Knockdown of Porcine Endogenous Retroviruses by RNA Interference in Chinese Experimental Miniature Pig Fibroblasts

Abstract Background The clinical application of porcine-derived xenotransplants is limited by the potential risk of infection due to the presence of porcine endogenous retrovirus (PERV) in tissues, organs, and cells. The establishment of pig fibroblasts with low PERV expression and without PERV-C can provide a nuclear donor to generate a safer transgenic pig. Methods In this study, we obtained Chinese Experimental Miniature Pig fibroblasts (CEMPF) with low expression of PERV and none of PERV-C. We designed small interfering RNA (siRNA) expressed as short hairpin RNAs (shRNA) based on the highly conserved gag and pol regions of PERV and screened for the most effective siRNA to inhibit PERV expression. The selected shRNA- pol3 fragment was introduced into the CEMPF to obtain an engineered CEMPF stably expressing shRNA- pol3. Results The PERV mRNA expression level in the engineered CEMPF was only 7.9% of that observed in fibroblasts from wild-type CEMPF, PERV P15E protein expression was significantly reduced. HEK293 cells cocultured with the supernate of the engineered CEMPF showed no PERV infection. Conclusions Engineered CEMPF, which possess no risk of PERV-A/C infection, can serve as a nuclear donor to generate xenograft donor pigs.