Analysis of biological selections for high-efficiency gene targeting.
A two-marker selection system that allows the efficient isolation of diploid gene knockouts by two sequential rounds of targeted homologous recombination has been developed. A systematic evaluation of the biological parameters that govern the selection process showed that a successful strategy must match the expression level of the target gene, the efficacy of the marker, and the selection stringency. An enrichment ratio of 5,000- to 10,000-fold, which resulted in a 30% targeting efficiency of the c-myc gene in a fibroblast cell line, has been achieved. Such efficiency brings the difficulty of gene targeting effectively down to the level of simple transfections, since only 10 to 20 drug-resistant clones need to be screened to recover several homologous hits. The general utility of the targeting strategy is of interest to investigators studying gene function in a large variety of mammalian tissue culture systems.
Hanson
K D
KD
Department of Molecular Biophysics and Biochemistry, Yale University School of Medicine, New Haven, Connecticut 06510.
Sedivy
J M
JM
D000818:Animals; D002478:Cells, Cultured; D018274:Electroporation; D005786:Gene Expression Regulation; D018390:Gene Targeting / Q000379:methods*; D016259:Genes, myc; D005822:Genetic Vectors; D066298:In Vitro Techniques; D051381:Rats; D011995:Recombination, Genetic; D015183:Restriction Mapping; D012641:Selection, Genetic