| There are still some problems in gene transfer in animals. For example, it is difficult and costly to predicate the efficiency of the gene transfer. Therefore, it is important to culture the fertilized eggs to morula or blastocyst stage for transferring and identify foreign gene integration in genome before the embryos transfer to receptors, in order to increase efficiency of producing transgenic animals. In this paper, the construct that sheep metallothionein (sMT) gene promoter directs pig growth hormone (PGH) gene was microinjected into the pronucleus of mouse fertilized eggs. Then, the effects of several culture media on overcoming the 2-cell block in embryo development were studied, the blastocysts containing foreign gene were measured by using PCR method. A total of 1697 eggs collected from 58 superovulation mice, of which I 279eggs was in pronucleus stage and microinjected with sMTPGH, and there were 392 eggs survived after microinjection and were cultured in vitro by three media. In the experiment, the fertilized eggs overcome the 2-cell block and developed to blastocyst stage in the modified M16(mMI6) media. In addition, the mM 16 contented epidermal growth factor (EGF) improved blastocyst rates at day 3 embryos cultured in vitro. The results showed that 12%, 68% and 900/a embryos passed the 2-cell block and developed to 4-cell stage, and 0, 20% and 43% developed to blastocyst stage in M16, mMl6 and mMl6+EGF media, respectively. It seems that taurine plays an important role in overcoming the 2-cell block during transgenic mouse embryo development and EGF maybe produce some material which can eliminate the toxin came from the embryos or the media. The transferred gene was measured by using PCR method in 57 blastocysts, 4 blastocyst retentions of foreign gene. The results showed that the rations of foreign gene integrated into genomes in blastocyst stage were 7%. This method might provide us a way to screen transgenic embryos when we use embryo section technique in farm animals. |
PDF Full Text Request