Type-A spermatogonia first appear at between 3-7 d postnatally in mice and are the only immortalized diploid cells that reproduce in adulthood in these animals. In our current study, we explored the feasibility of producing stable transgenic mice using these cells. Enhanced pEGFP-N1 plasmids were suspended in ExGen500 transfection reagent and injected at different angles into the testes of 7-d-old male ICR mice. The resulting type-A spermatogonia-mediated gene transfer （TASMGT） mice were then mated with normal females at different stages of sexual maturity （6, 12, and 24 wk）. The integration and expression of the introduced EGFP gene was evaluated in the F1 transgenic offspring by PCR and Southern blotting analysis. The foreign gene integration rates for a low-dose group （15 μL gene suspension injected into each testis） and a high-dose group （30 μL suspensions injected） at the three stages of female sexual maturity tested were 11.76% （2/17）, 14.29% （3/21）, and 11.11% （2/18）, and 5% （1/20）, 5.56% （1/18）, and 0 （0/17）, respectively. The average integration rates for these two dose groups were 12.5% （7/56） and 3.64% （2/55）, respectively, which was a significant difference （P0.05）. Semi-quantitative RT-PCR analysis further showed that the introduced GFP gene was expressed in 3/9 integration mice. In addition, GFP expression was observed in the sperm cells from the TASMGT mice, and also in the embryos and F2 pups from the F1 generation transgenic mice. Hence, although the foreign gene integration rate for TASMGT is not high and the transgenic offspring show as yet unexplained defects, our results indicate that this method is a potentially feasible and reproducible new approach to creating transgenic mice.
JU Hui-mingBAI Li-jingREN Hong-yanMU Yu-lianYANG Shu-linLI Kui
Polyunsaturated fatty acids （PUFAs） are essential for normal growth in mammals, especially the ω-3 PUFAs, which play important roles in preventing several life-threatening diseases, such as coronary heart disease and diabetes. In this study, we aimed to investigate whether the sFat-1 gene from Caenorhabditis briggsae could be functionally expressed in transgenic pigs, and whether the transgenic could synthesize high quality ω-3 PUFAs endogenously. In this study, a gene construct consisting of CMV promoter and 1.9 kb cDNA of ω-3 fatty acid desaturase gene （sFat-1） from C. briggsae was injected into the male pronucleus of pig embryos by microinjection. The piglets were screened for the transgene by PCR, Southern blot and reverse transcription-PCR analysis. Pigs that give positive results were mated with wild-type pigs to produce the next generation and the transmission of transgene was examined by PCR analysis. Fatty acids compositions of various tissues in the transgenic pigs were then analyzed by gas chromatograph. A total of 878 embryos were transferred into 42 recipients, among which 29 successfully got pregnant and gave birth to a total of 162 piglets, and 8 of them were identified to be transgenic. Fatty acid compositions in the transgenic pigs were altered, and the levels of ω-6：ω-3 ratios were decreased from 14.53 in the control to 2.62 in Fat-1 transgenic pigs. A number of primary sFat-1-transgenic pigs were bred in this study, which lays the foundation for cultivation of new varieties of transgenic pigs.
