Optimization Of The Culture System On Kunming Mouse ESC

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.