BackgroundThe mouse inbred line C57BL/6J is widely used in mouse genetics and its genome has been incorporated into many genetic reference populations. More recently large initiatives such as the International Knockout Mouse Consortium (IKMC) are using the C57BL/6N mouse strain to generate null alleles for all mouse genes. Hence both strains are now widely used in mouse genetics studies. Here we perform a comprehensive genomic and phenotypic analysis of the two strains to identify differences that may influence their underlying genetic mechanisms.ResultsWe undertake genome sequence comparisons of C57BL/6J and C57BL/6N to identify SNPs, indels and structural variants, with a focus on identifying all coding variants. We annotate 34 SNPs and 2 indels that distinguish C57BL/6J and C57BL/6N coding sequences, as well as 15 structural variants that overlap a gene. In parallel we assess the comparative phenotypes of the two inbred lines utilizing the EMPReSSslim phenotyping pipeline, a broad based assessment encompassing diverse biological systems. We perform additional secondary phenotyping assessments to explore other phenotype domains and to elaborate phenotype differences identified in the primary assessment. We uncover significant phenotypic differences between the two lines, replicated across multiple centers, in a number of physiological, biochemical and behavioral systems.ConclusionsComparison of C57BL/6J and C57BL/6N demonstrates a range of phenotypic differences that have the potential to impact upon penetrance and expressivity of mutational effects in these strains. Moreover, the sequence variants we identify provide a set of candidate genes for the phenotypic differences observed between the two strains.

As the human genome project approaches completion, the challenge for mammalian geneticists is to develop approaches for the systematic determination of mammalian gene function. Mouse mutagenesis will be a key element of studies of gene function. Phenotype-driven approaches using the chemical mutagen ethylnitrosourea (ENU) represent a potentially efficient route for the generation of large numbers of mutant mice that can be screened for novel phenotypes. The advantage of this approach is that, in assessing gene function, no a priori assumptions are made about the genes involved in any pathway. Phenotype-driven mutagenesis is thus an effective method for the identification of novel genes and pathways. We have undertaken a genome-wide, phenotype-driven screen for dominant mutations in the mouse. We generated and screened over 26,000 mice, and recovered some 500 new mouse mutants. Our work, along with the programme reported in the accompanying paper, has led to a substantial increase in the mouse mutant resource and represents a first step towards systematic studies of gene function in mammalian genetics.

Type 2 diabetes mellitus is the result of a combination of impaired insulin secretion with reduced insulin sensitivity of target tissues. There are an estimated 150 million affected individuals worldwide, of whom a large proportion remains undiagnosed because of a lack of specific symptoms early in this disorder and inadequate diagnostics. In this study, NMR-based metabolomic analysis in conjunction with multivariate statistics was applied to examine the urinary metabolic changes in two rodent models of type 2 diabetes mellitus as well as unmedicated human sufferers. The db/db mouse and obese Zucker (fa/fa) rat have autosomal recessive defects in the leptin receptor gene, causing type 2 diabetes. 1H-NMR spectra of urine were used in conjunction with uni- and multivariate statistics to identify disease-related metabolic changes in these two animal models and human sufferers. This study demonstrates metabolic similarities between the three species examined, including metabolic responses associated with general systemic stress, changes in the TCA cycle, and perturbations in nucleotide metabolism and in methylamine metabolism. All three species demonstrated profound changes in nucleotide metabolism, including that of N-methylnicotinamide and N-methyl-2-pyridone-5-carboxamide, which may provide unique biomarkers for following type 2 diabetes mellitus progression.

Two bottlenecks impeding the genetic analysis of complex traits in rodents are access to mapping populations able to deliver gene-level mapping resolution, and the need for population specific genotyping arrays and haplotype reference panels. Here we combine low coverage sequencing (0.15X) with a novel method to impute the ancestral haplotype space in 1,887 commercially available outbred mice. We mapped 156 unique quantitative trait loci for 92 phenotypes at 5% false discovery rate. Gene-level mapping resolution was achieved at about a fifth of loci, implicating Unc13c and Pgc1-alpha at loci for the quality of sleep, Adarb2 for home cage activity, Rtkn2 for intensity of reaction to startle, Bmp2 for wound healing, Il15 and Id2 for several T-cell measures and Prkca for bone mineral content. These findings have implications for diverse areas of mammalian biology and demonstrate how GWAS can be extended via low-coverage sequencing to species with highly recombinant outbred populations.

Genomic imprinting brings about allele-specific silencing according to parental origin. Silencing is controlled by cis-acting regulatory regions that are differentially marked during gametogenesis and can act over hundreds of kilobases to silence many genes. Two candidate imprinting control regions (ICRs) have been identified at the compact imprinted Gnas cluster on distal mouse chromosome 2, one at exon 1A upstream of Gnas itself and one covering the promoters for Gnasxl and the antisense Nespas (ref. 8). This imprinted cluster is complex, containing biallelic, maternally and paternally expressed transcripts that share exons. Gnas itself is mainly biallelically expressed but is weakly paternally repressed in specific tissues. Here we show that a paternally derived targeted deletion of the germline differentially methylated region at exon 1A abolishes tissue-specific imprinting of Gnas. This rescues the abnormal phenotype of mice with a maternally derived Gnas mutation. Imprinting of alternative transcripts, Nesp, Gnasxl and Nespas (ref. 13), in the cluster is unaffected. The results establish that the differentially methylated region at exon 1A contains an imprinting control element that specifically regulates Gnas and comprises a characterized ICR for a gene that is only weakly imprinted in a minority of tissues. There must be a second ICR regulating the alternative transcripts.