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This study investigated the effects of differentcultue systems, basal culture medium, cytokines, drugs, oxygen partial pressure and temperature on establishment of Kunming mouse embryonic stem cell (KM-ESC). The optimum culture condition that is appropriate for growing of KM-ESC was selected. The KM-ESC carrying GFP gene was established subsequently.This research compared the effects of 2 kinds of culture systems (KSR and N2B27) on KM-ESC. The results showed that: (i) in KSR culture system, ESC grew adherently and slowly and lots of cells on the fringe of colonies were differentiated or apoptosis fell off. ESC could be passaged to 6 generation. (ii) In N2B27 culture system, ESC grew suspendedly and fastly. Edge of colony was smooth. No cell was differentiated when ESC was passaged to 7 generation.On the base of N2B27 culture system, this study reasearched the effects of 4 basal culture medias (DMEM-high glucose,knockout DMEM,DMEM/F12,TCM-199), 3 cytokines (LIF, bFGF, EGF), 2 drugs (SU5402, heparin sodium),Oxygen Stress (5% O2, 20% O2) and temperature (37℃、39℃)on culture of KM-ESC. The results showed that: knockout DMEM and DMEM/F12 were more appropriate for the growing of ESC than DMEM with high glucose and TCM-199. (ii) LIF, bFGF and EGF siginificantly improved the growing of KM-ESC (P<0.05). (iii) Heparin sodium siginificantly improved the growing of KM-ESC (P<0.05). (iv)Effect of SU5402 on KM-ESC was not obviously. (v) 20% O2/ 39℃is beneficial to the colonies formation and proliferation of KM-ESC.Lentivivus vector with GFP gene was injected into the perivitelline space of zygotes. Then the injected couple was inoculated in N2B27 culture system. After culturation, KM-ESC was transefected successfully.
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