Efficient gene-driven germ-line point mutagenesis of C57BL/6J mice

Michaud, Edward J.; Culiat, Cymbeline T.; Klebig, Mitchell L.; Barker, Paul E.; Cain, KT; Carpenter, Debra J.; Easter, Lori L.; Foster, Carmen M.; Gardner, Alysyn W.; Guo, ZY; Houser, Kay J.; Hughes, Lori A.; Kerley, Marilyn K.; Liu, Zhaowei; Olszewski, Robert E.; Pinn, Irina; Shaw, Ginger D.; Shinpock, Sarah G.; Wymore, Ann M.; Rinchik, Eugene M.; Johnson, Dabney K.

doi:10.1186/1471-2164-6-164

Efficient gene-driven germ-line point mutagenesis of C57BL/6J mice

Journal Article · Sun Nov 20 23:00:00 EST 2005 · BMC Genomics

DOI:https://doi.org/10.1186/1471-2164-6-164· OSTI ID:1626444

Michaud, Edward J. ^[1]; Culiat, Cymbeline T. ^[2]; Klebig, Mitchell L. ^[3]; Barker, Paul E. ^[4]; Cain, KT ^[4]; Carpenter, Debra J. ^[4]; Easter, Lori L. ^[4]; Foster, Carmen M. ^[4]; Gardner, Alysyn W. ^[4]; Guo, ZY ^[5]; Houser, Kay J. ^[4]; Hughes, Lori A. ^[4]; Kerley, Marilyn K. ^[4]; Liu, Zhaowei ^[5]; Olszewski, Robert E. ^[4]; Pinn, Irina ^[4]; Shaw, Ginger D. ^[4]; Shinpock, Sarah G. ^[4]; Wymore, Ann M. ^[4]; Rinchik, Eugene M. ^[6] more »

Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). Life Sciences Division; Univ. of Tennessee, Knoxville, TN (United States). Graduate School of Genome Science and Technology; DOE/OSTI
Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). Life Sciences Division; Univ. of Tennessee, Knoxville, TN (United States). Graduate School of Genome Science and Technology
Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). Life Sciences Division; Univ. of Tennessee, Knoxville, TN (United States). Graduate School of Genome Science and Technology; Univ. of Tennessee, Knoxville, TN (United States). Dept. of Biochemistry, Cellular and Molecular Biology
Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). Life Sciences Division
SpectruMedix, State College, PA (United States)
Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). Life Sciences Division; Univ. of Tennessee, Knoxville, TN (United States). Graduate School of Genome Science and Technology; Univ. of Tennessee, Knoxville, TN (United States). Dept. of Biochemistry, Cellular and Molecular Biology; SpectruMedix, State College, PA (United States)

Background: Analysis of an allelic series of point mutations in a gene, generated by N-ethyl-N-nitrosourea (ENU) mutagenesis, is a valuable method for discovering the full scope of its biological function. Here we present an efficient gene-driven approach for identifying ENU-induced point mutations in any gene in C57BL/6J mice. The advantage of such an approach is that it allows one to select any gene of interest in the mouse genome and to go directly from DNA sequence to mutant mice. Results: We produced the Cryopreserved Mutant Mouse Bank (CMMB), which is an archive of DNA, cDNA, tissues, and sperm from 4,000 G1 male offspring of ENU-treated C57BL/6J males mated to untreated C57BL/6J females. Each mouse in the CMMB carries a large number of random heterozygous point mutations throughout the genome. High-throughput Temperature Gradient Capillary Electrophoresis (TGCE) was employed to perform a 32-Mbp sequence-driven screen for mutations in 38 PCR amplicons from 11 genes in DNA and/or cDNA from the CMMB mice. DNA sequence analysis of heteroduplex-forming amplicons identified by TGCE revealed 22 mutations in 10 genes for an overall mutation frequency of 1 in 1.45 Mbp. All 22 mutations are single base pair substitutions, and nine of them (41%) result in nonconservative amino acid substitutions. Intracytoplasmic sperm injection (ICSI) of cryopreserved spermatozoa into B6D2F1 or C57BL/6J ova was used to recover mutant mice for nine of the mutations to date. Conclusions: The inbred C57BL/6J CMMB, together with TGCE mutation screening and ICSI for the recovery of mutant mice, represents a valuable gene-driven approach for the functional annotation of the mammalian genome and for the generation of mouse models of human genetic diseases. The ability of ENU to induce mutations that cause various types of changes in proteins will provide additional insights into the functions of mammalian proteins that may not be detectable by knockout mutations.

View Accepted Manuscript (DOE)

Research Organization:: Oak Ridge National Laboratory (ORNL), Oak Ridge, TN (United States)

Sponsoring Organization:: USDOE Office of Science (SC), Biological and Environmental Research (BER). Biological Systems Science Division

Grant/Contract Number:: AC05-00OR22725

OSTI ID:: 1626444

Journal Information:: BMC Genomics, Journal Name: BMC Genomics Journal Issue: 1 Vol. 6; ISSN 1471-2164

Publisher:: SpringerCopyright Statement

Country of Publication:: United States

Language:: English

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59 BASIC BIOLOGICAL SCIENCES
allelic series
biotechnology & applied microbiology
freeze sperm
genetics & heredity
mutagenesis screen
sort intolerant from tolerant
temperature gradient capillary electrophoresis

Efficient gene-driven germ-line point mutagenesis of C57BL/6J mice

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