| In this study, mouse ES-D3 cells were used for optimizing ES-D3 cells feeder cellculture system, cryopreservation system and the plasmid pN1-EGFP transient transfectionsystem, in order to find the best culturing, cryopreservation and transfection system, andprovide bases for the later research on production of transgenic animals mediated byembryonic stem cells (ES). The specific test results were as follows:1. In this paper, mouse 13.5d old embryos were used to be digested by three differentmethods to obtain primary mouse embryonic fibroblasts (PMEF), and the effect of differenttemperature, time with mitomycin treatment, myeoplasma contamination on the MEFgrowth performance were investigated..The results showed that effects on cell survival rateand adherent rate of different digestion methods were significant When digested mouseembryos with a 5～10ml 0.25％trypsin-0.04％EDTA. 2Na, at 37℃, 5％CO2, saturatedhumidity incubator for 5～10 rain, cell survival rate (91.83±3.17％) and adherent rate(87.50±1.22％) were significantly higher than the other three groups. The mouse embryonicfibroblasts had typical features. The results showed that the MEF were temperaturesensitive. Under 37℃, cell adhesion rate (87.50±1.29％) was the highest, which indicated37℃was the best growth temperature. With the increase of temperature, adherent cells ratewas significantly decreased. At 37.8℃, cell adhesion rate was less than 30％. When treatedMEF with 10μg/mL mitomycin C to produce feeder cells, the optimal treatment time was2.5h. Time with Mitomycin C treatment have a great influence on the feeder ceils. Iftreatment time is too short, the cells growth can't be inhibited, or if the processing time istoo long, it will cause the death of the feeder cells, thus affecting the cultivation of ES cells.MEF cells of mycoplasma contamination were also detected, mycoplasma contaminationhad a significant effect on the growth performance of MEF, contaminated embryonicfibroblasts morphology were not typical, and apoptosis was severe. 2. ES cells cryopreservation efficiency were analysed with different varieties andconcentration of serum, and different transfection methods with the exogenous gene(pN1-EGFP) were also used to study the transfection efficiency on ES-D3 cells. The resultsshowed that the effect of different serum concentration on frozen efficiency of ES-D3 cellswas siguificant. When serum concentration in the frozen liquid was 60％, the rate of livecells after thawing was the best with typical cell clones shape and positive AKP. Undersuspension culture in vitro, the cells could be a typical EB-like organizations. Withdifferent serum varieties of domestic and imported fetal calf serum (FCS), the ES-D3 cellsfrozen efficiency was significantly different. When the ES-D3 cells were transfected withexogenous gene pN1-EGFP, the transfection efficiency of suspended transfection methodwas 88.72±0.91％. But alter transfection, ES-D3 clones forms were not typical, and easy todifferentiate. Adherent transfection, under the optimum conditions (DNA:Lipofectamine2000=2μg: 12μL), ES-D3 transfection efficiency could be achieved at74.45±2.16％. Transfection efficiency was significantly lower than suspension transfectionefficiency, but ES-D3 clones had typical morphology and differentiation phenomenon wasnot apparent alter transfection.
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