The transfer and stable integration of the HSV thymidine kinase gene into mouse cells.
Treatment of mutant mouse cells (Ltk-) deficient in thymidine kinase with Bam I restriction endonuclease-cleaved HSV-1 DNA results in the appearance of numerous surviving colonies which stably express thte tk+ phenotype. Through a series of electrophoretic fractionations in concert with transfection assays, we isolated a 3.4 kb fragment which contains the thymidine kinase gene and which alone is competent in the biochemical transformation of Ltk- cells. In this report, we have examined the distribution of tk sequences in the DNA of several transformed clones following stable gene transfer. A series of complementary experiments involving reassociation kinetics in solution and annealings with tk DNA to restriction-cleaved cellular DNA following electrophoresis and transfer to filters allow us to make the following general conclusions concerning the fate of the tk gene in all clones examined: the tk gene is present in all cells at a frequency of one copy per chromosomal complement; the tk gene is stably integrated in the DNA of all transformants; and integration is not site-specific and occurs at different loci in the DNA of all transformants examined. The existence of a single active tk gene in tk+ transformants now facilitates an analysis of the sequence organization of tk- mutant cells and provides a useful model system for studies on the transfer of cellular genes.
D002460:Cell Line; D002999:Clone Cells; D004247:DNA / Q000235:genetics*; D004262:DNA Restriction Enzymes; D005796:Genes; D005814:Genes, Viral; D009693:Nucleic Acid Hybridization; D018139:Simplexvirus / Q000235:genetics*; D013937:Thymidine Kinase / Q000096:biosynthesis*; D014162:Transfection
D004247:DNA; D013937:Thymidine Kinase; D004262:DNA Restriction Enzymes