Generating Mutant Mice via ENU Mutagenesis

ENU mutagenesis and breeding were performed as described on a pure C3HeB/FeJ (C3H) background (Hrabé de Angelis et al. 2000 (link); Sabrautzki et al. 2012 (link); Aigner et al. 2011 (link)). Briefly, C3H mice were originally purchased from the Jackson Laboratory (Bar Harbor, ME) and ENU (Serva Electrophoresis, Heidelberg, Germany) was applied in three weekly intervals by intraperitoneal injections of 90 mg/kg body weight to 10–12 wk old male mice (G0). G0 mice were mated with wild-type C3H females to produce F1 offspring. F1 males not showing any obvious phenotypic alterations were mated with wild-type C3H females to obtain the G2 generation. We either choose 6–8 female G2 mice for matings with their F1 father or performed intercross matings of G2 mice to produce at least 20 mice (G3 families). Phenotyping for dysmorphological alterations was performed according to a standardized protocol (Fuchs et al. 2000 (link)). A mutation was confirmed by showing a Mendelian distribution of expected homozygous mutant mice. The Scube3^N294K/N294K mouse line was maintained on the C3H genetic background for more than 10 generations.