【摘要】 为了为核移植法制作转人溶菌酶基因克隆牛提供供体细胞,研究体外分离培养牛皮肤成纤维细胞并进行了人溶菌酶基因转染。首先采用组织块贴壁法分离培养牛耳成纤维细胞,经传代纯化后,脂质法将含有双重筛选标记的人溶菌酶乳腺特异性表达载体转入体外培养的第4代牛皮肤成纤维细胞,500μg/mLG418筛选2周,G418减半继续培养2～3代,然后对转染筛选后的细胞进行PCR检测及核型分析。结果表明,转染24h后荧光显微镜下检测到有绿色荧光蛋白表达,传至15代的细胞核型正常,含有正常牛的二倍体核型(60,XX),PCR检测得到大约850bp的片段,说明目的基因已成功导入并表达。研究所获得的转基因牛耳成纤维细胞整合并表达了外源基因,同时保持了遗传稳定性,可作为体细胞核移植的供体细胞进行转基因克隆牛研究。更多还原

【Abstract】 In order to offer doner cells for bovine transgenic cloning by nuclear transfering, bovine fibroblast cells were isolated by attaching tissue explants from ear skin of a bovine and purified by passage. The plasmid containing genes of human lysozym and two selecting marks(the EGFP and Neor genes) which express specifically in mammary gland was transfected into forth passages bovine fibroblast by Lipofectmine TM2000, and G418 selection (500 μg/mL) was applied since then. After 2 weeks, positive colonies were maintained in culture medium containing 250 μg/mL G418 for 2 ~ 3 passages. A small part of the positive cells were analyzed by PCR for gene integration, and karyotype was analyzed. Results showed that EGFP was expressed 24 h after transfection under fluorescence microscopy. The karyotype of the 15th passages was normal and diploid (60,XX). The fragment of 850 bp was detected by PCR, demonstrating that the foreign gene was integrated into the genome and could be expressed. The results indicate that the obtained cells may be competent for bovine transgenic cloning.更多还原