Production of transgenic spermatozoa by lentiviral transduction and transplantation of porcine spermatogonial stem cells

Spermatogonial stem cells (SSCs) are adult stem cells that transmit genetic information from the parent to the next generation (progeny) in males, and thus, SSCs have used in germline-modification for generating transgenic animals. In this study, we demonstrated the feasibility of transgenic sperm production by employing an effective busulfan treatment method to prepare recipient pigs for the transplantation of genetically modified donor porcine SSCs. We purified SSCs from pig testis cells by sequentially employing Laminin-coated dishes and culture dishes. The purified cells were transduced with lentivirus expressing enhanced green fluorescent protein (eGFP) at a multiplicity of infection (MOI) of 6 for 9 h. eGFP transduced pig SSCs were then transplanted into the seminiferous tubules of 12 to 16-week-old recipients born to busulfan-treated sows. We obtained six recipient pigs after transplantation and maintained them for more than 6 months. The collected viable spermatozoa from 2 out of 6 recipients were positive for eGFP gene expression in polymerase chain reaction. eGFP-expressing spermatozoa appeared morphologically normal under the microscope. When spermatozoa from these recipients were used for intra cytoplasmic sperm injection, eGFP expression could be detected in the embryos. Furthermore, eGFP colonies were derived from donor-transduced SSCs observed in the recipients’ testes. In summary, we demonstrated the successful production of functional-transgenic spermatozoa by transplantation of porcine SSCs where the transgenic was transduced by employing the lentiviral vector system.