Effect Of GGTA1KO/hCD55 Gene Modified Porcine Urothelial Cell CYP3A29 Gene Knockout On The Binding Of IgG And IgM In Human Peripheral Blood

Objective:To investigate the effect of GGTA1KO/hCD55 gene modified porcine urothelial cell CYP3A29 gene knockout on the binding of IgG and IgM in human peripheral blood,which it provides experimental basis for finding new potential xenoantigens besides α-gal,Sda and Neu5GC.Methods:(1)Porcine urinary transitional epithelial cells modified with GGTA1KO/hCD55 gene were isolated and cultured in vitro,and identified by immunofluorescence assay.(2)CRISPR/Cas9 gene editing technique was used to knock out the CYP3A29 gene of GGTA1KO/hCD55 gene modified porcine urinary transitional epithelial cells.The results were verified by Western-blot analysis and PCR.(3)The experiment was divided into three groups:wild-type porcine urinary transitional epithelial cell group(WT)GGTA1KO/hCD55 porcine urinary transitional epithelial cell group(GGTA1KO)GGTA1/CYP3A29KO/hCD55 porcine urinary transitional epithelial cell group(CYP3A29KO),human peripheral blood complement-dependent cytotoxicity(CDC)assay was performed.Results:(1)In cell immunofluorescence urinary transitional epithelial cells was observed in the experiments of specific protein markers proteinuria plaque UroplakinⅢ and epithelial cells of protein marker AE1 positive,confirmed GGTA1KO/hCD55 pig urinary transitional epithelial cell isolation and culture of success.(2)PCR results indicated that the CYP3A29KO bands in the experimental group were smaller than those in the negative control group,and the gene size was smaller.Western-blot analysis showed that no bands corresponding to CYP3A29 gene were found in the CYP3A29KO group compared with the control group.These results indicated that CYP3A29 gene was successfully knocked out in GGTA1KO/hCD55 porcine urinary transitional epithelial cells.(3)In CDC experiment,the average cytotoxicity of human IgG to urinary transitional epithelial cells in CYP3A29KO group,GGTA1KO group and WT group was 38.75%,43.63%and 53.61%,respectively.The mean cytotoxicity of human IgM to urinary transitional epithelial cells in CYP3A29KO group,GGTA1KO group and WT group was 38.61%,42.73%and 52.79%,respectively.The cytotoxicity of human lgG and lgM to urinary transitional epithelial cells in CYP3A29KO group was lower than that in GGTA1KO group and WT group(P<0.05).Conclusion:(1)The CYP3A29 gene of porcine urinary transitional epithelial cells modified by GGTA1KO/hCD55 gene was successfully knocked out.(2)The CYP3A29 gene knockout of GGTA1KO/hCD55 modified porcine urothelial cells reduced the cytotoxic effect of human peripheral blood on the porcine urothelial epithelial cells.(3)CYP3A29 may be the fourth heteroantigen in GGTA1KO/hCD55 modified pigs besides α-gal,Sda and Neu5GC.