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Using selectable genes as proof of principle, a new high-throughput genotype-based mutation screen in mouse embryonic stem (ES) cells was developed [Chen et al. (2002) Nat. Genet. 24, 314-317]. If expanded to nonselectable genes, this approach would allow one to proceed quickly from sequence to whole-animal phenotypes. Here data are presented showing that a screen of a cryopreserved library of clonal, germ line competent, N-ethyl-N-nitrosurea (ENU) mutagenized ES cells can identify a large series of allelic mutations in Smad2 and Smad4, two nonselectable genes of the transforming growth factor beta superfamily of signaling molecules. Whole animal phenotypic analyses of some of these alleles provided evidence for novel developmental processes mediated by these components of transforming growth factor beta signaling, demonstrating the utility of non-null alleles created by chemical mutagens. The accurately assessed mutation load of the ES cell library indicates that it is a valuable resource for developing mouse lines for genetic and functional studies. This methodology can conceptually be applied for the generation of an allelic series of subtle mutations at any locus of interest in the mouse.

proceedings of the national academy of sciences of the united states of america

An allelic series of mutations in Smad2 and Smad4 identified in a genotype-based screen of N -ethyl- N - nitrosourea-mutagenized mouse embryonic stem cells