International Mammalian Genome Society

The 16th International Mouse Genome Conference (2002)


EJ Michaud
Oak Ridge National Laboratory and The University of Tennessee, Knoxville

1,2) Culiat CT, 1) Larimer FW, 1) Cain KT, 1) Carpenter DJ, 1) Easter LL, 1) Foster CM, 1) Gardner AW, 1) Houser KJ, 1) Hughes LA, 1) Kerley MK, 1) Lu T-YS, 1) Olszewski RE, 1) Pinn I, 1) Shaw GD, 1) Shinpock SG, 1) Wymore AM, 1) York ML, 1,2) Baker EJ, 1,2) Snoddy JR, 1,2) Johnson DK, and 1,2,3) Rinchik EM
1) Life Sciences Division, Oak Ridge National Laboratory, 2) The University of Tennessee-Oak Ridge National Laboratory Graduate School of Genome Science and Technology, 3) Department of Biochemistry, Cellular, and Molecular Biology, The University of Tennessee

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 consider genome-wide, gene (sequence)-driven approaches to mouse germline mutagenesis. Such gene-driven strategies allow one to perform whole-genome mutagenesis, and then screen 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 have been generating a cryopreserved bank of DNA, tissues (for RNAs and proteins), and sperm from 5,000 C57BL/6JRn 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 both regulatory regions and coding sequences of virtually every gene in the genome.  High-throughput Temperature Gradient Capillary Electrophoresis (TGCE) is used to identify mutations by heteroduplex analysis in pre-selected genes in the CMMB DNA panel, and mutant stocks are recovered by in vitro fertilization or intracytoplasmic sperm injection from the parallel bank of frozen sperm.  We will present progress on (i) production of the 5000-member CMMB (now ~75% complete); (ii) methods used for mutation screening by high-throughput TGCE; (iii) our current estimate of the per-base-pair mutation frequency in the CMMB; and (iv) reconstitution of mutant stocks. [Research sponsored by the Laboratory Directed Research and Development Program of Oak Ridge National Laboratory, and by the Office of Biological and Environmental Research, U.S. DOE, under Contract No. DE-AC05-00OR22725 with UT-Battelle, LLC.]

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