In vivo Testis Transfection Efficiently Produced Transfected Sperm Cells in Ram but not Rooster

We report a method for gene transfer via in vivo testis-mediated gene transfer (TMGT) in sheep. A non-viral vector, pDB2, which carried an enhanced green fluorescent protein (EGFP) transgene under control of a human cytomegalovirus (CMV) promoter, was mixed with the TransIT transfection reagent. The lipoplex mixture was injected intra-testicularly or into the cauda epididymis of 12 and 5 rams, respectively, as well as the intra-testis of 5 mature roosters. Each injected rooster was crossed with four virgin hens and their hatched chicks were assessed for the presence of the EGFP transgene. After 60 days, both rams and roosters were investigated for the transgene. Unlike roosters, polymerase chain reaction (PCR) analysis of sperm cells which were collected from the epididymis and seminiferous revealed that more than 50% of sperm samples from the intratesticular rete-injected group were PCR-positive for the EGFP transgene. Transgene uptake was also observed in seminiferous tubules and epididymis of the intratesticular rete and the cauda epididymis groups, respectively. In conclusion, the combined approach of TMGT and lipofection can lead to ovine sperm transfection. This approach has potential to be combined with the clustered regularly interspaced short palindromic repeat (CRISPR)-Cas technology and used for on-farm gene editing of sheep species.