Deoxyribonucleic acid-mediated gene transfer in mammalian cells: molecular analysis of unstable transformants and their progression to stability.
To elucidate mechanisms involved in deoxyribonucleic acid-mediated gene transfer, we transferred the herpes simplex virus thymidine kinase gene (TK) into mouse Ltk- cells. Independent TK+ clones (transformants) and derivatives of each were tested for phenotypic expression and the presence and arrangement of TK sequences. Initially, transformants expressed viral TK unstable, with 10% of the cells in each generation losing both the TK+ phenotype and virally derived TK sequences. After a prolonged period in culture, stable subpopulations arose from which the TK+ phenotype and viral sequences were no longer lost at detectable frequency. Analysis of unstable cell populations indicated that individual viral deoxyribonucleic acid molecules were reduced in size, but were linked to other deoxyribonucleic acid to form molecules large enough to be precipitated in a Hirt fractionation. We term these molecules transgenomes. Analysis of independent unstable subclones derived from the primary transformants demonstrated that individual transgenomes could contain multiple copies of the viral TK sequences. Recipient cell lines frequently possessed more than one type of transgenome and possibly multiple copies per cell of each type. Stable derivatives possessed only one of the transgenomes present in the unstable parent, and these sequences were associated with a recipient cell chromosome.
D000818:Animals; D002460:Cell Line; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics*; D005814:Genes, Viral; D051379:Mice; D010641:Phenotype; D010957:Plasmids; D018139:Simplexvirus / Q000201:enzymology / Q000235:genetics; D013937:Thymidine Kinase / Q000235:genetics; D014162:Transfection
D004247:DNA; D013937:Thymidine Kinase