Mutational specificity studies of endogenous mammalian cell loci: methodological aspects

Abstract

We report here the details of a modified cloning method first described by Seed et al. (1983) for use in an extensive study of mutational specificity at the aprt locus of Chinese hamster ovary cells. The technology depends on homologous recombination between a suppressor probe plasmid and the desired insert in a genomic library constructed in a double amber mutant bacteriophage λ vector. Recombinant phage form blue plaques on a non-suppressor, lacZ amber host. We have determined the sensitivity of the method. Homologous recombination frequency is in the order of 10 −3, 6–7 orders of magnitude above non-homologous events. Recombination efficiency is unaffected when the target phage is diluted 100,000-fold with parental vector. A background of plaques is observed along with the desired blue plaques. Although the color discrimination permits us to easily avoid these background plaques, we have characterized the frequency and the nature of these events.