| Embryonic stem (ES) cells, derived from preimplantation embryos, are undifferentiated, immortal cells capable of differentiating into derivatives of all three embryonic germ layers.Pluripotential ES cells have been used extensively in studies of embryogenesis. gene function, and development in the mouse. ES cells transferred to a mouse blastocyst can contribute substantially to all differentiated cell types in the fetus, including the germ line. Consequently, gene targeting within ES cells has enabled both Whole-animal studies of gene function and the production of mouse models of human genetic diseases and abnormalities. ES cells have also been used to stud}' the differentiation on various cell types and tissues in vitro, such as neural cells, hematopoietic lineages, and cardiomyocytes. ES-derived cells have been successfully transplanted into fetal and adult mice. Where they have demonstrated morphological and functional integration. ES cells would have broad applications in basic research and transplantation therapies. The major object of this paper is to isolate and clone ES cells from C57BU6 strain mouse as well as demonstrating pluripotem of ES cells from various aspects. The main contents as follows.(1) Derivation of ES cells from the mouse blastocysts.Three days after ovulation, 535 blastocysts were recovered by a surgical uterine flush technique from C57BL/6 mice and played on passage 1 to passage 5 mouse embryonic fibroblasts feeder layer mitotically inactivated with mitomycin C (Img/lOOml for 4 hours). After 4 days of growth, the intact inner cell mass(lCM) was separated with 0.25% trypsin-EDTA and again played on feeder in Dulbecco's modified Eagle medium(4500mg of glucose per liter) supplemented with 15% newborn bovine serum, 0.1 mM nonessential amino acids, 0.1 mM 2-mercaptoethanol. 2mM glutamine, 50 units/ml of penicillin, and 50 units/ml of streptomycin. Cultures were grown in 5% COi, 95% humidity and 37癈 in incubator. After 7 to 10 days of culture, 15 ES cell colonies were selected and explanded. If ES cells were plated at low density in the absence of fibroblasts on four-well tissue culture plates in the same medium as that used for initial cell line isolation, these ES cells were induced to generate epithelial-like cells and endodermal-like cells. If ES cells were plated at high density. They wouldaggregate to formate cystic embryoid bodies.(2) Aggregation of ES cells and tetraploid embryosThe oviducts of superovulated Kunming white females were flushed 44-46 hours after treatment with human chorionic gonadotropin to collect 1074 late two-cell-stage embryos. The embryos were placed twenty at a time between two platinum electrodes laid 1mm apart in 0.3M mannitol in the electrode chamber. The blastomeres were fused by a short electric pulse(80V for 50 u sec) applied by a pulse generator. Fusion of blastomeres should be completed in 20-60 minutes. After 25 hours of culture, most of the tetraploid embryos developed to the four-cell stage. Zonae pellucidae of the 387 four-cell-stage tetraploid embryos were removed by treatment with acid Tyrode's buffer. The embryos were plated on ES cell layer. After 40 hours of coculture, 248 embryos aggregated with ES cells were collected and transferred into the uteri of twenty four 2.5-day pseudopregnant recipients. Ten recipients were pregnant, but no live fetuses were born. Three pregnant recipients were routinely subject to a Caesarean section on day 18 of pregnancy and got seven abnormal fetuses. The results demonstrated that the ES cells derived from C57BL/6 mice were pluripotential to a certain extent. The low efficient production of completely ES cell-derived animals was attributed to the bad recipient embryos and too high a contribution from the ES cells. At the same time, the tetraploid embryos were plated on embryonic fibroblasts feeder layer and formatted ES-like colonies, but the colonies would lysis after 5 to 6 days of growth.(3) Enucleation of ES cellsSome of the most basic and interesting problems in eukaryotic cells are th...
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