USE OF RETROVIRAL SHUTTLE VECTOR FOR INFECTION OF ONCOGENES INTO HUMAN CELLS

将外源基因导入逆转录病毒穿梭载体提供了 高感染多种人类细胞类型的机会 频率V-Ha-ras癌基因的编码区被克隆到 定向插入小鼠逆转录病毒穿梭载体pZip-neo SV(X)。 含有Ha-ras编码区的细菌克隆的质粒DNA被 磷酸钙分离及转染NIH3T3细胞的研究 沉淀法。每6个克隆中就有1个是生物学上的 活跃于转化灶的诱导。 质粒DNA(pZip-neo-ras和pZip-neo-sar定位)为 磷酸钙沉淀法导入Psi-2细胞 方法。将G418抗性克隆转入psi-2细胞，分离并鉴定 他们的培养上清液被滴定以用于病毒生产。病毒滴度约为 10~5次方病毒/毫升。随后的感染 用Psi-2细胞产生重组病毒的PSI-Am细胞产生病毒 股票的5×10的三次方-10的四次方病毒/毫升。

The introduction of foreign genes into a retroviral shuttle vector offers the opportunity of infecting a variety of human cell types at high frequency. The coding region of the v-Ha-ras oncogene was cloned in both orientations into the murine retroviral shuttle vector pZip-neo SV(X). Plasmid DNA of bacterial clones containing the Ha-ras coding region was isolated and transfected into NIH 3T3 cells by the calcium phosphate precipitation method. One out of six clones transfected was biologically active in induction of transformed foci. The plasmid DNAs (pZip-neo-ras and pZip-neo-sar orientation) were transfected into Psi-2 cells by the calcium phosphate precipitation method. G418 resistant clones of transfected Psi-2 cells were isolated and their supernatant was titered for virus production. Virus titers of about 10 to the fifth power virus/ml were achieved. Subsequent infection of Psi-am cells with recombinant virus produced from Psi-2 cells yielded virus stocks of 5 x 10 to the third power - 10 to the fourth power virus/ml.