三种反义c-myc逆转录病毒表达载体的纯化、病毒包装、滴度测定及整合检测

为了增强逆转录病毒构建体对靶细胞的转染能力 ,本实验使用 Wizard TMDNA纯化系统对构建成功的三种反义 c- myc逆转录病毒表达载体进行了纯化 ,并经脂质体介导包装 PA317细胞 ,使用 NIH 3T3细胞测定了 PA317抗性细胞克隆的病毒滴度 ,用 neo基因 PCR扩增检测外源基因的整合情况。结果显示纯化后的DNA达到了真核细胞转染的要求 ,但其包装滴度的高低还受靶细胞生长状态、 PA317抽样量的高度影响 ,而neo基因 PCR检测是一种敏感、经济和可靠的基因整合检测法

Purification Packaging, Titer and Detection of C-myc Retrovirus Expression Vector

In order to obtain high transfective ability of retroviral constructure, Wizard TM DNA purification system was applied to three kinds of antisense c myc retroviral expression vectors which used to transfect PA317 package cells with lipofectine, then viral titers were assayed and external gene integration were examined by neo gene PCR amplification. The results indicated that the purified DNA accorded with the demands of eukaryonic cell transfection, however, the viral titers were influenced by the growth of target cells and the number of PA317 sampling in the meanwhile. Neo gene PCR amplification was also indicated a sensitive, economical and reliable test for gene integration.