Uracil incorporation into a gene targeting construct reduces the frequencyof homologous and nonhomologous recombinants in human cells

Gene targeting allows the introduction of specific modifications into the e ukaryotic genome by homologous recombination, but its efficiency is low in many mammalian systems. We are exploring different ways to increase the eff iciency of gene targeting and we report here the effect of uracil incorpora tion in the targeting construct. Plasmids containing uracil substituting fo r a fraction of thymine residues are hyperrecombinogenic in some bacterial systems. To test whether a similar stimulation of recombination occurs in m ammalian cells, we have prepared a uracil-rich HPRT targeting construct and quantified its homologous and nonhomologous recombination frequencies comp ared to the same plasmid lacking uracil. The uracil-rich plasmid led to red uctions in both homologous and nonhomologous recombination in human cells. (C) 2000 Elsevier Science B.V. All rights reserved.