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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
In order to enhance the ability of retrovirus constructs to transfect target cells, we successfully purified three constructed antisense c-myc retroviral vectors using the Wizard TM DNA purification system and liposome-mediated packaging PA317 cells, the titer of the PA317-resistant cell clone was measured using NIH 3T3 cells, and the integration of exogenous genes was detected by neo gene PCR amplification. The results showed that the purified DNA reached the requirements of eukaryotic transfection, but the packaging titers were highly influenced by the target cell growth state and the amount of PA317 sample. The PCR detection of neo gene was a sensitive, economical and reliable The gene integration test