绵羊胎儿成纤维细胞体外培养及转基因研究

Study on in vitro Cell Culture and Transgene for Sheep Fetal Fibroblasts

目的 用增强型绿色荧光蛋白 (EGFP)基因转染体外培养绵羊胎儿成纤维细胞 ,探讨绿色荧光蛋白对绵羊胎儿成纤维细胞生物学特性的影响。方法 体外分离培养绵羊胎儿成纤维细胞 ,经脂质体介导EGFP基因转染第一代成纤维细胞 ,G4 18筛选 10～ 12d ,挑选转基因单克隆细胞 ,传代培养 ,进行细胞形态观察、生长曲线以及染色体核型分析 ,并进行了培养细胞性别鉴定。结果 整合有EGFP基因的绵羊胎儿成纤维细胞生物学行为与未转染外源基因的细胞无明显差别 ,根据荧光强度可直接反应外源基因的表达量。结论 EGFP基因作为体内报告基因可用于转基因细胞的研究 ,并将整合有EGFP基因的转基因细胞为克隆动物提供核供体奠定了基础。

Objective Sheep fetal fibroblasts(SFF) were transfected by enhanced green fluorescent protein(EGFP) gene to investigate the effect of the foreign gene on the biological characteristics of SFF. Methods SFF were dispersed and cultured in vitro.pEGFP-C1 was transfected to the passage 1 SFF by liposomes.The SFF were incubated in medium containing G418 for 10-12 days.Resistant colonies were picked and subcultured until use.The SFF were examined by cell morphology,growth curve or the nuclear type of chromosome.Sex-determining region Y gene(SRY) of the SFF was identified.Results The biological characteristics did not differ significantly between the resistant colonies and the control cells.Expressive amount of foreign gene was revealed by fluorescent intensity.Conclusion The present research can provide a support for the transgenic cell with EGFP as the report gene in vitro.Transgenic donor cells integrated with EGFP gene provide an efficient new strategy for the production of transgenic farm animals.