amplified using iQTM SYBR green supermix (Biorad, CA, USA). The primer sequences used in real-time RT-PCR were as follows and quantified using the expression level of GAPDH. The results are shown in Fig.
GalT expression confirmation: 5'-CCAGATGGAAGGCTCCAGTG-3 'and 5'-AACGCAGAGGACCCAGCTCTA-3'
GAPDH: 5'-TCGGAGTGAACGGATTTG-3 'and 5'-CCTGGAAGATGGTGATGG-3'
As shown in Fig. 9, it was confirmed that the transformed cell lines G103 and G107 showed about half or less GalT expression as compared with the control (WT).
Next, expression of CD39 gene was confirmed by RT-PCR in the same cell line. The results are shown in Fig.
As shown in FIG. 10, it was confirmed that the exogenous hCD39 gene was expressed at a remarkably high level in the G103 and G107 transformed cell lines, and this expression was not observed in the control group.
5-2. Transformation In the pig ? 1,3- GT And hCD39 gene Expression analysis
The expression of GalT and CD39 genes in the piglets (P1 and P2) screened in Example 4 was analyzed in the same manner as in Example 5-1. mRNA was obtained from piglet ear skin fibroblasts. The results are shown in Figs. 11 and 12. Fig.
As shown in Figs. 11 and 12, it was confirmed that GalT expression was markedly decreased and hCD39 expression was high in fibroblasts of transgenic pigs (P1 and P2).
In order to confirm the above results at the protein level, western blotting was performed according to a conventionally known method. More specifically, after separating proteins from porcine fibroblasts, 20 의 of protein was electrophoresed on 8% sodium dodecylsulfite polyacrylamide gel and transferred onto PVDF membranes (Invitrogene). The membrane was blocked with blocking buffer (1 x TBS, 0.1
As shown in Fig. 13, the protein expression of hCD39 was observed in the fibroblasts of the transgenic piglets (P1 and P2), and the expression was not observed in the control group.
5-3. FACS analysis
To detect cell surface protein expression, flow cytometry was performed. First, the transgenic pig's fibroblasts were washed with HBSS (Hank's balanced salt solution) and trypsinized to obtain a single cell suspension. The cells were then resuspended in 100 μL PBS and then incubated with 10 μL of fluorescein isothiocyanate (FITC) -conjugated anti-CD39 (Abcam, MA, USA) for 1 hour on ice. 1 μL of Alexa Fluor 488-conjugated anti-isolectin GS ib4 (Life technology, NY, USA) was added to the cells and incubated again on ice for 1 hour. This was analyzed with a flow cytometer (BD FACSvantage SE, Germany). The results are shown in Fig. 14 and Fig.
As shown in Figs. 14 and 15, GalT was down-regulated in transgenic piglets (P1 and P2), and hCD39 was expressed on the cell surface.
5-4. human Complement Serum-mediated cytotoxicity assay
The inhibition of the complement mediated immune rejection was confirmed using the fibroblasts and wild-type fibroblasts obtained from the piglets (P1 and P2) screened in Example 4 above. First, 1 x 10 4 fibroblasts per well were seeded in 96-well plates and cultured for 24 hours. The cultured cells were cultured in a standard culture medium filled with 50% human serum (Innovative research, MI, USA) for 30, 60, 90, 120, 180 and 240 minutes. Then, 10 μL of CCK-8 solution (Dojindo, Japan) was added and further cultured for 3 hours. The absorbance at 450 nm wavelength was analyzed using a microplate reader. The results are shown in Fig.
As shown in Fig. 16, the survival rate of wild-type fibroblasts decreased from 56% to 45.7%, while the survival rate of transformed pigs P1 was 62.3% and the survival rate of P2 was 58.2% Respectively.
