Study On Sperm-mediated Gene Transfer Of FecB Transgenic Tai Hang Goat

Sperm-mediated gene transfer is the simple and efficient method of transgenic animal which refers to the sperm as the carrier of foreign genes into oocytes when fertilization, the fertilized egg contains the foreign gene. Liposomes can be used in transgenic research owing to its function that could carry the foreign gene into the cell interior by membrane fusion. This study focus on the influencing factors of the sperm binding with the foreign genes in the process of the sperm-mediated gene transfer and preliminary studies of sperm-mediated gene transfer of FecB transgenic Tai hang goat, the results are as follows:1. Washing the sperm fully by centrifugation before mixed the sperm and liposome-DNA complexes could significantly improve the liposome-mediated transfection efficiency of exogenous DNA with sperm. The positive rate of sperm transfection of washing group before DNaseI digestion was 38% while the positive rate of sperm transfection of the control group was 14%. The positive rate of sperm transfection of washing group after DNaseI digestion was 21% while the positive rate of sperm transfection of the control group was 5%, the difference was significantly between the treatments (P <0.01).2. Liposome-mediated the goat sperm transfect exogenous DNA could be detected after DNaseI digestion indicating that the liposome-mediated method allows the exogenous DNA into sperm by membrane fusion. 18.3% of the sperm were found with exogenous DNA by the use of fluorescence in situ hybridization. The ratio increased to 38.7% after lipofectamine2000 mediated. The statistical analysis shows that it was significantly difference (P <0.01).3. In this experiment, the co-culture method and liposome-mediated transfection of the goat sperm with the exogenous DNA could be detected after DNaseI digestion indicating that the sperm have the ability that binding of exogenous DNA spontaneously.4. Treat the 18 ewes with estrus synchronization and 14 of them were estrus, the estrus rate was 77.8%. Incubated the sperm with FecB gene, 8 ewes were pregnant after artificial insemination, the pregnancy rate was 51%. None positive lamb was found after PCR.