Plasmid rescue - a tool for reproducible recovery of genes from transfected mammalian cells?
The efficient rescue of plasmids containing the thymidine kinase gene (tk) of Herpes simplex virus type I from genetically transformed mouse cells by transformation of bacteria is described. Rescued plasmids contain insertions of calf DNA used as a carrier in the transfection but usually lack portions of plasmid DNA. Deletions generally concern the region spanning from around the PvuII site of pBR322 to within the tetracycline resistance coding sequence, whereas the extent of tk sequence deletion varies, depending on the site of its integration (BamHI or PvuII) into the plasmid. Modelling the rescue process by transformation of bacteria with a mixture of original plasmids and sheared mouse cell DNA clearly demonstrates that deletions are caused by the presence of the mammalian DNA and they probably occur during re-transformation of bacteria before the onset of tetracycline gene expression. Plasmids lacking the Tcr region are reproducibly rescuable without deletion. Methods for reproducible re-isolation of transferred genes from mammalian cells are discussed.
D000818:Animals; D004247:DNA / Q000235:genetics; D004262:DNA Restriction Enzymes; D004926:Escherichia coli / Q000235:genetics; D007739:L Cells / Q000201:enzymology; D051379:Mice; D008854:Microscopy, Electron; D010957:Plasmids; D013937:Thymidine Kinase / Q000235:genetics*; D014162:Transfection
D004247:DNA; D013937:Thymidine Kinase; D004262:DNA Restriction Enzymes