关键词: 小鼠; 胚胎成纤维细胞; 分离培养

Abstract: To establish a stable culture system of mouse embryonic fibroblast(MEF),in order to construct of MEF feeder layers in vitro and somatic cell nuclear transfer technique nuclei donor build platform. The mouse primary embryonic fibroblasts were isolated from mouse fetus of Kunming white at different gestational ages. According to the influence of different serum and trypsin digestion time on MEF, the growth behavior and the characteristics of MEF were studied. Results showed that, in the form of four gestational ages 8.5 d, 10.5 d, 12.5 d, 14.5 d trials, the optimal gestational age for isolation of MEF was 12.5 d;in the form of five serum concentrations 7%, 9%, 10%, 11%, 13% trials, add 11% tire bovine serum concentration was the best, multiplication factor from 3 to 5 stable at around 1.35. These results indicated that the use of 12.5 d fetal rats, trypsin digestion 5 to 10 min and in serum containing 11% DMEM medium MEF cells in training. The original generation and nestin was better. MEF (3 to 5 generations) were quite suited to be a feeder layer and somatic cell nuclear transfer technique nucleic donor.

Key words: mouse; embryonic fibroblast; isolation and culture