From the above experimental results, it was found that the transformed pig of the present invention inhibits the activity of
<110> Optipharm.CO., LTD <120> Transgenic cloned piglets for xenotransplantation and producing method thereof <130> 1-10 <160> 4 <170> Kopatentin 2.0 <210> 1 <211> 1844 <212> DNA <213> Artificial Sequence <220> <223> human CD39 <400> 1 catggaagat acaaaggagt ctaacgtgaa gacattttgc tccaagaata tcctagccat 60 ccttggcttc tcctctatca tagctgtgat agctttgctt gctgtggggt tgacccagaa 120 caaagcattg ccagaaaacg ttaagtatgg gattgtgctg gatgcgggtt cttctcacac 180 aagtttatac atctataagt ggccagcaga aaaggagaat gacacaggcg tggtgcatca 240 agtagaagaa tgcagggtta aaggtcctgg aatctcaaaa tttgttcaga aagtaaatga 300 aataggcatt tacctgactg attgcatgga aagagctagg gaagtgattc caaggtccca 360 gcaccaagag acacccgttt acctgggagc cacggcaggc atgcggttgc tcaggatgga 420 aagtgaagag ttggcagaca gggttctgga tgtggtggag aggagcctca gcaactaccc 480 ctttgacttc cagggtgcca ggatcattac tggccaagag gaaggtgcct atggctggat 540 tactatcaac tatctgctgg gcaaattcag tcagaaaaca aggtggttca gcatagtccc 600 atatgaaacc aataatcagg aaacctttgg agctttggac cttgggggag cctctacaca 660 agtcactttt gtaccccaaa accagactat cgagtcccca gataatgctc tgcaatttcg 720 cctctatggc aaggactaca atgtctacac acatagcttc ttgtgctatg ggaaggatca 780 ggcactctgg cagaaactgg ccaaggacat tcaggttgca agtaatgaaa ttctcaggga 840 cccatgcttt catcctggat ataagaaggt agtgaacgta agtgaccttt acaagacccc 900 ctgcaccaag agatttgaga tgactcttcc attccagcag tttgaaatcc agggtattgg 960 aaactatcaa caatgccatc aaagcatcct ggagctcttc aacaccagtt actgccctta 1020 ctcccagtgt gccttcaatg ggattttctt gccaccactc cagggggatt ttggggcatt 1080 ttcagctttt tactttgtga tgaagttttt aaacttgaca tcagagaaag tctctcagga 1140 aaaggtgact gagatgatga aaaagttctg tgctcagcct tgggaggaga taaaaacatc 1200 ttacgctgga gtaaaggaga agtacctgag tgaatactgc ttttctggta cctacattct 1260 ctccctcctt ctgcaaggct atcatttcac agctgattcc tgggagcaca tccatttcat 1320 tggcaagatc cagggcagcg acgccggctg gactttgggc tacatgctga acctgaccaa 1380 catgatccca gctgagcaac cattgtccac acctctctcc cactccacct atgtcttcct 1440 catggttcta ttctccctgg tccttttcac agtggccatc ataggcttgc ttatctttca 1500 caagccttca tatttctgga aagatatggt atagggatcc accggatcta gataactgat 1560 cataatcagc cataccacat ttgtagaggt tttacttgct ttaaaaaacc tcccacacct 1620 ccccctgaac ctgaaacata aaatgaatgc aattgttgtt gttaacttgt ttattgcagc 1680 ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc 1740 actgcattct agttgtggtt tgtccaaact catcaatgta tcttaacgcg taaattgtaa 1800 gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta aatc 1844 <210> 2 <211> 2967 <212> DNA <213> Artificial Sequence <220> <223> left arm <400> 2 aattcatgat tattatcctc caagcctgtt