DOE Human Genome Program Contractor-Grantee Workshop VIII
February 27-March 2, 2000  Santa Fe, NM

104. Mouse Genetics and Mutagenesis for Functional Genomics: Phenotype-Driven Regional Mutagenesis and Genomics at the Oak Ridge National Laboratory

E. M. Rinchik^1,2, D. A. Carpenter¹, E. J. Michaud¹, Y. You¹, P. R. Hunsicker¹, L. B. Russell¹, D. R. Miller¹, M. L. Klegig², and D. K. Johnson¹

¹Life Sciences Division, Oak Ridge National Laboratory, P.O. Box 2009, Oak Ridge, TN 37831-8077 and ²Department of Biochemistry, Cellular, and Molecular Biology, University of Tennessee, Knoxville, TN 37996

rinchikem@ornl.gov

A major goal of the mouse-genetics program at ORNL is to apply our experience in chemical germ-cell mutagenesis, mutation recovery and propagation, and broad-based phenotype screening, for creating a large, user-friendly mouse-mutation resource that can be used by the wider biological community for functional annotation of human DNA sequence. Our current overall program expands previous work that molecularly characterized regions of mouse Chromosome (Chr) 7 while also recovering N-ethyl-N-nitrosourea (ENU)-induced, recessive single-gene mutations. For example, in one screen of a ~5-cM Chr-7 region (human 11p and 15q homologies), simple phenotype-screening criteria had ascertained 19 new mutations in 1218 gametes, and, recently, broadly based phenotype-screening has yielded seven additional heritable mutations (including two subtle behavioral ones) in ~450 additional gametes, with another 13 subtle variants undergoing heritability testing. All mutations are being placed, by a simple set of genetic complementation crosses with overlapping deletions, into the rich DNA-sequence and expression map evolving for this region.

Mutations within two additional regions [mid-Chr 7 (human 15q homology), and mid-to-distal Chr 15 (human 8q, 22q, and 12q homologies] are being recovered using dominantly and recessively marked inversion chromosomes in three-generation screens, which allows easy detection and low-cost maintenance of chromosomally "pre-mapped" deleterious recessive mutations without any molecular genotyping. In parallel, deletions are being developed in embryonic stem cells for use as finer-mapping and gene-identification reagents. Our experimental design also provides for the generation of multiple mutant test-class mice of a singular genotype for comprehensive multi-site phenotype screening (e.g., across the Tennessee Mouse Genome Consortium) and for establishment of aging colonies to be screened for later-onset recessive phenotypes. It also provides a facile means for placing any mutation on a number of inbred genetic backgrounds to analyze modifier effects in genetic-network analyses. We estimate that approximately 8-10% of the genome will be covered by our screens in the near term, with even wider coverage possible as additional genetic reagents are created.