Efficient gene-driven germ-line point mutagenesis of C57BL/6J mice.
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.
Michaud
Edward J
EJ
Life Sciences Division, Oak Ridge National Laboratory, P,O, Box 2008, Oak Ridge, TN 37831, USA. michaudejiii@ornl.gov
Culiat
Cymbeline T
CT
Klebig
Mitchell L
ML
Barker
Paul E
PE
Cain
K T
KT
Carpenter
Debra J
DJ
Easter
Lori L
LL
Foster
Carmen M
CM
Gardner
Alysyn W
AW
Guo
Z Y
ZY
Houser
Kay J
KJ
Hughes
Lori A
LA
Kerley
Marilyn K
MK
Liu
Zhaowei
Z
Olszewski
Robert E
RE
Pinn
Irina
I
Shaw
Ginger D
GD
Shinpock
Sarah G
SG
Wymore
Ann M
AM
Rinchik
Eugene M
EM
Johnson
Dabney K
DK
D000818:Animals; D003433:Crosses, Genetic; D015925:Cryopreservation; D004247:DNA / Q000378:metabolism; D004252:DNA Mutational Analysis; D018076:DNA, Complementary / Q000378:metabolism; D030541:Databases, Genetic; D005038:Ethylnitrosourea / Q000494:pharmacology; D005260:Female; D005821:Genetic Techniques; D005838:Genotype; D018095:Germ-Line Mutation; D006720:Homozygote; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008957:Models, Genetic; D016296:Mutagenesis; D009153:Mutagens; D009154:Mutation; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D010641:Phenotype; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D020554:Sperm Injections, Intracytoplasmic; D013094:Spermatozoa / Q000378:metabolism; D014018:Tissue Distribution
D018076:DNA, Complementary; D009153:Mutagens; D004247:DNA; D005038:Ethylnitrosourea