Effect of vector topology on homologous recombination at the CHO aprt locus

Abstract

The Chinese hamster ovary aprt gene was used as a model for studying the effect of vector topology on gene targeting frequency. A single recombination vector containing 2.7 kb of isogenic DNA homologous to the aprt gene was digested with eight separate restriction enzymes to generate a variety of both replacement- and insertion-type recombination substrates. The frequency of homologous recombination, normalized by cotransfection with a linearized neo' marker, was assayed by the correction of a mutant hemizygous aprt allele and was not found to reflect vector topology. Southern analysis of representative recombination products suggests that the gene targeting events occurred predominantly by double crossover/gene conversion.