cctcctccag cccatctgag aaaatactac 60 aacccccctg cttaagcaga aatcttgggt cttccttgtc tcatctctga taacaaaatt 120 accaaccacg tcctatcaat tctctctcca aagtatatat atatatattt ttttttaatt 180 ttttcccgct gtacagcatg gggatcaagt tattcttaca tgtatatttt ccccccaccc 240 tttgttccgt tgcaatatga gtatctagac atagttctca atgctactca gcaggatctc 300 cttgtaaatc taagttgtat ctgataaccc caagctcccg atccctccca ctccctccct 360 ctcctgtcgg gcagccacaa gtctattctc caagtccatg attttctttt ctgtggagat 420 ggtcatttgt gctggatatt agattccagt tataagtgat atcatatggt atttgtcaaa 480 gtatattattt tatttttctt tgtctttttg tcttttgtct tttttttttt tgttgttgtt 540 gttgttgttg ttgttgctat tacttgggcc gctcccgcgg catatggagg ttcccaggct 600 aggagttgaa tcggagctgt agccaccggc ctacgccaga gccacagcaa cgcgggatcc 660 gagccgcgtc tgcaacctac accacagctc acggcaacgc tggatcctta acccactgag 720 caagggcagg gaccgaaccc gcaacctcat ggttcctagt cggattcgtt aaccactgcg 780 ccacgacggg aactcccaaa gtatattttg aatcaagcca ccctttgagc caggccacct 840 cctctttatg gtcatgagaa cggtctgccc ttgtcctttt ctccattctc cacactcagc 900 acccagatgg gtctctctag gtgaagttgg atcaggggat tctccagctt tagatgcttt 960 ttgggattcc ccaccctact ttccatacct ttccaggttc tgactgcctc tgcccccctt 1020 ctgactgcct agcaccagcc actcaagggg gacagtgtca gtcactattt ttttcttgtc 1080 caggtttttt gcttttgttt ttttcaaaca cgagcagctc tttctcttgt ctgcctggta 1140 tagatgctgt ttccaaaata ttctcatccc ttctcacggc ccttgtcatc ctttcccatc 1200 ctatcttcat cccttgggaa gctctaaagt catctcccca aattgaaggg tgactaaaga 1260 gtttcccaga aggaaaaact gagtttccaa ctactacact gacttgcaag aaatgtttgt 1320 gtcttcatta aatgaaaaag aaaaaactgt aacaagatat gagaaaatac agaaaggaaa 1380 taataagact agaaaagtca aatatatagt gaaggtgttg catcaaacac ttaaataaac 1440 tagtacagat gttaaaagac taaattatat agttgaagga tagctgtgaa gatgtaaact 1500 atgacatcta aaacacaaaa tgttggcgtt cccgtcacgg cacagtggaa acgaatccga 1560 ctaggaacca tgaggttgca ggttcaattc ctgcccttgc tcagtgggtt aaggatccgg 1620 tgttgccgtg agctgtggtg taggtagcca atgaggcttg gatcccgcgt tgctgtggct 1680 ctggtgtagg ccggtggcta cagctccgat tcgaccccta gcctgggaac ctccatatgc 1740 cgcgggagcg gccctaaaaa gacaaataga cctaaaaaga aaaaaatcac aagacccaca 1800 aaatgttgcg aatcagtcct ctactagtat tatgtaattg tgcaagtttt ccttttatgt 1860 ctgttaatat ttgcgttcta gatgtaggtg ctctgatatc gtgtgcatat atgttaacca 1920 atgttatgtc ttcctctggt attgatccct ttgttattat gtaatgccct actttatctt 1980 ttgttacatt ctttgtttat gagtattgct gatatgtggc tagctgccac acttttcttg 2040 tcctttccat ttgaaataaa tatctttcta tctccaccca aattaaagta ctccgcaacc 2100 tgttattcca cccagcatcc cttccctctt caactacaat ttcatgcagc gatcaagaaa 2160 tacgaatgta ccgactgttt gccacttgtg tgggtgcatt ggggaaaagc tgggtgggaa 2220 gtggcagagc ctagattata aaggaccagg gtgagagttc ccattgtggc tcagctgaaa 