In producing mutant mice by gene targeting in embryonic stem (ES) cells, the efficient isolation of the homologous recombinants is still a critical step. We previously reported on a negative selection using the diphtheria toxin A (DT-A) fragment gene for homologous recombinants (1). It was efficient but limited to gene loci expressed in ES cells. For wider applicability of this negative selection to many gene loci not expressed or expressed at low levels in ES cells, we exploited a novel targeting vector composed of a polyA-less neo gene, a mRNA destabilizing signal, a pausing signal for RNA polymerase II from the minute virus of mice, and the DT-A gene. There was about a 30-fold decrease in frequency of G418-resistant colonies with this strategy against that using only the neo gene in the vector, and homologous recombinants were obtained at frequencies of more than 1/50 among G418 resistant cells at fyn, csk, c-mos, and insulin receptor substrate-1 gene loci.