The Study On Early Transgenic Embryos Culture And Identification In KM Mice

The objective of this experiment is to establish an effective method for selecting transgenic embryos before being transferred to recipient. SPF KM mice were selected as experimental animals. By studying on nest culture method, selection of culture media, reliability analysis of pig oviduct epithelium cells co-culture system, an effective system for culture of early embryos in vitro was built. On the base of experiments above, the effects of microinjection and the collection time on the development of embryos were studied, and the retention of HSA gene in mouse embryos cultured in vitro was detected. The results were follows:1. All embryos cultured in the single dish in which the suspensions were not covered with mineral oil were blocked at 2-cell stage. However, 36% of embryos cultured in the single dish in which the suspensions were covered with mineral oil developed to blastocyst stage. Whether the suspensions in 35mm dish in nest culture method was covered with mineral oil or not, the developmental rate of embryos had no significant difference. Compared with the nest culture method, the developmental rate of embryos in Brinster's method was significant lower. The results above showed that the nest culture method is effective for early embryo culture in vitro.2. Comparison of the development of embryos cultured in media WM, mWM, CZB and TE, it was found that mWM, CZB and TE effectively overcame the two-cell block of mouse embryos, and further supported 76%, 45% and 52% embryos developed to stage respectively. However, the blastocyst rate of embryos cultured in TE was significantly higher than mWM and CZB. The effects of 2.5mmoL taurin combined with glucose at different concentrations on the early development embryos in vitro were compared, and the results showed that 2.5mmoL taurin combined with 10mg/100mL glucose was the best combination, it made 78% embryos develop to blastocyst stage.3. There was no significant difference between the blastocyst rate of embryos co-cultured with primary and fist generation pig oviduct epithelium cells derived from pig oviduct during same estrous cycle. Blastocyst rate of embryos co-cultured with pig oviduct epithelium cells came from pig oviduct during diestrus was significantly lower than metestrous. 4. The blastocyst rate of transgenic embryos collected at 24~26 hours and 18~22 hours after hCG-treated reached 45% and 20% respectively. 5. Comparison of the development of embryos microinjected with HSA gene and the development of embryos without microinjection, the results showed that the developmental rate of embryos microinjected was significantly lower than that of embryos without microinjection when considering number of 1-cell embryos as the parameters, but there has a insignificant difference between the developmental rates of embryos with microinjecting and embryos without microinjection when considering number of 2-cell embryos as the parameters. This illustrated that microinjection was one main reason that decreased the developmental rate of 2-cell embryos, but it did not affect the development of transgenic embryos behind 2-cell stage.6. The retention of HSA gene in mouse embryos was detected by PCR, and the results showed that with development of early embryos microinjected with HSA, rate of transgenic embryos was decreasing gradually.