2280 tgaatctgac tagcatccat gaggacgaag gtttgatccc tggcctcaat cagtgggtta 2340 aggatctggc gttgctgtcc gtgagttgtg gtgtagttcg cagacaaggc gtggacttag 2400 tgtggctgtg gctgtggcat aggctagtgg ctacagctct gattcgaccc ctagcctggg 2460 aatctctata tgctgtgagt gtggccctaa aatttaaatg aaattaaata aaggaccagg 2520 gtatattttt ctttgaggat aaggtacata gtcagtatat cagggacagt agacctagga 2580 aacggatgct tcctctagtc tgtgatgcga ggtggggcat ctgagttggg ggcggctgga 2640 gcccttaggg accattaact aaacccgtca ctctcccaca tctcggtgga ccttgggatc 2700 agtcaggatg cttccccttt gagcctcaaa atggccttag tatccttccc aacccagacg 2760 gccctgtcag ttcattgact tggctaattt gccagtgtag gcctatgcaa attaaggtag 2820 aacgcactcc ttagcgctcg ttgactattc atcaactttt ccttttagaa aagatattgg 2880 tataagcact tcttaaaaaa ccatattcca ctctgggtgt atttaatcta attttccctt 2940 ctccttttct tttcccagga gaaaata 2967 <210> 3 <211> 4011 <212> DNA <213> Artificial Sequence <220> <223> right arm <400> 3 ctgtcaatgc tgcttgtctc aactgtaatg gttgtgtttt gggaatacat caacaggtaa 60 ttatgaaaca tgatgaaatg atgttgatga aagtctcctc taatctccta gttatcagcc 120 aagtcaccag cttgcattaa aagtaggatt cactgacacc gtaaagaaag cattccagag 180 agttgccgtt gtggctcagg ggcagcaaac ccaattagga tccaagagga ggtgggtttg 240 atccctggcc ttgctctttg gcttaaggat ccggcattgc cgtgacctgt ggtgtaggtt 300 gcagatgcag ctcggatctg gcattgctgt ggctgtggcg taggctggtg gcttcagctc 360 cagtttgacc cctagcctgg gaacttccat atcccacact tgcggcccta aaaagcaaag 420 aaagaaagaa aatattctac ccttcctgta tccctgagcc cttaaatacc gtctttaaag 480 tcattagatc ttcaagtacc ttccagctaa ttaattatct tccttcctgc catgttgcca 540 ttgtcctgat ttttatacct ctgcagttct gggtaggcta gagccagaaa taataaggtc 600 atgttaagac caagatataa tattaaatta tttatatgac cagatatgga agttaccttg 660 agaactttca gacaggaatt ccatgagaaa tacaccctga tttttgcaat cctaaaatat 720 ttgcagagtt taaaggaaca actcaagttg ttgacttttg ctgcaaaaca cactgagtcg 780 ctggtgattc atttgtgcct ggctaaactt ttgggtgttt tgtctttttt ttttaactct 840 ggaaagcaaa atgaattaaa catttctgag ttttcaaatt catcagtgga ttcaccccaa 900 atatttgacg ctgcttcttt gcttttggaa actacgatgc cttggagatt ccagctggag 960 acgcttctga cagaaagaaa tgtctgcaag cagctaccaa aatgcatgat ggctttgact 1020 taagaggtat tgataccgct tggactttct ttcaaaaagg ccaccttaca acttggcctg 1080 aaggcattcc cgtggtggtg cagcggaaaa tgaatctgac taggaacccc gaggttgtgg 1140 gttcaatccc tggccttgct cagtggctta aggatcgggt gttgaagtaa gctgtggtgt 1200 agattgcaga cgcagcttgg atctggtgtt gctgtggctt tggtgtaggc cggcagctac 1260 agctccactt ggacccctag tctgggaacc tccatatgcc acaggtgtgg ccctaaaagg 1320 aaaaaagaca acaaacaaac aaaaaaccaa aaaacaactt ggcctggaga gctatgtcat 1380 caccattgat attttgatgg gtagtgtttt agtagcccct caagttcagg atgatggcct 1440 ggattaacgt tagaatgtct cttaaattct aagacttgat