International Mammalian Genome Society

H2 ENU-Generated Mutations in Mouse Embryonic Stem Cells: A Novel Gene-Based Mutagenesis Approach

Yijing Chen¹, Della Yee¹, Kathy Dains², Alu Chatterjee², Rick Woychik² and Terry Magnuson¹. ¹Case Western Reserve University, 10900 Euclid Ave. Cleveland, OH 44106; ²Parke-Davis Laboratory for Molecular Genetics, 1501 Harbor Bay Parkway, Alameda, CA 94502

The approaching of the post-genomics era has heightened the need to fill in the gap between obtaining gene sequence and understanding gene function. The challenge is to develop efficient mutagenesis methods for generating mutations necessary for the study of gene function. The high animal maintenance cost has limited large scale whole animal mutagenesis to a few centers with necessary breeding facilities. Our laboratory is exploring the potential of generating mutations in mouse embryonic stem (ES) cells with ENU, the most potent mutagen in mouse germ cells. Using the selectable Hprt locus, we characterized the mutation frequency of ENU in mouse ES cells. We also showed that this frequency could be up-regulated by modulating the DNA repair activities. Sequencing of the Hprt mutants revealed missense, nonsense and splice mutations causing exon deletions and mutations affecting the transcript level, demonstrating the ability of ENU to create an allelic series critical for a complete understanding of gene functions. ENU generated mutant ES cell clones have also been demonstrated to be germline competent. We are currently assessing the relationship between mutation load and germline competency. The efficient mutation frequency and the potential of high through-put automation should make this ES cells based mutagenesis approach applicable to creating allelic series in genes and/or domains of interest. In combination with other types of mutagenesis approaches, this novel approach will allow us to apply the power of genetics to gain insight into the biological functions of genes.