Efficient and fast targeted production of murine models based on ENU mutagenesis

Abstract

Mice with targeted genetic alterations are the most effective tools for deciphering organismal gene function. We generated an ENU-based parallel C3HeB/FeJ sperm and DNA archive characterized by a high probability to identify allelic variants of target genes as well as high efficiencies in allele retrieval and model revitalization. Our archive size of over 17,000 samples contains approximately 340,000 independent alleles (20 functional mutations per individual sample). Based on an estimated number of approximately 30,000 mouse genes, the parallel sperm/DNA archive should permit the identification and recovery of ten or more alleles per average target gene which translates to a calculated 99% success rate in the discovery of five allelic variants for any given average gene. The low rate of unrelated ENU-induced passenger mutations has no practical impact on the analysis of the allele-specific phenotype at the G3 generation because of dilution and free segregation of such unrelated passenger mutations. To date 39 mouse models representing 33 different genes have been recovered from our archive using in vitro fertilization techniques. The generation time for a murine model heterozygous for a mutation in a gene of interest is less than 2 months, i.e., three to four times faster compared with current embryonic stem-cell-based technologies. We conclude that ENU-based targeted mutagenesis is a powerful tool for the fast and high-throughput production of murine gene-specific models for biomedical research.

Analysis of breeding and pathology helps refine management practices of a large-scale N′-ethyl-N′-nitrosourea mouse mutagenesis programme

“…In our larger sample size, we can confirm a relationship between cumulative dose and infertility rates suggested in smaller cohorts of these strains. Indeed, a more recent study of comparable population size, but in C3HeB/FeJ mice, 14 shows a similar finding to ours and is also in agreement with previously published rates in smaller cohorts of this inbred strain. 8 We were particularly interested in our study to extend this analysis and to explore the components of lifetime fecundity: regained fertility and lifetime productivity.…”

Dual function of the UNC-45b Chaperone with myosin and GATA4 in cardiac development

“…To reduce the possibility of the complex mutant phenotypes being caused by a second mutation, the experiments reported here were performed on the progeny of heterozygous mutant mice that underwent six backcrosses, which would make the possibility of mutations in the Gata4 locus on chromosome 17 co-segregating with the two UNC-45b mutations in the C3H strain on chromosome 11 of the order of 1 in 10 6 (Augustin et al, 2005;Keays et al, 2006). The two mutations in the inbred C3H strain arose from distinct pools of sperm mutagenized with ethylnitrosourea, indicating their independent origin.…”

“…To further minimize the possibility of presence of secondary mutations, heterozygous males were backcrossed with wild-type females until G6. G6 animals were used for all final analyses (Augustin et al, 2005;Keays et al, 2006). The heterozygotes showed normal behavior, morphology, growth, and fertility.…”

“…Mutant mice were generated at Ingenium Pharmaceuticals, Martinsried, Germany using N-ethyl-N-nitrosourea (ENU) (Augustin et al, 2005;Keays et al, 2006). Two mutations were identified by PCR screening in exons coding for sequences in the central and carboxyl-terminal UCS regions.…”

Thematic review series: Systems Biology Approaches to Metabolic and Cardiovascular Disorders Approaches to lipid metabolism gene identification and characterization in the postgenomic era

“…With the availability of mouse genome sequence and highthroughput mutation screening via PCR coupled with high-temperature gradient capillary electrophoresis, it is now possible to screen cryopreserved sperm from ENUmutagenized mice for mutations in any gene of interest (115)(116)(117). Sperm containing mutations of interest are then used to generate live mice by in vitro fertilization.…”