Study On The Production Of Chimeras Derived From The Transgenic Embryonic Stem Cells By Blastocyst Injection

Production of chimeras is the only method proving whether the embryonic stem(ES) cell can be cultured in vitro and maintained the ability of germ line differentiation, which has an impotent place in establishment of ES cell system, introduction of foreign genes into ES cells and production of germ line chimeras, also plays as a key step to obtain transgenic mouse through embryonic stem(ES) cell. The experiment used ES cells line(S8 cells) transfected by LacZ gene, which was cultured on mouse embryonic feeder layers made by fibroblast of mouse embryo, and through blastocyst injection, the Sg cells that had been digested into single cell were injected into the blastocyst of the outbreed mouse straining Kunming albino females as the provider for recipient -embryo which had grown for 3.5 days post coitum(d.p.c), then transplanted the manipulated embryos into the uterus of 2.5 d.p.c. pseudopregant Kunming albino female recipients to allow the embryos to develop in vivo after recovering culture, which offsprings were identified one week after the chimera mouse being born according to the contribution of ES cells to tissues of chimeric mice by coat color. The experiment was treated total 597 embryo with Sg cells of 8-13 generation through blastocyst injection, there were 585 embryo which got blastocyst cavity again after recovery culture of 1-3 hours, not only the figure of cells were clear, but also the juncture between the cells of feeder layers could be seen clearly, the surviving rate of embryo achieved 97%. All 37 liver-born offsprings were obtained after 17- 19 days of gestation by using embryonic transfer(the fetiferous rate was 16%), among which 8 are chimeras (the generative rate of chimera was 21.6%)with coat color. The result showed that the chimeras mouse can be generated by blastocyst injection using the ES (Sg) cells, and all of them occurred the sex-converted. This experiment was firstly shown that chimeras could be obtained in china at present by using S8 calls transfected LacZ gene.