gagccagcag gaccattttg 1500 gccacttaga aaggaactgc atcttcaggt ccatcagtag aaggaggatt ctctagggag 1560 ttctctctta gctcagcggg ttcaagaatt cagtcttgtc cctacagcag ctcaggtgac 1620 tgctatggct tggctttgat ccctggccca ggaatttctg catgctgcag gtgcagccaa 1680 aaaaaaaaaa aaaaaaaaaa aaaaaggagg aggtggattc cctagaataa gaagctgtca 1740 ttcctttgga tgcttcatag atctaaccac ttctggaaca gttattccct ctcattctga 1800 agaactcatt ttaagaaaaa caagacgagc tagagagtga acaaatgtct acaaaccaac 1860 cttttcgaat tgaggaaact gtggtacttc ctctgaagaa aagatgacag cgttggatgc 1920 agagaccctg gggctccctt aggtacttga ggactgagga gatattctca gtggaggctg 1980 gagctaggct gcctggggtt ggtcctgtgc caccacttcc ctcctctgtg actttgggca 2040 agtttcccta tctttaaaaa tggggatgat agtagtacct gcttcatagg gttgttggat 2100 gt; ccaccaccac tatcaccatc tgtccggagg gcagcatagg acaggagatt ttggcaaata 2220 gaaggaagag ttctaggagt tcccgttgtg gtgcagggga aatgaatcca actaggaact 2280 aggagatttc gggttcaatc ccgcgcctcg ctcagtgggt taaggatcca gtgttgccat 2340 gagctgtggt gtagattgca gacatggcta ggatctggag ttgctatggc tgtggtgtaa 2400 gctggcagct gtagctcgga ttctacccct agcctgggaa tttccgtatg ccacaggttt 2460 ggccctacaa agaaaaaaga aaaagaaaaa gaaaaaattc taggggctga aagaatctaa 2520 cagaagagca agttccccat ggggttcctg acctgagttg agatgcttgt gtaggcaacc 2580 ttcaagctct gaactcttga ttgttttgaa ttgcagccag agttatactt ccatattttg 2640 ggtacttcac aaaattaaaa cacagaagcc aaaggcccag aagtgcatat tggtgctggc 2700 ctcccataaa gagggttgtt ttgcagtgct gggcacactc tctcttcaca gtaactggag 2760 cagattctgg ctgctcttca gggccgtagt ctggcaccca gactgcagcc acatcattct 2820 tcaatgtgag gaatctattt gaacatctgc aaggggttta aaaggcagga gattctttgc 2880 caccttgtga attggtctga ggtgagctga gggcactaac cttagacagg tgggtagcac 2940 tgtagctaaa gaggattaca ggagttcctg ttgtggctta gtggtaacaa atccaactag 3000 tatccatgag gattcaggtt cgatccctgg cctcgctcag tgggtcaggt atccggtgtt 3060 gctgtggctg tggtgtaggc tggcagcttc agctctgatt tgacccctag cctgggaact 3120 tccatgtgct gtaggtaagg cccttgaaaa aaaaaaaaaa agagatttac aaaataactc 3180 catcaaacac atacagctgt ttaagaatgt catccaggac agcatttggt taaaggctag 3240 atgaaaaaaa aaaaaaaatc ttagaatttt atttatttat tttttctttt tagggccaga 3300 cctgtggcct atggaaatgc ctgggctagg ggtggaatca gagctgccta caccacagcc 3360 atagccacgc cagatccaag ccccgtctgt gacctacacc acagctcatg gcaaacactg 3420 gatccttaat ccactgagtg aggccaggaa ttgaacccac attctcatgg atgctagttg 3480 ggttcttaag ccactgagcc acaagcttag aattttagag gtggaagaaa ctttaagagc 3540 tataataaag taatgatggt gatggtgatt ttgatgttag cggctactag ttattgagtg 3600 tttgcttgtg ccaggaactc cactgttcat tccctcctgt ttttaaaaca gccctggaag 3660 gtcagtgtta gtccacattt ctagatgagg aatactgagt ttccacaata ttaaatgtga 3720 acgttcaagg tcacattttt aggaagattt aggtccaggg ctgtctgact