SV40 T基因转化的山羊乳腺上皮细胞系及其生物学特性

目的建立能用于乳腺特异表达基因构件质量检验的山羊乳腺上皮细胞系.方法根据已发表的SV40病毒T基因序列设计引物,以整合有SV40 DNA早期基因区的COS-1细胞基因组DNA为模板,用高保真PCR扩增SV40 T基因.将获得的SV40 T基因克隆入真核表达载体,并用获得的重组表达质粒转染山羊原代乳腺上皮细胞.经有限稀释和反复传代后获得转化细胞克隆,对其生物学特性进行研究.结果扩增出序列正确的SV40T基因,重组质粒转染获得的转化细胞的对数生长期为接种后第4天,细胞群体倍增时间为23.5*!h,克隆形成率为26.7%.DNA斑点杂交试验证明转化细胞的基因组中整合有SV40 T基因,染色体核型分析试验表明转化细胞的核型无明显异常,裸鼠接种试验证明转化细胞不能形成肿瘤,软琼脂集落形成试验表明转化细胞在软琼脂中不能生长.部分细胞克隆已在体外传30代以上,保持正常乳腺上皮细胞的形态特征,在胶原基质上能形成腺泡样结构.结论本研究获得的SV40 T基因转化的山羊乳腺上皮细胞具有转化细胞系的生物学特性.

Establishment and Characterization of SV40 T Gene-Transformed Dairy Goat Mammary Gland Epithelial Cell Lines

Objective To establish goat mammary gland epithelial cell lines for evaluation of mammary gland-specific expression components.Methods A pair of primers were synthesized according to the previously published DNA sequence and used to amplify the SV40 T gene from COS-1 cells by high fidelity PCR.The SV40 T gene was subcloned into eukaryotic expression vector pTarget with its neo gene deleted and the resulting recombinant vector pTarget-LT was transfected into primary goat mammary gland epithelial cells.The transformed cell clones were obtained by limited dilution and repeated passage.Results An expected PCR product of 2.5?kb was obtained,which was 99% identical in sequence to the previously published SV40 T gene.Following transfection and repeated in vitro passage,49 clones were obtained with a clone forming rate of 26.7% and a logarithmic growth period at the forth day post-plating.Dot blotting analysis showed that the SV40 T gene was integrated into the transformed cell genome.The transformed cells had a normal karyotype with no tumorigenicity to nude mice and were not able to grow in soft agar culture.One of the transformed cell clone had been passed for more than 30 generations,which maintained normal morphology of mammary gland epithelial cells and was able to form glandulous structures on collagen matrix.Conclusion These results showed that the transformed mammary gland epithelial cell line shared similar biological characteristics of other transformed cell lines.