Abstract:

o prepare mouse fibroblast feeder layers and to establish C57BL/ 6 mouse embryonic stem cell(ESC) lines and to provide  basis for the further establishment of human ESC lines.Methods The mouse embryonic fibroblasts(MEF) were isolated from the mouse embryos at 13.5-14.5 d by primary tissue digestion method and the 3.5 d blastulae of C57BL/6 mice were cultured on the mouse fibroblast feeder layer incubation;the inner cell mass(ICM) was separated and amplified and passaged over 40 generations.The growth of the MEF  colonies was observed.The ESC colonies were identified byalkaline phosphatase(AKP) staining,early embryo-specific surface antigen(SSEA-1) and octamer-blinding transcription factor 4(OCT-4) staining,and differentiation experiments in vivo.Results The  ESC after  isolation and culture could stably passage to more than 40 generations and showed acolony-like growth.The ESC showed a red-brown positive expression after AKP, SSEA-1 and OCT-4 staining.The teratomas formed after ESC was inoculated in nude mice.HE staining showed that there were three germ layer tissue components in ESC.Conclusion The C57BL/6 mouse ESC lines are successfully established.

Key words: embryonic fibroblast;C57BL/6 mouse;embryonic stem cell;alkaline phosphatase