tgggtaacct 3780 gggtaaccct tcctttagtc aaggtttcca ttgttcaggc gatggacaag taggtgaaat 3840 gccttaacag tgaacttatg tctaacttct aattagaact cagatcttct gattcatcat 3900 ctggggctcc ttctggagct ggttgttcat gccaaatgct gcgaggggta cagtgtgcgc 3960 caaggagaat ccctaccctc aaggggttat gctgtagatg gagcaggcag a 4011 <210> 4 <211> 3728 <212> DNA <213> Artificial Sequence <220> <223> hCD39 casette <400> 4 catggaagat acaaaggagt ctaacgtgaa gacattttgc tccaagaata tcctagccat 60 ccttggcttc tcctctatca tagctgtgat agctttgctt gctgtggggt tgacccagaa 120 caaagcattg ccagaaaacg ttaagtatgg gattgtgctg gatgcgggtt cttctcacac 180 aagtttatac atctataagt ggccagcaga aaaggagaat gacacaggcg tggtgcatca 240 agtagaagaa tgcagggtta aaggtcctgg aatctcaaaa tttgttcaga aagtaaatga 300 aataggcatt tacctgactg attgcatgga aagagctagg gaagtgattc caaggtccca 360 gcaccaagag acacccgttt acctgggagc cacggcaggc atgcggttgc tcaggatgga 420 aagtgaagag ttggcagaca gggttctgga tgtggtggag aggagcctca gcaactaccc 480 ctttgacttc cagggtgcca ggatcattac tggccaagag gaaggtgcct atggctggat 540 tactatcaac tatctgctgg gcaaattcag tcagaaaaca aggtggttca gcatagtccc 600 atatgaaacc aataatcagg aaacctttgg agctttggac cttgggggag cctctacaca 660 agtcactttt gtaccccaaa accagactat cgagtcccca gataatgctc tgcaatttcg 720 cctctatggc aaggactaca atgtctacac acatagcttc ttgtgctatg ggaaggatca 780 ggcactctgg cagaaactgg ccaaggacat tcaggttgca agtaatgaaa ttctcaggga 840 cccatgcttt catcctggat ataagaaggt agtgaacgta agtgaccttt acaagacccc 900 ctgcaccaag agatttgaga tgactcttcc attccagcag tttgaaatcc agggtattgg 960 aaactatcaa caatgccatc aaagcatcct ggagctcttc aacaccagtt actgccctta 1020 ctcccagtgt gccttcaatg ggattttctt gccaccactc cagggggatt ttggggcatt 1080 ttcagctttt tactttgtga tgaagttttt aaacttgaca tcagagaaag tctctcagga 1140 aaaggtgact gagatgatga aaaagttctg tgctcagcct tgggaggaga taaaaacatc 1200 ttacgctgga gtaaaggaga agtacctgag tgaatactgc ttttctggta cctacattct 1260 ctccctcctt ctgcaaggct atcatttcac agctgattcc tgggagcaca tccatttcat 1320 tggcaagatc cagggcagcg acgccggctg gactttgggc tacatgctga acctgaccaa 1380 catgatccca gctgagcaac cattgtccac acctctctcc cactccacct atgtcttcct 1440 catggttcta ttctccctgg tccttttcac agtggccatc ataggcttgc ttatctttca 1500 caagccttca tatttctgga aagatatggt atagggatcc accggatcta gataactgat 1560 cataatcagc cataccacat ttgtagaggt tttacttgct ttaaaaaacc tcccacacct 1620 ccccctgaac ctgaaacata aaatgaatgc aattgttgtt gttaacttgt ttattgcagc 1680 ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc 1740 actgcattct agttgtggtt tgtccaaact catcaatgta tcttaacgcg taaattgtaa 1800 gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta aatcccggtg aagttcctat 1860 actttctaga gaataggaac ttcggatcct ggcagggcct gccgccccga cgttggctgc 1920 gagccctggg