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Knockout of GGTA1 Gene by Promoter-trap Strategy in Wuzhishan Miniature Pig

Author: ZhengDaoShan
Tutor: MaWenLi
School: Southern Medical University,
Course: Biochemistry and Molecular Biology
Keywords: Miniature Pig Knockout GGTA1 gene Hyperacute rejection
CLC: Q78
Type: Master's thesis
Year: 2011
Downloads: 31
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Abstract

In 1954, the first cases of the same kind of human kidney transplants done in the United States, opening a new era in human medicine, Joseph Murray, the pioneer of the study also received the 1990 Nobel Prize for Medicine and Physiology Prize. With the continuous advancement of modern medicine, the survival rate of organ transplantation is a substantial increase in the number of surgery has also grown dramatically. The human allogenic organ transplants have saved thousands of lives of patients with organ failure, known as the \However, with demand growth in the number of organ transplant organ shortage problem has become increasingly prominent, has become a major bottleneck restricting the development of clinical organ transplantation. Xenotransplantation can ease the the organ shortage of the status quo, porcine xenotransplantation become generally accepted by the scientific community in anatomy, physiology and human similarities, the most appropriate sources of xenotransplantation. However, the pig organs used in clinical trials is facing many problems, one of the most critical obstacles to hyperacute rejection (the Hyperacute Rejection HAR). HAR is mainly caused by a1, 3 - galactose transferase enzyme (al ,3-galactosyltransferase, GGTA1) gene encodes the formation of aGal epitope knockout GGTA1 gene can eliminate aGal epitopes in order to overcome HAR, since 2003 The GGTA1 gene abroad has been reported missing pig organs for xenotransplantation experimental and successfully overcome HAR. However, the late start of the domestic research in this area, have not been found the GGTA1 genetic knockout cloned pigs born reported. Wuzhishan inbred miniature pigs of nurturing highly inbred populations. Gene highly homozygous, ease of operation of the genetic engineering; blood physiological and biochemical indicators of human proximity the organ sizes similar to humans, is more desirable xenotransplantation donor. Constructed in this study the enzyme neomycin phosphotransferase (neomycin phosphotransferase, neo)-deficient targeting vector selection marker gene promoter, neo gene the upstream join Kozak sequence to increase translation efficiency. Linearized targeting vector electricity transfection method to import fetal fibroblast cells after transfection by G418. PCR method to detect drug-resistant cell clones point. Transfection screening 499 G418-resistant cell clones were detected by PCR, two cell clones homologous recombination. 1 the GGTA1 / - cell clones for donor cell nuclear transfer reconstructed embryos were transplanted to two receptor sows in natural estrus, a receptor pregnancy, childbirth two cloned pigs, respectively, the number WZSP -KO01 and WZSP-KO02, after PCR and Southern identification are the GGTA1 single allele knockout cloned pigs. In this study, cultivate GGTA1 gene knockout Wuzhishan miniature pig for xenotransplantation research platform. The study mainly divided into the following three parts: the first part: the the promoter defects targeting vector pBS-GGTAl-SKO's build GGTA1 gene primers were designed according to the NCBI database, LAF / LAR and SAF / SAR were amplified 5684bp homologous homologous to the short arm of the long arm, 1692bp. Nearly inbred Wuzhishan miniature pig genome as a template, PCR amplified a 5684bp The the homologous long arm (Long Arm, LA) homologous to the short arm of 1692bp (Short Arm, SA), the LA and SA were cloned into T vector pEASY -T1 Simple sequencing the reorganization the carrier pEASY-T1-LA, pEASY-T1-SA. Targeting backbone vector pBluescript II SK () and pEASY-T1-SA Kpn Ⅰ and Bgl Ⅱ double digestion, gel electrophoresis recycling vector backbone PBS and SA fragment with T4 DNA ligase, by restriction enzyme digestion and sequencing obtained after the carrier PBS-SA. The carrier PBS-SA and pIRESneo Sma Ⅰ and Xho Ⅰ double digestion, gel electrophoresis recycling PBS-SA and neo fragment with T4DNA ligase obtained by restriction enzyme digestion and sequencing carrier PBS-SA- neo. Carrier