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

Authors

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

Document Type

Article

Publication Date

11-21-2005

Abstract

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.

Recommended Citation

BMC Genomics 2005, 6:164 doi:10.1186/1471-2164-6-164