Phenotype-driven mouse ENU mutagenesis screens

In the past decade, forward genetic screens in the mouse have come into their own as a practical method for exploring the genetic basis of many biological processes. By looking directly for disruption in a process of interest, genetic screens have always been powerful, but completion of the genome sequence has made mouse forward genetic screens practical, as well. The sequenced genome means we can map and sequence more efficiently than before, so small focused screens are now within the reach of even small labs. N-Ethyl-N-nitrosourea (ENU) is the preferred mutagen in forward genetic screens, because it is extremely potent in the premeiotic male germ line, where it induces point mutations. This last point is crucial, as point mutations lead to all classes of mutations (e.g., null, hypomorphs, neomorphs, antimorphs, and hypermorphs), which is why forward genetic screens can implicate a gene in a particular process when a targeted deletion may not. Point mutations often mimic human disease states, yielding highly relevant animal models. Since mammals reproduce, lactate, behave, develop, and protect themselves from infection differently from other vertebrates, mammalian forward genetic screens are uniquely informative. In fact, in the past decade, forward genetics has uncovered mutations demonstrating that certain genes exist only in mammals, that specific mechanisms function only in mammals, and that particular biological processes may exist only in mammals; hence screens focused on these processes have identified unsuspected genes. As powerful as the approach is, forward genetics remains a method for the committed; the process of screening requires organization and tenacity. This chapter is intended to help those who are ready to make the commitment by providing practical advice. To this end I detail the issues surrounding screen design and screen execution, as well as mutation identification and confirmation.