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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~(TM)DNA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NIH3T3细胞测定了PA317抗生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:1.纯化后的DNA260mm和280nm的OD值均大于1.8,且在琼脂脂糖电泳电中未有明显的RNA出现,说明DNA达到了真核细胞转染的要求,经灭菌和浓度调整后即可用于细胞转染.2.经测定,PA317的病毒滴度达到10~4级,这主要是因为病毒的感染效率不仅直接受病毒载体类型的影响,而且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.3.目的基因整合的检测;在所有抽取的转基因克隆细胞的基因组DNA中均能扩增出一条特异的、长430bp的neo基因片断电泳条带,而未转染组则无此条带.在检测基因转染后是否发生了染色体整合上,可用用Southem杂
In order to enhance the ability of retrovirus constructs to transfect target cells, three antisense c-myc retroviral vectors were successfully constructed using Wizard ~ (TM) DNA Purification System and purified by lipids PA317 cells were cultured in vitro and the titer of PA317 cells was measured by NIH3T3 cells.The PCR products of neo gene were used to detect the integration of foreign genes.The results showed that the OD260mm and 280nm Greater than 1.8, and in agarose electrophoresis no significant RNA appeared, indicating that the DNA reached the eukaryotic transfection requirements, after sterilization and concentration can be used for cell transfection.2.According to the determination, PA317 Of the virus titer reached 10 to 4, mainly because the virus infection efficiency is not only directly affected by the type of viral vectors, but also the level of its packaging titers are also highly affected by the target cell growth status, PA317 sample size. Purpose gene integration detection; in all the extracted transgenic cloned cells can amplify a specific genomic DNA fragment 430 bp long neo gene electrophoresis bands, but not in the untransfected group was not detected in the detection of gene Is it transfected? Chromosomal integration of birth, can be used with hybrid Southem