International Mammalian Genome Society

The 15th International Mouse Genome Conference (2001)


Dr Yijing Chen
University of North Carolina-Chapel Hill
Department of Genetics
Campus Box 7264
Chapel Hill
NC 27599

Co-Authors:  Vivian J, Yee D, Schneider E, and Magnuson T
Institution:   University of North Carolina-Chapel Hill

ENU-generated allelic series of mutations of smad4 in es cells

A comprehensive collection of mouse mutants is critical for the functional annotation of the mammalian genome. Chemical mutagenesis in mouse ES cells is a rapid and cost-effective means to generate germline mutations. Taking advantage of the high mutation rate and high throughput mutation detection technology, we developed a genotype-based method to create allelic series of mutations in any genes of interest. We generated a cryopreserved ES cell bank composed of over 2000 clones mutagenized with ENU. DNA and RNA are isolated from individual clones for amplifying regions of interest. Mutations were identified with the DHPLC technology. The functional consequence of the mutation is assessed through germline transmission. Smad4 is central to the TGF-b-related signaling and is implicated in numerous developmental and disease processes. Targeted mutations in Smad4 cause early lethality, making studying its later functions difficult. Screening the mutant ES cell bank, we identified a total of 16 Smad4 mutations of which 10 are non-silent.  So far, three mutant lines have passed through the germline and more chimeras are being tested. The genetics analyses of this allelic series of Smad4 mutations will allow a fine-tuned dissection of its functions.  We are also successful in creating allelic series of mutations in other loci, demonstrating the general utility of our ES cell based approach, which will be an important addition to the existing functional genomic tools.

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