| The technology of embryonic stem(ES) cell and animal cloning is two high technique which develop rapidly in an area of medicine and biology. It has great significance for the studies on basic theories of medicine and biology, and it will be applied wildly in many aspects. In order to establish the technique of isolating ES cell, and test the developmental ability of reconstructed embryos derived from ES cell and oocytes of intra- or inter-species, mouse ES cell were isolated from mouse blastocysts, and were used as donor nuclear, and were transferred into oocytes of various strains. The factors that influenced the development of reconstructed oocytes were determined. The developmental ability of embryos reconstructed with mouse ES cells and rabbit oocytes was observed and analyzed.The methods of Evans and Martin were changed slightly and used to isolate the mouse ES cell in my experiment. In brief, the intact blastocysts were plated on STO feeder layer treated with mitomycin, and were cultured in the media supplymented with BRL condition medium. Two mouse ES lines was established, and were cultured more than 30 passages, which showed AKP and SSEA-1 strong positive. Karyotypes of two ES lines were normal 40XY. ES cells of diploid were more than 90%. One chimaeric mouse was produced by injection of ES cells into blastocyst of C57BL/6J. The chimaeric rate of fur colour was about 15%. Fl generations of germ-transmission derived from ES cell were not obtained when chimaeric mouse mated with KunMing Albino mice and C57BL/6J mice. The results showed that ES cells derived from KunMing Albino mouse remained undifferentiated state when they were cultured in vitro for a long time, and karyotypies were stable, but the ability of germ-transmission have not been verified.There were great effects of the genetic constitution of ES cell on the development of embryos reconstructed with ES cells in intraspecies. In order to know if the genetic constitution of recipient oocytes influenced the development of embryos reconstructed from ES cell, Oocytes of C57BL/6J and KunMing Albino were used as recipient, respectively. There was no significant difference between the fusion rates of reconstructed embryos. The cleavage rate of embryos reconstructed with C57BL/6J oocytes was significant high than that of KunMing Albino, but the former only developed to 4-cell stage; the later could develop to blastocyts. It is implied that the oocytes of inbred mouse cannot support the long-term development of KunMing ES cell. Meanwhile, there was different in the development of embryosreconstructed with various strain oocytes.The activation methods, passages and types of ES cells were comparing in mouse cloning. Two types of fibroblast cells were used as donor control, one derived from ear skin of an adult KunMing albino mouse, and the other derived from a mouse fetus. Three types of cells were transferred into perivitelline space under zona pellucida of rabbit oocytes respectively. The reconstructed oocytes were fused and activated by electric pulses, and cultured in vitro. The developmental rate of reconstructed oocytes derived from embryonic stem cells was 16. 1%, which was significantly higher than that of both the adult mouse fibroblast cells (0-3. 1%,/KO.05) and fetus mouse fibroblast cells (2.1-3.7%, /XO. 05). Chromosome analysis confirmed that blastocyst cells were derived from ES donor cell. These observations show that reprogramming is easier in interspecific embryos reconstructed with ES cells than that reconstructed with somatic cells, and that ES cells have the higher ability to direct the reconstructed embryo development normally than fibroblast cells.Oocytes were reconstructed with outbreeding Kunming albino mouse ES cells and enucleated rabbit oocytes, and the effects of the passages of ES cells and 6-DMAP on the development of interspecific reconstructed oocytes were analyzed. The interspecific reconstructed ES-rabbit oocytes were activated either by combined two set electric pulses and 6-DMAP or by two set electr...
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