SLA Haplotype Typing Of GGTA1 Gene Knockout Bama Minipigs And Preparation Of HEPCR Gene Transgenic Swine

Pig is the ideal donor for xenotransplantation.The hyperacute reaction(HAR)of pig to human organ transplantation caused by ?-1,3 galactosidase(Gal)has been solved basically.Then the cellular rejection,thrombosis and inflammation become the most important issues.Swine leukocyte antigen(SLA)is one of the main factors that cause the cellular rejection.Human endothelial protein C receptor(EPCR)can effectively reduce the thrombosis and inflammation caused by pig to human organ transplantation.This study mainly researching the health level of ?-1,3 galactosidase transferase gene(GGTA1)knockout Bama minipig(BMP),As well as SLA alleles and haplotypes,Screening the donor with low cellular rejection for xenotransplantation.Prepare the transgenic hEPCR pigs in order to reduce inflammation and thrombosis caused by xenotransplantation.There are three research contents in this paper:1.Study on the breeding and health level of GGTA1 gene knockout Bama minipig.The GGTA1 gene knockout(GTKO)BMP made by our group has been bred to F3 generation.In this study,the genotype,litter size,blood routine and blood biochemical indexs of GTKO Bama pigswere continuously tracked through three generations.The typing of GGTA1 gene knockout was identified by sequencing of PCR products.?-1,3-Gal was detected by flow cytometry after incubation Fluoresce in isothiocyanate-Griffonia simplicifolia Isolectin B4(FITC-GSIB4)with peripheral blood mononuclear cells(PBMC).Reproductive performance was evaluated by litter size.Health status was assessed by blood routine and blood biochemical indexes.The results showed that the inheritance of GGTA1 gene was consistent with Mendelian law;PBMC of piglet with GGTA1 double knocked(GGTA1-/-)genotype presented low fluorescence expression detected by flow cytometry.The litter size of the Bama minipig gilt with GTKO was 7.38±1.64,and the litter size of sow was 9.43±1.68,which had no significant difference in the fecundity with Ordinary BMP;Meanwhile no obvious difference were found in blood routine and blood biochemical indexescompared with wild-type BMP.In short,this study established GTKO BMP pedigree with stable hereditary,normal fertility and healthy physiological indicators.The GGTA1-/-Bamaminipigs effectively solved the hyperacute rejection,and can be used as a good donor for xenotransplantation research.2.SLA genotyping of GGTA1 knockout Bama minipigs.This research mainly focuses on the 5 loci of SLA gene(SLA-1,SLA-2,SLA-3,SLA-DRB1 and SLA-DQB1).The direct sequencing of DNA products method was used to obtain GTKO Bama minipig's SLA alleles and haplotypes.Complement dependent cytotoxicity(CDC)test was used to detect PBMC death rates of different SLA haplotypes in response with human serum.The results showed that 15 alleles in 5SLA locus of the GGTA1 gene knockout Bama minpigs.There were 5 SLA haplotypes in the GGTA1 knockout Bama minpigs,Hp-80.27,Hp-81.27,Hp-83 a.45,Hp-82.44 and Hp-83 b.10c respectively,among which SLA-2*05:07(SEQ ID NO:KX022946)and SLA-3*03:10(SEQ ID NO: KX022947)were newly found.The cell death rate of the four hybrid haplotypes(Hp-80.27/81.27,Hp-82.44/83 b.10c,Hp-80.27/83 b.10c and Hp-83 a.45/83 b.10c)in CDC test were less than 27.5%,the lower cell death rate in Hp-80.27/81.27 and Hp-82.44/83 b.10c were less than 17%.The results showed that PBMC of different SLA haplotypes in response with human serum results were different.Hp-80.27/81.27 and Hp-82.44/83 b.10c haplotypes had lower cell death rates and were more suitable for heterologous organ transplantation studies.3.Preparation of hEPCR pigs.In this study,we constructed the hEPCR gene expression vector p CAGGS-hEPCR-Puro,which is widely expressed in the promoter,was used to transfect the Bama and Wuzhishan hybrid porcine fetal fibroblasts.The transgenic cloned pigs were prepared by somatic cell cloning technology.The cloned piglets was identificated by PCR technique,The expression of hEPCR in various organs of transgenic pigs was analyzed by real-time quantitative PCR(q RT-PCR),Western Blot and immunohistochemistry.In this study,p CAGGS-hEPCR-Puro vector was successfully constructed,and 71 effective clones were screened,two hEPCR positive pigs were obtained.The expression of hEPCR gene was found in the liver,kidney,heart,spleen,pancreas,lung and aorta.The expression level of hEPCR gene was the highest in the aorta,pancreas and lung.Western Blot and immunohistochemistry showed that hEPCR protein was expressed in the ear,aorta,pancreas,heart and kidney.In this study,we successfully produced transgenic hEPCR gene pigs,and hEPCR gene has a high expression in the main organs and tissues of cloned pigs,which provide a good donor for heterologous organ transplantation studies.