International Mammalian Genome Society

logo18th International Mouse Genome Conference

17-22 October 2004, Seattle, USA


POSTER 130 - GENOME-WIDE MUTATION DISCOVERY BY TGCE IN GENE-DRIVEN APPROACH OF ENU-MUTAGENESIS

Sakuraba Y, Sezutsu H, Takahasi R, Uchiyama M, Tsuchihashi K, Fujimoto N, Ichikawa R, Kaneko S, Goda N, Ikeda A, Karashima Y, Inoue M, Kaneda H, Minowa O, Wakana S, Noda T, Shiroishi T, Gondo Y

RIKEN GSC, Yokohama, Japan

It has become practical to generate and recover mutant mice having ENU-induced point mutations in a specific target gene by reverse genetics. We have prepared frozen sperms and genomic DNA from over 7,000 G1 male mice for the gene-driven approach of ENU mutagenesis. How to find the mutations is one of the most important issues in the gene-driven approach. We have adopted a Temperature Gradient Capillary Electrophoresis (TGCE) method. Multiplexing of the TGCE enabled the high-throughput search of point mutations. We have found over 100 mutations from 130 Mb analysis using the TGCE method. Forty-four mutations will alter the amino acid sequences; 36 missense, 2 nonsense, 1 “make-sense” (Stop to Tryptophan) and 5 splicing signal mutations. Eight mutations were found in candidate regulatory regions. The differences of mutation spectra between TGCE and direct-sequencing methods as well as between gene-driven and phenotype-driven screens will be discussed.

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