Sperm As Vector For Gene Transfer By Intratestis Injection

Sperm-mediated gene transfer approach transfer foreign gene to embryos through fertilization. It has many merit, such as simplicity, hight efficency, not require costly equipment et al., while other transgenic method have different defect, for example being the most widely-used method for transgenesis, microinjection requires costly sophisticated equipment and special experimental skills. Though successfully applied to mice, the experiment efficiency of microinjection is much lower when applied to other species, such as livestock or marine animals. And it's not fitful to mammalian animal for not having clear pronuclear. In vitro sperm mediated gene transfer had high unstable transgenic rate, and had many factors affect its transgenic rate. Different lab's results had great difference, even paradox. Intratestis injection as method to transfer foreign gene into sperm can avoid inhibitory factor and complicated dealing of sperm, ensuring fertilization and embryo development. Goat is the first choice for produce mammalian bioreactor. While there had not research report of goat in vivo sperm-mediated gene transfer abroad. Our research can provide some reference for other gene transfer protocol and transgenic research.Our experiment mainly investigated spermatozoa transfection rate's relationship with injection position, dose, and sperm collection time, and possibility of in vitro fertilization to produce transgenic embryos following intratestis injection.Two local male goats were selected; seminal duct and testis tissue were selected for injection DIG labeled plasmid pEGFP-N1. Sperm was collected following 20, 30, 40 days after injection. The integration of pEGFP-N1 in the sperm was determined by PCR and Southern blotting. Transfection rate was determined by DIG Labeling and Detection Kit, results shows that testis tissue injection has the higher and long-last transfection rate. Other four local male goats were selected; 3,4,5,6 ml of 1.0~1.5μg/μl pEGFP-N1 plasmid DNA were injected into 4 goat of bilateral testes tissue. Sperm was collected after 20,30,40,50 days. Sperm of 40 days following testis tissue injection capacitation were induced by IA...