High-throughput site-directed mutagenesis in ES cells

Introduction of nonselectable mutations into the genome of embryonic stem c ells by homologous recombination allows to investigate the function of gene s at the molecular level and has been achieved, however, at very low effici encies by the Hit and Run, Tag and Exchange, and Double Replacement strateg ies. Comparing those strategies at a single locus with vectors derived from a single fragment of the desmin gene led to the improvement of two strateg ies by employing a new selection cassette and modified selection procedures . Modified strategies resulted in the introduction of nonselectable point-m utations in 53% of the Hit and Run derived embryonic stem cell clones and i n 0.7% of the Tag and Exchange clones. Efficiency of intrachromosomal recom bination at Hit alleles outscored replacement-type recombination at the tag ged alleles making the modified Hit and Run strategy the method of choice f or the efficient introduction of nonselectable point mutations into the gen ome of embryonic stem cells. (C) 2001 Elsevier Science.