Isolation and culture of murine embryonic stem cells of Kunming species / 中国组织工程研究

ABSTRACT

BACKGROUND: Hundreds of mouse embryonic stem cell lines are established presently. Most of these cell lines are collected from 129, C57BL/6J and BALB/C mice. Success rate of creating embryonic stem cell lines with Kunming mice is very low. OBJECTIVE: To explore the method of isolation and culture of murine embryonic stem cells of kunming species to increase the efficiency of establishment of embryonic stem cell lines from Kunming species mouse. DESIGN, TIME AND SETTING: The cell experiment was performed at the Chongqing Key Laboratory of Neurology from April 2006 to June 2007. MATERIALS Blastocysts of 3.5 days and 4 days from Kunming species mouse were collected. METHODS: The blastocysts of 3.5 days and 4 days from Kunming species mouse were cultured on the fibroblast cell feeder layers for 4-5 days, and the cells from inner cell mass were isolated and subsequently cultured in vitro in 2.5 g/L trypase-0.4 g/L ethylenediamine tetraacetic acid (EDTA) and 1 g/L trypase-0.2 g/L EDTA at room temperature for 2-4 minutes. Embryonic stem cell colony was mechanically straggled. MAIN OUTCOME MEASURES: Colony growth was observed and determined by alkaline phosphatase staining, OCT-4 staining and karyotype analysis. RESULTS: Adherence rate, inner cell mass frequency and cloning efficiency of 4 day post coitum (dpc) embryo were higher than those of 3.5 dpc embryo (P