Mutagenesis

The random nature of ENU-induced mutations facilitates a phenotype driven approach to mutation detection. High throughput phenotyping of mice from dominant and recessive mutagenised pipelines have generated a wide range of models across a number of disease areas. We are scrutinising pedigrees of mice in a phenotype driven recessive screen to identify mutants resulting in chronic, late-onset, or age-related disease.

Phenotype driven screens make no assumptions about the nature of the genetic change underlying observed phenotypes, and thus provide a powerful tool for identifying novel genes and pathways contributing to disease pathogenesis. We will focus on recessive aging screens using a standard Harwell design employing C57BL/6J and C3H mice (see above) to generate G3 pedigrees. As with the current pedigrees employed in the vision screen, we will use a C3H strain carrying a wild-type allele at the rd1 (Pde6b) locus. Moreover, these pedigrees will be integral to the age-related deafness screen (see below) so we will screen G2 females to ensure no G3 offspring are homozygous for the Ahl1 allele at the Cdh23 locus that predisposes BL/6 (but not C3H) to age-related hearing loss. In total some 250 pedigrees will be generated and aged during the next five years. In general, G3 progeny will be assessed using batteries of tests taking into account the disease domain of interest. Because of the age-related nature of the screen we will initially generate relatively large pedigrees comprising of approximately 100 G3 animals, so enhancing our ability to identify, confirm and map phenodeviants without recourse to further breeding of G1 males, and further aging of G3 animals. The application of next generation sequencing will facilitate the first-pass identification of mutants in the ageing screen. However, for all pedigrees we will preserve the sperm from the founder G1 male, and if necessary we can expand the pedigree for further mapping and characterisation. Moreover, depending upon the age of onset it may be possible to freeze sperm from affected G3 males. Mutant G3 progeny can be outcrossed and intercrossed to generate G5 progeny for additional mapping resource. At defined time points urine and blood samples will be stored in a biobank for retrospective screening once a phenotype of interest has been identified.

The DMAT interacts closely with the Bioinformatics group to provide a rapid pipeline from phenotype identification to sequencing results, and are jointly developing tools and processes to maximise the potential of NGS with regard to mutant identification. Each mutagenised mouse that is sequenced will contain multiple mutations, particularly if G1 mice are sequenced, and hence we are developing a database of sequencing results for further interrogation to screen for mutations in specific genes of interest.

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