ccttcacccg aacttggggg gtggggtggg gaaaaggaag taacgcgggc 1980 gtattggccc caatggggtc tcggtggggt atcgacagag tgccagccct gggaccgaac 2040 cccgcgttta tgaacaaacg acccaacacc gtgcgtttta ttctgtcttt ttattgccgt 2100 catagcgcgg gttccttccg gtattgtctc cttccgtgtt tcagttagcc tccccctagg 2160 gtgggcgaag aactccagca tgagatcccc gcgctggagg atcatccagc cggcgtcccg 2220 gaaaacgatt ccgaagccca acctttcata gaaggcggcg gtggaatcga aatctcgtga 2280 tggcaggttg ggcgtcgctt ggtcggtcat ttcgaacccc agagtcccgc tcagaagaac 2340 tcgtcaagaa ggcgatagaa ggcgatgcgc tgcgaatcgg gagcggcgat accgtaaagc 2400 acgaggaagc ggtcagccca ttcgccgcca agctcttcag caatatcacg ggtagccaac 2460 gctatgtcct gatagcggtc cgccacaccc agccggccac agtcgatgaa tccagaaaag 2520 cggccatttt ccaccatgat attcggcaag caggcatcgc catgggtcac gacgagatcc 2580 tcgccgtcgg gcatgctcgc cttgagcctg gcgaacagtt cggctggcgc gagcccctga 2640 tgctcttcgt ccagatcatc ctgatcgaca agaccggctt ccatccgagt acgtgctcgc 2700 tcgatgcgat gtttcgcttg gtggtcgaat gggcaggtag ccggatcaag cgtatgcagc 2760 cgccgcattg catcagccat gatggatact ttctcggcag gagcaaggtg agatgacagg 2820 agatcctgcc ccggcacttc gcccaatagc agccagtccc ttcccgcttc agtgacaacg 2880 tcgagcacag ctgcgcaagg aacgcccgtc gtggccagcc acgatagccg cgctgcctcg 2940 tcttgcagtt cattcagggc accggacagg tcggtcttga caaaaagaac cgggcgcccc 3000 tgcgctgaca gccggaacac ggcggcatca gagcagccga ttgtctgttg tgcccgtcat 3060 agccgaatag cctctccacc caagcggccc ggagaacctg cgtgcaatcc atcttgttca 3120 atcatgcgaa acgatcctca tccctgtctc ttgatcgatc tttgcaaaag cctaggcctc 3180 caaaaaagcc tcctcactac ttctggaata gctcagaggc cgaggcggcc tcggcctctg 3240 cataaataaa aaaaattagt cagccatggg gcggagaatg ggcggaactg ggcggagtta 3300 ggggcgggat gggcggagtt aggggcggga ctatggttgc tgactaattg agatgcatgc 3360 tttgcatact tctgcctgct ggggagcctg gggactttcc acacctggtt gctgactaat 3420 tgagatgcat gctttgcata cttctgcctg ctggggagcc tggggacttt ccacacccta 3480 actgacacac attccacagc tggttctttc cgcctcagga ctcttccttt ttcaatatta 3540 ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa 3600 aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccaccta agcttgaagt 3660 tcctatactt tctagagaat aggaacttcg cgctataact tcgtataatg tatgctatac 3720 gaagttat 3728
Claims (12)
Wherein the knock-in vector is characterized in that the human-derived CD39 gene represented by SEQ. ID. NO. 1 has been deposited in a pig-derived alpha-1,3-galactosyltransferase gene.
(b) transplanting the transformed cell line of step (a) into an enucleated oocyte and then fusing to produce a nuclear transfer embryo; And
(c) transplanting the nuclear transfer embryo of step (b) into a tubal duct of a surrogate mother to produce a live egg.
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