In recent years, chemical mutagenesis has become an increasingly popular method to disrupt gene functions due to its high efficiency of inducing mutations throughout the genome. Mutagenesis of embryonic stem (ES) cells offers the possibility of gene-driven approaches which, however, require efficient mutation detection procedures to screen archives of mutated samples for lesions in particular genes.

We have developed an approach that focusses on the detection of splice mutations in highly pooled cDNA samples using exon-skipping PCR primers. As a proof of concept, splice mutants for the Kit gene were isolated from a library comprising approximately 40,000 ES cell clones treated with N-ethyl-N-nitrosourea (ENU) followed by transmission through the mouse germline. The approach will be useful for the production of mouse models for human disease-related splice mutations and as a general gene disruption strategy.

In a complementary approach, we employed mutation detection with CEL I endonuclease from celery to identify point mutations in pools of genomic DNA. In a pilot screen based on pooled DNA samples of mutagenized ES cell clones, we identified several point mutations in the Sult1a1 locus demonstrating the efficiency of this screening strategy in the mouse.

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