International Mammalian Genome Society

logo18th International Mouse Genome Conference

17-22 October 2004, Seattle, USA

POSTER 85 - Functional Annotation of Human Genes by Gene-Driven ENU Mutagenesis in Mice

Michaud EJ 1,2, Culiat CT 1,2, Barker G 1, Cain KT 1, Carpenter DJ 1, Easter LL 1, Foster CM 1, Gardner AW 1, Geiger J 3, Guo ZY 3, Houser KY 1, Hughes LA 1, Kerley MK 1, Klebig ML1,2, Liu Z 3, Olszewski RE 1, Pinn I 1, Shaw GD 1, Shinpock SG 1, Wymore AM 1, Johnson DK 1,2, Rinchik EM 1,2,4

1Life Sciences Division, Oak Ridge National Laboratory, Oak Ridge, TN, United States 2The University of Tennessee–Oak Ridge National Laboratory Graduate School of Genome Science and Technology, Oak Ridge, TN; United States, 3SpectruMedix, PA, United States 4Department of Biochemistry, Cellular, and Molecular Biology, The University of Tennessee, Knoxville, TN, United States

The availability of the complete DNA sequence of the mouse genome, coupled with the development of high-throughput methods for rapid detection of single-nucleotide polymorphisms (SNPs), have made it practical to implement genome-wide, gene-driven approaches to mouse germline mutagenesis. Such gene-driven strategies enable the performance of whole-genome mutagenesis, and screening for alterations in any pre-selected gene(s). To complement embryonic stem-cell-based gene-driven mutagenesis resources, such as gene-trap libraries and banks of N-ethyl-N-nitrosourea (ENU)-mutagenized ES cells, we generated a cryopreserved bank of DNA, tissues (for RNAs and proteins), and sperm from 4,000 C57BL/6J mice that each carry a unique load of paternally induced ENU mutations. This ORNL Cryopreserved Mutant Mouse Bank (CMMB) is a source of induced, heritable SNPs in virtually every gene in the genome. The ability to produce an allelic series of mutations in any gene, where each mutation may have different degrees of severity of the mutant phenotype, is invaluable in discovering the full scope of biological functions of a gene. High-throughput Temperature Gradient Capillary Electrophoresis (TGCE) is used to identify mutations in any gene by heteroduplex analysis in pre-selected genes in the CMMB DNA/RNA panel, and mutant stocks are recovered by intracytoplasmic sperm injection (ICSI) from the parallel bank of frozen sperm. Thus, the CMMB will provide mouse models of a wide range of altered proteins for phenotypic, gene/protein-network, and structural biology-type analyses. We will present progress on the identification of ENU-induced mutations in the 4,000-member CMMB by TGCE, and the recovery of mutant mice by ICSI.

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