Identification of an imprinting control region affecting the expression of all transcripts in the Gnas cluster.

Genomic imprinting results in allele-specific silencing according to parental origin. Silencing is brought about by imprinting control regions (ICRs) that are differentially marked in gametogenesis. The group of imprinted transcripts in the mouse Gnas cluster (Nesp, Nespas, Gnasxl, Exon 1A and Gnas) provides a model for analyzing the mechanisms of imprint regulation. We previously identified an ICR that specifically regulates the tissue-specific imprinted expression of the Gnas gene. Here we identify a second ICR at the Gnas cluster. We show that a paternally derived targeted deletion of the germline differentially methylated region (DMR) associated with the antisense Nespas transcript unexpectedly affects both the expression of all transcripts in the cluster and methylation of two DMRs. Our results establish that the Nespas DMR is the principal ICR at the Gnas cluster and functions bidirectionally as a switch for modulating expression of the antagonistically acting genes Gnasxl and Gnas. Uniquely, the Nespas DMR acts on the downstream ICR at exon 1A to regulate tissue-specific imprinting of the Gnas gene.

A gene-driven approach to the identification of ENU mutants in the mouse.

The construction of parallel archives of DNA and sperm from mice mutagenized with ethylnitrosurea (ENU) represents a potentially powerful and rapid approach for identifying point mutations in any gene in the mouse genome. We provide support for this approach and report the identification of mutations in the gene (Gjb2) encoding connexin 26, using archives established from the UK ENU mutagenesis program.

A mutation in Af4 is predicted to cause cerebellar ataxia and cataracts in the robotic mouse.

The robotic mouse is an autosomal dominant mutant that arose from a large-scale chemical mutagenesis program. It has a jerky, ataxic gait and develops adult-onset Purkinje cell loss in the cerebellum in a striking region-specific pattern, as well as cataracts. Genetic and physical mapping of the disease locus led to the identification of a missense mutation in a highly conserved region of Af4, a putative transcription factor that has been previously implicated in leukemogenesis. We demonstrate that Af4 is specifically expressed in Purkinje cells, and we hypothesize that the expression of mutant Af4 leads to neurodegeneration. This function was not identified through knock-out studies, highlighting the power of phenotype-driven mutagenesis in the mouse to identify new pathways involved in neurological disease.

A dominant mutation within the DNA-binding domain of the bZIP transcription factor Maf causes murine cataract and results in selective alteration in DNA binding.

The murine autosomal dominant cataract mutants created in mutagenesis experiments have proven to be a powerful resource for modelling the biological processes involved in cataractogenesis. We report a mutant which in the heterozygous state exhibits mild pulverulent cataract named 'opaque flecks in lens', symbol Ofl. By molecular mapping, followed by a candidate gene approach, the mutant was shown to be allelic with a knockout of the bZIP transcription factor, Maf. Homozygotes for Ofl and for Maf null mutations are similar but a new effect, renal tubular nephritis, was found in Ofl homozygotes surviving beyond 4 weeks, which may contribute to early lethality. Sequencing identified the mutation as a G-->A change, leading to the amino-acid substitution mutation R291Q in the basic region of the DNA-binding domain. Since mice heterozygous for knockouts of Maf show no cataracts, this suggests that the Ofl R291Q mutant protein has a dominant effect. We have demonstrated that this mutation results in a selective alteration in DNA binding affinities to target oligonucleotides containing variations in the core CRE and TRE elements. This implies that arginine 291 is important for core element binding and suggests that the mutant protein may exert a differential downstream effect amongst its binding targets. The cataracts seen in Ofl heterozygotes and human MAF mutations are similar to one another, implying that Ofl may be a model of human pulverulent cortical cataract. Furthermore, when bred onto a different genetic background Ofl heterozygotes also show anterior segment abnormalities. The Ofl mutant therefore provides a valuable model system for the study of Maf, and its interacting factors, in normal and abnormal lens and anterior segment development.

Novel phenotypes identified by plasma biochemical screening in the mouse.

We used ENU mutagenesis in the mouse for the rapid generation of novel mutant phenotypes for both gene function studies and use as new animal models of human disease (Nolan et al. 2000b). One focus of the program was the development of a blood biochemistry screen. At 8-12 weeks of age, approximately 300 ml of blood was collected from F1 offspring of ENU mutagenized male mice. This yielded approximately 125 ml of plasma, used to perform a profile of 17 standard biochemical tests on an Olympus analyzer. Cohorts of F1 mice were also aged and then retested to detect late onset phenotypes. In total, 1,961 F1s were screened. Outliers were identified by running means and standard deviations. Of 70 mice showing consistent abnormalities in plasma biochemistry, 29 were entered into inheritance testing. Of these, 9 phenotypes were confirmed as inherited, 10 found not to be inherited, and 10 are still being tested. Inherited mutant phenotypes include abnormal lipid profiles (low total and HDL cholesterol, high triglycerides); abnormalities in bone and liver metabolism (low ALP, high ALP, high ALT, and AST); abnormal plasma electrolyte levels (high sodium and chloride); as well as phenotypes of interest for the study of diabetes (high glucose). The gene loci bearing the mutations are currently being mapped and further characterized. Our results have validated our biochemical screen, which is applicable to other mutagenesis projects, and we have produced a new set of mutants with defined metabolic phenotypes.

Towards a mutant map of the mouse--new models of neurological, behavioural, deafness, bone, renal and blood disorders.

With the completion of the first draft of the human genome sequence, the next major challenge is assigning function to genes. One approach is genome-wide random chemical mutagenesis, followed by screening for mutant phenotypes of interest and subsequent mapping and identification of the mutated genes in question. We (a consortium made up of GlaxoSmithKline, the MRC Mammalian Genetics Unit and Mouse Genome Centre, Harwell, Imperial College, London, and the Royal London Hospital) have used ENU mutagenesis in the mouse for the rapid generation of novel mutant phenotypes for use as animal models of human disease and for gene function assignment (Nolan et al., 2000). As of 2003, 35,000 mice have been produced to date in a genome-wide screen for dominant mutations and screened using a variety of screening protocols. Nearly 200 mutants have been confirmed as heritable and added to the mouse mutant catalogue and, overall, we can extrapolate that we have recovered over 700 mutants from the screening programme. For further information on the project and details of the data, see http://www.mgu.har.mrc.ac.uk/mutabase.