Knockout Of GGTA1 Gene By Promoter-trap Strategy In Wuzhishan Miniature Pig

The first successful human kidney transplant performed in the United States in 1954 has opened a new era in medicine.Joseph E. Murray, pioneer of this research, was awarded the 1990 Nobel Prize for Physiology or Medicine for the discovery concerning organ and cell transplantation in the treatment of human disease. With the substantial development of modern medicine, the survival rate of organ transplantation has increased significantly. Human allogeneic organ transplantation, known as "the summit of the 21st century medicine", has saved hundreds of thousands of patients with organ failure.However, as the requirement for organ transplantion has surged, the worsening problem of organ donor shortage becomes a major concern in the field of transplantation. Donor organ shortage is a major bottleneck restricting the development of clinical organ transplantation. Luckily,The situation of the severe shortage of organs could be solved by production of genetically engineered pigs as xenotransplant donors.The pig organs used in clinical trials will face with many problems,in which the first barrier to successful xenotransplantation is hyperacute rejection (HAR). HAR is a rapid and massive humoral immune response directed against the pig carbohydrate Galal,3-Gal epitope,thought to be the major xenoantigen,which is synthesized by al,3-galactosyltransferase (GGTA1). Producing of GGTA1 knockout cloned pigs may prove to be a more effective approach to overcome HAR. Although the production of GGTA1 gene knockout pigs has been reported previously, domestic related research in GGTA1 knockout cloned pig remains blank.The objective of our study was to disrupt GGTA1 gene in Wuzhishan Miniature Pig (WZSP), which is highly inbred and easy for genetic engineering, as well as whose physiological parameters such as blood character and organ size is similar to the human. For these reasons mentioned above, the WZSP is an ideal donor for xenotransplantation.In this study, the exon4 locus of the GGTA1 gene was efficiently targeted with a promoterless construct containing a Kozak translation initiation site to initiate Iranslation of the neomycin resistance reporter gene. The construct was linearized and electroporated into the porcine fetal fibroblasts cells. Two GGTA1 gene knockout cell clones were identified by PCR from 499 neomycin resistant colonies. Subsequently, one cell clones were used for nuclear transfer. Reconstructed embryos established with positive cells were transferred into naturally two cycling surrogate sows. One early pregnancy was established,which gave birth to two live piglets numbered WZSP-KO01 and WZSP-KO02 respectively. GGTA1 gene knockout pigs were detected by two polymerase chain reaction screening and further confirmed by Southern blot analysis. In the research, we have produced two GGTA1 gene knockout pigs and developed a basic platform for xenotransplantation research.This study is divided as the following three parts:PartⅠ:The construction of the GGTA1 promoterless knockout vectorIn this chapter, the primers for amplifying homology long arm and short armof GGTA1 gene (LAF/LAR and SAF/SAR) was designed based on the sequence of GGTA1 gene from NCBI database. The long arm was 5684bp while the short arm was 1692bp, both of which were cloned into pEASY-T1 Simple vector and sequenced. The recombinant vector pEASY-T1-LA and pEASY-T1-SA was established, respectively. Targeting backbone vector pBluescriptⅡSK (+) and pEASY-T1-SA were digested with KpnⅠand BglⅡand the corresponding fragments were recovered after gel electrophoresis, with which the recombinant vector PBS-SA was established.Plasmid PBS-SA and pIRESneo was digested with SmaⅠand XhoⅠrespectively and the corresponding fragments were recovered after gel electrophoresis. with which the recombinant vector PBS-SA-neo was established. Plasmid vector PBS-SA-neo and pEASY-T1-LA were digested with NotⅠand ClaⅠ, the corresponding fragments were recovered after gel electrophoresis. The recombinant vector pBS-GGTA1-SKO was established with the fragments.The full length of the targeting vector pBS-GGTAl-SKO is about 12Kb, of which include 5684bp homologous long arm and 1692 bp the short arm.PBS-GGTA1-SKO plasmid was digested with different restriction enzymes and the correct sizes of the corresponding fragments were obtained.PartⅡ:Screening and identification of transfected fetal fibroblast.Targeting Vector pBS-GGTA1-SKO was linearized with ClaⅠ. Fetal fibroblast cells were electroporated (program TO16) using NucleofectorⅡ.The electroporated cells were cultured for 24 hours in the normal medium and then added with G418 to a final concentration at 250μg/mL. Selection cultures were carried out for 10-15 days until the cell colonies were isolated.The resistant cell clones were cultured in 24-well plates; each cell clone was devided into two holes of a 24-well plates. One hole clone was taken for PCR Screening, while the other was cryopreservation by freezing. Positive colonies for GGTA1 gene disruption were seqcenced.In total, electroporations were performed with 1.5×107 primary cells and 499 G418 resistant colonies were obtained from the fetal fibroblast cell, of which two colonies were positive for GGTA1 gene disruption (G12,G13 respectively). In order to identify whether the homologous recombination event has occurred, two pairs of primers (P1F/P1R and P2F/P2R) were designed and 1890 bp and 5895bp targeted sequences were amplified by PCR. The homologous recombination event was further confirmed by DNA sequencing. FartⅢ:Nuclear transfer and identification of cloned pigsGGTA1+/- donor cell was inserted into the perivitelline space of an enucleated oocyte. Fusion of donor cell-oocyte complex was conducted in a fusion medium using electric stimulation. The fused and activated embryos were cultured for 1-2 days until embryo transfer. Reconstructed embryos were transferred into recipient oviducts. One early pregnancy was established. Two piglets numbered WZSP-KO01 and WZSP-KO02 were obtained.Genomic DNA of piglets tissues was extracted and precipitated with isopropyl alcohol.The DNA was then digested with Nco I and separated on a 0.8% agarose gel. Following electrophoresis, the DNA was transferred to a nylon membrane for southern bloting.Disruption of the GGTA1 gene was confirmed by PCR using two pairs of primers (P1F/P1R and P2F/P2R) designed to amplify 1890 bp and 5895bp targeted fragments, respectively. Southern hybridization analysis of cloned piglets indicated one allele of GGTA1 genes was replaced by targating vector. The 8kb band represents the wildtype GGTA1 gene and while 5.4kb band represents the targeted GGTA1 locus.In conclution, the promoterless vector targeting the exon4 locus of GGTA1 gene was constructed successfully. Two positive colonies for GGTA1 gene disruption has been confirmed after screening of transfected fetal fibroblast. Two cloned piglets that possess an GGTA1 knockout allele were produced and a basic platform for developing GGTA1 null pigs has been established.