Abstract:

BACKGROUND: Up to date, hundreds of mouse embryonic stem cell (ESC) line has been established. Mouse of Kunming line is commonly used in experiment, and rare reports have addressed the success establishment.
OBJECTIVE: To establish simple, efficient protocol for the derivation of ESCs from Kunming mice, and to elevate the success rate of establishment.
METHODS: Mouse ESCs were incubated with the media containing serum surrogate or fetal bovine serum. Females of Kunming mice were superovulated with an injection of priganant mare’s serum gonadotropin and human chorionic gonadotropin. Blastocysts were collected by flushing the uterus at 3.5 days after copulation. Embryos were transferred in dish with fresh mouse embryonic fibroblasts feeder layer. Blastocysts detachment and colony forming efficiency of inner cell mass were observed. 4-6 days after culture, inner cell masses were digested mechanically in 0.05% trypsin-0.02%ethylenediamine tetraacetic acid.The disaggregated inner cell masses were transferred into a new feeder layer. Typical ESCs morphology was observed through an inverted microscope.
RESULTS AND CONCLUSION: Pregnant mice of 3.5 days were the best choice to derive ESCs. Inner cell masses were separated through cutting in drops of 0.05% trypsin-0.02% ethylenediamine tetraacetic acid. The disaggregated inner cell masses grew more rapidly and maintained less differentiation. The ESCs were passaged many times in fetal bovine serum medium. This medium is more suitable to separate ESCs from Kunming mouse.