Study On The Modification Of αGal Epitope And α1, 3-galactosyltransferase Gene On Porcine Somatic Cells

Transplantation of cells,tissues and organs is a well-known and accepted life-saving procedure for several diseases.Currently,the number of patients awaiting transplantation is continuously increasing,and shortage of available deceased organ donors is the major limitation for allotransplantation.Research for alternative sources of tissues is ongoing and xenogeneic organs or cells provide an attractive solution. Because of its physiological and anatomical similarities to human,short gestation period,production of large litters,offspring grow rapidly,can be easily bred under qualified specific pathogen-free conditions,and carring fewer ethical objections than the use of nonhuman primates,the pig has been identified as the most suitable donor animal for xenotransplantation.Because of immunological rejections including hyperacute rejection(HAR), acute vascular rejection(AVR),acute cell-mediated rejection(ACR)and chronic rejection,the transplanted pig organ will be rapidly rejected by recipient after xnotransplantation.Both HAR and AVR are mediated by xenoreactive antibody reacting with the epitope galactose-α1,3- galactose(αGal),a common carbohydrate structure on the surface of almost all mammalian cells with the exception of humans, apes,and Old World monkeys.Synthesis of theαGal epitope is catalyzed byα1,3-galactosyltransferase(GGTA1 orα1,3GT).Because no functional copy of the GGTA1 gene exists,there is noαGal expressed on the surface of human cells.It has been reported that,in humans,up to 1%of the total circulating IgG is anti-αGal natural antibody.A number of strategies have been used to removeαGal epitopes from porcine cell surface.These methods include treatment of pig organs withα-galactosidase to removeαGai epitopes;overexpression ofα1,2-fucosyitransferase orα2,3-sialyltransferase in pig cells to compete with GGTA1;and disruption of GGTA1 gene.The former two methods only partially or temporarily remove theαGal from the surface of the xenografts,and the residualαGal molecules are still sufficient to activate the complement cascade and cause destruction of the grafts.Complete elimination ofαGal epitopes from the donor organs should be achievable by knockout of the GGTA1 gene.The birth of the first homozygous GGTA1 knockout(GT-KO) pigs,not expressing the major xenoantigen,was reported by PPL therapeutics and the Pittsburgh team in 2003.China still falls behind in this field.The present study includes two parts.The first part was designed to modifyαGal epitopes on porcine red blood cells(pRBCs).pRBCs was treated in vitro with recombinant alpha-galactosidase(r-AGL)and then transfused to rhesus monkey,to investigate the possibility of transfusing AGL-treated pRBCs to rhesus monkeys.The second part was to modify GGTA1 gene in pig somatic cells.To construct a pig GGTA1 gene knockout vector with positive and negative selection markers,and use it to disrupt the GGTA1 gene in porcine kidney cell line PK-15 cells.The genotype of GGTA1(+/-)PK-15 cells was identified.First part:Transfusion ofα-galactosidase treated porcine red blood cells to rhesus monkey.Objective:To investigate the possibility of transfusing alpha-galactosidase-treated pig red blood cells(pRBCs)to rhesus monkeys.Methods:pRBCs were treated in vitro with recombinant alpha-galactosidase (r-AGL).The effect of the treatment was measured by flow cytometry and pRBCs were cross-matched with rhesus monkey sera.Rhesus monkeys were treated with immunosuppressant(cobra venom factor and Dexamethasone)and the pRBCs were labeled by FITC before transfusion.The survival rates of AGL-treated pRBCs and unmodified pRBCs were measured by flow cytometry.Blood and urine biochemical analyses of receipted monkeys were performed.Results:AGL could effectively removeαGal xenoantigens on pRBCs membrane and reduce the hemagglutination.Flow cytometry data showed that the survival time of unmodified pRBCs was less than 30 minutes and the survival rate of AGL-treated pRBCs is 29%at 2h after transfusion.The modified pRBCs were detectable in rhesus monkey blood at 8h after transfusion.Conclusion:These results suggest that the transfusion of AGL-treated pRBCs could prevent the hyperacute rejection mediated by natural antibodies and prolong the survival time of xenograft.Second part:Disruption GGTA1 gene with positive-negative targeting vector in porcine kindey cell line PK-15 cellsObjective:To construct a pig GGTA1 gene knockout vector with positive-negative selection markers,and disrupt the GGTA1 gene in porcine somatic cells.Methods:Two GGTA1 genomic fragments were amplified by PCR from genomic DNA of primary porcine fetal fibroblasts(PFFb).The targeting vector was designed to be able to use positive-negative selection.The short arm is a 2-kb fragment containing most part of exon 9 of GGTA1 gene.It was inserted into the XbaI-ClaI site at 3'end of the positive selection marker neo sequence in pLoxp plasmid.The long arm is a 5.4-kb fragment containing part of exon 8,intron 8 and part of exon 9 of GGTA1 gene.It was inserted into the NotI site at the 5' end of the neo sequence in the plasmid.The 2.7-kb negative selection marker tk was at the 3' end outside of the short arm in the vector.The 1.8-kb neomycin phosphotransferase(neo) gene will insert into the 5' end of exon 9 to disrupt the GGTA1 gene once homologous recombination event occurs.After linearization at SalI site in the 5' end of the long arm,the targeting vector PSL/GT has 7.1-kb recombination arm.The SalI-linearized targeting vector was transfected into PK-15 cells by lipofectamine 2000.Cells were selected in medium containing G418 plus gancyclovir. The drug-resistant colonies were amplified and genomic DNA was prepared for identifying.The colonies were firstly screened by PCR analysis with Neol(a sequence from the 3' end of the neo gene)and P16(a sequence from the 3' end of exon 9 in sequences located outside the short recombination arm)as forward and reverse primers.Cell colonies in which a 2.5-kb DNA fragment could amplified were potential recombinants in GGTA1 locus.The PCR positive colonies were further identified by Southern blot.A probe outside the short homologous arm on exon 9 was used for hybridization.The colonies would be heterozygous for knockout at the GGTA1 locus and thus would have one nomal copy and one disrupted copy of the GGTA1 gene.The genomic DNA was degested with BstEⅡand used for Southern blot analysis.The GGTA1(+/-)cells should show two band:a 7-kb band of the size expected for the endogenous GGTA1 allele and a 8.8-kb band for insertion of the neo sequence at the GGTA1 locus,GGTA1+/+ cells only one 7-kb band.Results:A porcine GGTA1 gene targeting vector PSL/GT with positive-negative marker was constructed and was used to disrupt GGTA1gene in PK-15 cells.436 colonies were picked up after cultured in G418 and gancyclovir.31 were positive according to PCR and only one was GGTA1(+/-)cell according to Southern blot.Conclusion:We constructed an effective porcine GGTA1 gene targeting vector PSL/GT with positive-negative markers.The recombinant vector was transfected into porcine kidney cell line PK-15 cells and heterozygous GGTA1(+/-)PK-15 cells were obtained.This work provides a basis for future studies to generate GT-KO pigs for xenotransplantation.