PBSSA-neo and pEASY-T1-La respectively Not the Ⅰ and Cla Ⅰ bis digested, gel electrophoresis PBS-SA-neo and LA fragment was recovered, with T4 DNA ligase, transformed screening recombinant plasmid was extracted by enzymatic Wuzhishan miniature pig GGTA1 gene knockout vector pBS-the GGTA1-SKO cut identification. Targeting vector pBS-GGTA1-SKO whole length to 12Kb, including 5684bp homologous to the long arm of the LA and the the 1692bp homologous to the short arm of SA pBS-GGTA1-SKO plasmid obtained by different restriction endonuclease correct size ranging fragment. Part II: Wuzhishan miniature pig fetal fibroblasts transfected screening and identification by restriction endonuclease Cla I digested plasmid pBS-GGTA1-SKO linearization. Cells using the Amaxa Nucleofector Ⅱ nuclear the transfected instrument and supporting transfection reagent transfection. Before transfection, cells were inoculated 6-well plates until the cells grew to 80-90% confluence, using electrical transfection method of the linearized targeting vector pBS-GGTA1-SKO accordance Nucleofector Ⅱ nuclear transfected instrument procedures T016 transfected To Wuzhishan porcine fetal fibroblast cells, 24h after adding G418 (250μg/mL) screened at 10-15 days, and then transmitted having a resistant cell clones were amplified in 24-well plates, cells per well in a 1:2 spread another 24-well plate, take the 1 hole cells used for PCR amplification, another 1 hole cryopreservation spare. Locus DNA extracted resistant cell clones identified by PCR and sequencing of the positive cell clones homologous recombination zone. Targeting vector were co-transfected cells 1.5 × 107, G418-resistant cell clones obtained after screening 499 To verify cell clones point whether homologous recombination, based on the sites homologous recombination using two pairs of primers were these clones The points of the two PCR detection, PCR identification of the primers used for P1F/P1R and P2F/P2R result, two clone points can be amplified out of the correct size PCR product of 1890bp and 5895bp, respectively named as G12 and G13. These two positive clones for DNA sequencing, sequencing results confirmed that the cells correct homologous recombination. Part III: nuclear transfer, embryo transfer and cloning pigs identified GGTA1 / - cells as donor cells, blind suction method using micromanipulation to go nuclear and electric fusion method for nuclear transfer cloned embryos were cultured in vitro for 1-2 days 1-2 cell stage reconstructed embryos were transplanted to two recipient pigs the transplant surgical method tubal deep transplantation, transplant about 200 embryos per head. The 1-2 cell stage reconstructed embryos transplanted to two receptor sows, 30 days B-test, a receptor gestation, the pregnancy rate was 50%. Surrogate sow farrowing a total of 2, respectively numbered WZSP-KO01, and WZSP-KO02. Extract the cloned pigs organization genomic DNA, genomic DNA as a template obtained by PCR using digoxigenin-labeled probes. Nco Ⅰ digested genomic DNA 12h (20μg), electrophoresed on a 0.8% agarose gel overnight, the DNA in the gel was transferred to nylon membrane fixed hybridization. After PCR and southern identification of two cloned pigs are the GGTA1 single allele knockout pig, PCR identified primers used for P1F/P1R and P2F/P2R were amplified PCR products of the correct size for 1890bp and 5895bp, respectively. Southern identification of positive clone pigs two hybridization bands: the GGTA1 single knockout cloned pigs Southern hybridization to two bands of wild-type GGTA1 gene homologous recombination-based the GGTA1 gene appears 5.4kb at 8kb at two with The brightness ratio of 1:1. Without causing homologous recombination the GGTA1 genes only an 8 kb hybridization with, confirmed that the the two cloned pigs are GGTA1 single allele knockout cloned pigs. In summary, this study successfully constructed Wuzhishan miniature pig gene GGTA1 part exon4 regional knockout deficient promoter targeting vector, transfection screened positive cell clones point two single allele knockout, nuclear transfer and embryo transfer two the GGTA1 single allele knockout Wuzhishan miniature pig, the establishment of a the xenotransplantation research foundation platform, to provide the basic research platform among cloned pigs to cultivate suitable for human organ transplants GGTA1 biallelic knock.

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