Recombination in mouse L cells between DNA introduced into cells and homologous chromosomal sequences

Abstract

In this paper, we show that DNA added to mouse L cells by the calcium phosphate method can be inserted into the genome of those cells by homologous recombination. 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 (tk delta 5') and is in the cell's chromosome, and the other is missing its 3' portion (tk delta 3') and is in the introduced DNA. Gene reconstruction by homologous insertion is relatively inefficient; approximately one Tk+ transformant is produced per 10(6) cells per 4 micrograms of added tk DNA, a frequency of about 10(-5) that of normal tk gene transformation. The Tk+ transformants produced by homologous recombination contain Sma I and Pvu II fragments that are diagnostic of the intact tk gene, contain a herpesvirus-specific thymidine kinase activity, and can transfer the Tk+ phenotype to Tk- cells by DNA-mediated gene transfer. Two surprising observations 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 complementary 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.