Porcine α-1, 3-galactosyltransferase Gene Knock-out And HLA-G1 Gene Knock-in In Somatic Cells

xenotransplantation faces several immunologic barriers: hyperacute rejection (HAR),acute vascular rejection (AVR) and chronic rejection mediated by cell. The major barrier, HAR, was mediated by porcine endothelial cell which has a-Gal epitope produced by a-1, 3-galactosyltransferase (a-1,3- GT), due to xeno-antibodies in human which are directed against the a-Gal epitope. By far, knocking out a~l,3-GT was considered as the best way to overcome HAR. HLA-G belongs to non-classical HLA-1 antigen, it combines with NK cell and CTL's membrane receptor KIR and then starts the inter cell inhibition signal pathway to inhibit the cytotoxic effect of NK and CTL. To solve these two barriers, we using the ninth exon of a-l,3-GT gene to construct the gene knock out vector, and HLA-G 1 gene was cloned into it. That will knock-out a~l,3-GT gene and knock in HLA-G 1 gene using somatic cell targeting method. This will provide a basis for Xenotransplantation and the research of transgenic animal.We have previously cloned porcine a-1 3- galactosyltransferase genomic gene, which includes parts of intron VIII and part of the exon IX. .In this experiment, we have cloned the segment of 1.9kb exon IX from porcine genome by using the PCR method, which was cloned into pGEM-Teasy vector and identified by DNA sequencing. The segments of 4.5kb and 1.9kb were used as 5'and 3'homologous targeting arm respectively, the plasmid pGEM-5zf as the basic vector, the neo gene as positive selected mark ,the GFP gene as negative selected mark.We constructed the gene targeting vector pZJ according to the strategy of positive-negative selection(PNS).It will knock out a-1, 3-galactosyltransferase gene and express the HLA-G1 gene. The size of vector is 15.5 Kb in length.Fibroblast cells were isolated from 3 pig fetuses at day 38 of gestation. 3 cell lines was established in vitro cell culture system. Only one of it grows well during gene targeting operation. The sex of these cell lines was identified by Sry-PCR.Using lipotransfection method to transfer pZJ into pig fetal fibroblast cell. After 7 days G418 selection, 30 neo-positive cell clones were obtained, among them, 12 of which is non-green ones.The genomic DNA samples from 7 cell clones were identified by PCR method. The result shows that 3 cell clones are site-specific recombinant. These suggest primarily our targeting vector is successful in the gene targeting.The similar study was carried in bovine fibroblast cell with the same vector. After 7 days G418 selection and 7 cell clones grow well. Two of cell clones were identified site-specific recombinant by PCR method.Successful completion of this project will establish a necessary accumulation for the next study on the cell level and for production of gene-targeting cloned animal.