Sasaki K, Mizusawa H, Ishidate M, Tanaka N
Department of Cell Biology, Hatano Research Institute, Food and Drug Safety Center (FDSC), Kanagawa, Japan.
Somat Cell Mol Genet. 1992 Nov;18(6):517-27. doi: 10.1007/BF01232648.
We attempted to establish the optimum conditions for the calcium phosphate (CaPO4) precipitation protocol by counting G418 resistant (G418r) colonies after transfection of pSV2-neo DNA into BALB 3T3 cells. The amount and molecular size of carrier DNA, number of plating cells, treatment period of DNA-CaPO4 precipitates and expression time of G418 selection were found to be important factors in the induction of G418r colonies. Six G418r clones were derived from BALB 3T3, NIH 3T3 and FRSK cells, and cocultured with G418 sensitive (G418s) parent cells in G418 medium. The colony formation capacity of all G418r cell clones decreased with the increasing number of plated G418s cells. Cell-cell contact appeared to be necessary to reduce the colony formation of G418r cells, and contact-dependent G418r cell killing was probably not related to gap junction formation. Contact-mediated cell killing is a likely explanation for the observation that induction of G418r colonies is often reduced under conditions of high-density plating, long treatment of DNA-CaPO4 precipitates, and long expression time of G418 selection. These results suggest that in some instances transfection efficiency using pSV2-neo DNA should be carefully evaluated because culture conditions can mask the induction of G418r colonies.
我们试图通过将pSV2-neo DNA转染到BALB 3T3细胞后计数G418抗性（G418r）菌落，来确定磷酸钙（CaPO4）沉淀方案的最佳条件。发现载体DNA的量和分子大小、铺板细胞数量、DNA-CaPO4沉淀物的处理时间以及G418选择的表达时间是诱导G418r菌落的重要因素。从BALB 3T3、NIH 3T3和FRSK细胞中获得了六个G418r克隆，并在G418培养基中与G418敏感（G418s）亲代细胞共培养。所有G418r细胞克隆的集落形成能力随着接种的G418s细胞数量的增加而下降。细胞间接触似乎是减少G418r细胞集落形成所必需的，并且接触依赖性G418r细胞杀伤可能与间隙连接形成无关。接触介导的细胞杀伤可能是对以下观察结果的一种解释：在高密度铺板、DNA-CaPO4沉淀物长时间处理以及G418选择长时间表达的条件下，G418r菌落的诱导常常会降低。这些结果表明，在某些情况下，使用pSV2-neo DNA的转染效率应仔细评估，因为培养条件可能会掩盖G418r菌落的诱导。
