Wong B Y, Chen H, Chung S W, Wong P M
Morse Institute for Molecular Genetics, Department of Microbiology and Immunology, State University of New York, Brooklyn 11203.
J Virol. 1994 Sep;68(9):5523-31. doi: 10.1128/JVI.68.9.5523-5531.1994.
Retroviral gene transfer efficiently delivers genes of interest stably into target cells, and expression cDNA cloning has been shown to be highly successful. Considering these two advantages, we now report a method by which one can identify genes stimulating cell growth through functional analysis. The first step requires the construction of a retroviral cDNA expression library and the optimization of transfection of vector DNA into virus packaging cells. The second step involves the cocultivation of target cells with libraries of retrovirus-producing cells, resulting in the amplification of target cells transduced with a gene(s) stimulating cell growth. Under standardized conditions of transfection, we detected an average of 4,000 independent clones per dish, among which expression of a retroviral beta-galactosidase gene at an abundance of 0.2% could be detected. Next, we demonstrated the augmentation of the sensitivity of the assay by retroviral infection and functional analysis. We did this by cocultivating factor-dependent (FD) cells with dishes of GP/E cells transfected with plasmids containing various molar ratios of pN2-IL3 DNA and retroviral library cDNA and by determining the highest dilution of pN2-IL3 which still resulted in the conversion of FD cells to factor independence. The retroviral interleukin-3 gene at an abundance as low as 0.001% could be detected. Indeed, we were able to detect from FD cells the development of factor-independent colonies with different phenotypes after retroviral transfer of cDNAs from an immortalized hemopoietic stem cell line. Thus, the combination of a standardized high-efficiency DNA transfection and retrovirus-mediated gene transfer should facilitate the identification of genes capable of conferring to target FD cells a detectable new function or phenotype. By scaling up the size of the experiment realistically during screening, the assay can detect cDNA at an abundance of lower than 0.0001%.
逆转录病毒基因转移能够有效地将感兴趣的基因稳定地传递到靶细胞中，并且已证明表达性cDNA克隆非常成功。考虑到这两个优点，我们现在报告一种方法，通过该方法可以通过功能分析鉴定刺激细胞生长的基因。第一步需要构建逆转录病毒cDNA表达文库，并优化载体DNA转染到病毒包装细胞中的过程。第二步涉及将靶细胞与产生逆转录病毒的细胞文库共同培养，从而导致被刺激细胞生长的基因转导的靶细胞扩增。在标准化的转染条件下，我们每培养皿平均检测到4000个独立克隆，其中可以检测到丰度为0.2%的逆转录病毒β-半乳糖苷酶基因的表达。接下来，我们通过逆转录病毒感染和功能分析证明了该检测方法灵敏度的提高。我们通过将因子依赖性（FD）细胞与用含有不同摩尔比的pN2-IL3 DNA和逆转录病毒文库cDNA的质粒转染的GP/E细胞培养皿共同培养，并确定仍能导致FD细胞转化为因子非依赖性的pN2-IL3的最高稀释度来实现这一点。可以检测到低至0.001%丰度的逆转录病毒白细胞介素-3基因。实际上，我们能够从FD细胞中检测到在从永生化造血干细胞系逆转录病毒转移cDNA后具有不同表型的因子非依赖性集落的形成。因此，标准化的高效DNA转染和逆转录病毒介导的基因转移相结合，应有助于鉴定能够赋予靶FD细胞可检测的新功能或表型的基因。通过在筛选过程中实际扩大实验规模，该检测方法可以检测到丰度低于0.0001%的cDNA。
