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.

Nell1-deficient mice have reduced expression of extracellular matrix proteins causing cranial and vertebral defects.

The mammalian Nell1 gene encodes a protein kinase C-beta1 (PKC-beta1) binding protein that belongs to a new class of cell-signaling molecules controlling cell growth and differentiation. Over-expression of Nell1 in the developing cranial sutures in both human and mouse induces craniosynostosis, the premature fusion of the growing cranial bone fronts. Here, we report the generation, positional cloning and characterization of Nell1(6R), a recessive, neonatal-lethal point mutation in the mouse Nell1 gene, induced by N-ethyl-N-nitrosourea. Nell1(6R) has a T-->A base change that converts a codon for cysteine into a premature stop codon [Cys(502)Ter], resulting in severe truncation of the predicted protein product and marked reduction in steady-state levels of the transcript. In addition to the expected alteration of cranial morphology, Nell1(6R) mutants manifest skeletal defects in the vertebral column and ribcage, revealing a hitherto undefined role for Nell1 in signal transduction in endochondral ossification. Real-time quantitative reverse transcription-PCR assays of 219 genes showed an association between the loss of Nell1 function and reduced expression of genes for extracellular matrix (ECM) proteins critical for chondrogenesis and osteogenesis. Several affected genes are involved in the human cartilage disorder Ehlers-Danlos Syndrome and other disorders associated with spinal curvature anomalies. Nell1(6R) mutant mice are a new tool for elucidating basic mechanisms in osteoblast and chrondrocyte differentiation in the developing skull and vertebral column and understanding how perturbations in the production of ECM proteins can lead to anomalies in these structures.

Identification of mutations from phenotype-driven ENU mutagenesis in mouse chromosome 7.

We have used the new high-throughput mutation-scanning technique temperature-gradient capillary electrophoresis (TGCE) for the identification of point mutations induced by N-ethyl-N-nitrosourea (ENU) in the mouse genome. TGCE detects the presence of heteroduplex molecules formed between a wild-type gene segment and the corresponding homologous segment containing an induced mutation or a naturally occurring single nucleotide polymorphism (SNP). Partially denatured heteroduplex molecules are resolved from homoduplexes by virtue of their differential mobilities during capillary electrophoresis conducted in a finely controlled temperature gradient. Simultaneous heteroduplex analysis of 96 amplicons ranging from 150 to 600 bp in size is achieved in approximately 45 min without the need for predetermining the melting profile of each fragment. Initially, we exploited known mouse mutations to develop TGCE protocols for analyzing unpurified PCR samples amplified from crude tail-DNA preparations. TGCE was then applied to the rapid identification of three new ENU-induced mutations recovered from regional mutagenesis screens of a segment of mouse Chromosome 7. Enzyme assays and quantitative reverse transcription-PCR (qRT-PCR) methods validated these new mutations. Our data demonstrate that rapid mutation scanning with TGCE, followed by sequence verification only of detected positives, is an efficient approach to the identification of point mutations in the mouse genome.