Recombination in mouse L cells bel and homologous chromosomal sequ (homologous recombination/thymidine kinase gene/gene transfer/Dl)

In this paper, we show that DNA added to mouse L cells by the calcium phosphate method can be insert- ed into the genome of those cells by homologous recombina- tion. The insertion event is detected because it reconstructs a functional thymidine kinase (tk) gene from two defective genes that share 320 base pairs of homology. One of the genes is missing its 5' portion (tkA5') and is in the cell's chromosome, and the other is missing its 3' portion (tkA3') and is in the introduced DNA. Gene reconstruction by homologous inser- tion is relatively inefficient; approximately one Tk+ transfor- mant is produced per 106 cells per 4 ,ug of added tk DNA, a frequency of about 10-5 that of normal tk gene transforma- tion. The Tk+ transformants produced by homologous recom- bination contain Sma I and Pvu II fragments that are diagnos- tic of the intact tk gene, contain a herpesvirus-specific thymi- dine kinase activity, and can transfer the Tk+ phenotype to Tk- cells by DNA-mediated gene transfer. Two surprising ob- servations made in the course of these studies were that only 1 of 10 Tk- cell lines containing defective tk genes could be transformed to Tk+ by homologous insertion of the comple- mentary defective tk gene and that relatively little illegitimate insertion of introduced tk DNA into cellular DNA was detected in those cells that were transformed to Tk+ by homologous recombination.