逆转录病毒载体介导的LacZ基因在NIH-3T3细胞中的稳定表达(英文)

Stable Expression of LacZ Gene in NIH-3T3 Cell Mediated by Retroviral Vector

精原干细胞(SSCs)介导的转基因技术很可能成为制作转基因动物及治疗雄性不育的一条新途径。为了研究逆转录病毒载体介导法转染体外培养SSCs的可行性,用脂质体介导法将携带LacZ基因的重组逆转录病毒载体pLNCL导入包装细胞PA317,用含G418的培养液筛选得到5株稳定转染的产毒细胞。收集这些克隆的产毒上清,过滤后进行倍比稀释,用NIH-3T3细胞通过X-gal染色测定其浓缩前病毒滴度。结果显示,PA3173培养上清中病毒的浓缩前滴度最高,达1.1×103CFU/mL。再将筛选到的稳定转染的NIH-3T3细胞培养至单层,进行X-gal染色检测β-半乳糖苷酶的表达。结果显示,大多数稳定转染的NIH-3T3细胞均为X-gal+,表明这些细胞成功表达了目的基因LacZ。本研究结果为后期工作中用该载体感染体外培养SSCs奠定了基础。

The transgenic technique mediated by spermatogonial stem cells (SSCs) might be a new way for the production of transgenic animals and the therapy of male sterility. To investigate the possibility of in vitro transfection of SSCs mediated by retroviral vector, recombined retroviral vector pLNCL carrying LacZ gene was introduced into packaging cell PA317 by liposome-mediated transduction. Five stable virus-producing cell lines were screened with the media containing G418. Then the virus-containing supernatant was collected from these clones and filtered to prepare a serial dilution. The pre-concentrated viral titer of the supernatant was determined with NIH-3T3 cell by X-gal staining. It's demonstrated that PA3173 gave the highest pre-concentrated viral titer of 1.1×103 CFU/mL. Subsequently, the stable transfected NIH-3T3 clones were screened and cultured to confluence. The X-gal staining was proceeded to detect the expression of β-galactosidase. The results showed that most of the stably transfected NIH-3T3 cells were X-gal+, which suggested that LacZ gene was expressed successfully in these cells.