ANALYSIS OF BIOLOGICAL SELECTIONS FOR HIGH-EFFICIENCY GENE TARGETING

A two-marker selection system that allows the efficient isolation of d iploid gene knockouts by two sequential rounds of targeted homologous recombination has been developed. A systematic evaluation of the biolo gical parameters that govern the selection process shelved that a succ essful strategy must match the expression level of the target gene, th e efficacy of the marker, and the selection stringency. An enrichment ratio of 5,000- to 10,000-fold, which resulted in a 30% targeting effi ciency of the c-myc gene in a fibroblast cell line, has been achieved. Such efficiency brings the difficulty of gene targeting effectively d own to the level of simple transfections, since only 10 to 20 drug-res istant clones need to be screened to recover several homologous hits. The general utility of the targeting strategy is of interest to invest igators studying gene function in a large variety of mammalian tissue culture systems.