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

Journal Article · Sat Jan 01 00:00:00 EST 2005 · BMC Genomics

OSTI ID:978244

Michaud, III, Edward J ^[1]; Culiat, Cymbeline T ^[1]; Klebig, Mitch ^[1]; Barker, Gene ^[1]; Cain, K T ^[1]; Carpenter, Debra J S ^[1]; Easter, Lori L ^[1]; Foster, Carmen M ^[1]; Gardner, Alysyn Wallace ^[1]; Guo, ZY ^[2]; Houser, Kay J ^[1]; Hughes, Lori A ^[1]; Kerley, Marilyn K ^[1]; Liu, Zhaowei ^[2]; Olszewski, Robert Edward ^[1]; Pinn, Irina ^[1]; Shaw, Ginger D ^[1]; Shinpock, Sarah G ^[1]; Wymore, Ann ^[1]; Rinchik, Eugene M ^[1] ; Johnson, Dabney K ^[1]

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1. ORNL
2. SpectruMedix, State College, PA

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.

• Research Organization: Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States); Mouse Genetics Research Facility
• Sponsoring Organization: USDOE Laboratory Directed Research and Development (LDRD) Program; USDOE Office of Science (SC)
• DOE Contract Number: DE-AC05-00OR22725
• OSTI ID: 978244
• Journal Information: BMC Genomics, Vol. 6, Issue 164
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
99 GENERAL AND MISCELLANEOUS//MATHEMATICS, COMPUTING, AND INFORMATION SCIENCE
AMINO ACIDS
BIOLOGICAL FUNCTIONS
DISEASES
DNA
ELECTROPHORESIS
FEMALES
FUNCTIONALS
GENE MUTATIONS
GENES
GENETICS
MALES
MICE
MUTAGENESIS
MUTANTS
MUTATION FREQUENCY
MUTATIONS
PROGENY
PROTEINS
SCREENS
SPERMATOZOA
STRUCTURAL CHEMICAL ANALYSIS
TEMPERATURE GRADIENTS