Background: The 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. Results: We 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. Conclusions: Comparison 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.

Two closely related β subunit mRNAs (xo28 and xo32) were identified in Xenopus oocytes by molecular cloning. One or both appear to be expressed as active proteins, because: ( i ) injection of Xenopus β antisense oligonucleotides, but not of sense or unrelated oligonucleotides, significantly reduced endogenous oocyte voltage-gated Ca ²⁺ channel (VGCC) currents and obliterated VGCC currents that arise after injection of mammalian α ₁ cRNAs (α _1C and α _1E ); ( ii ) coinjection of a Xenopus β antisense oligonucleotide and excess rat β cRNA rescued expression of α ₁ Ca ²⁺ channel currents; and ( iii ) coinjection of mammalian α ₁ cRNA with cRNA encoding either of the two Xenopus β subunits facilitated both activation and inactivation of Ca ²⁺ channel currents by voltage, as happens with most mammalian β subunits. The Xenopus β subunit cDNAs (β3xo cDNAs) predict proteins of 484 aa that differ in only 22 aa and resemble most closely the sequence of the mammalian type 3 β subunit. We propose that “α ₁ alone” channels are in fact tightly associated α ₁ β3xo channels, and that effects of exogenous β subunits are due to formation of higher-order [α ₁ β]β _n complexes with an unknown contribution of β3xo. It is thus possible that functional mammalian VGCCs, rather than having subunit composition α ₁ β, are [α ₁ β]β _n complexes that associate with α ₂ δ and, as appropriate, other tissue-specific accessory proteins. In support of this hypothesis, we discovered that the last 277-aa of α _1E have a β subunit binding domain. This β binding domain is distinct from the previously known interaction domain located between repeats I and II of calcium channel α ₁ subunits.

Steve Brown and colleagues report an analysis of 20 phenotyping tests, including 413 data parameters, across 449 mutant mouse alleles. They identify widespread pleiotropy and assign putative functions to genes that lacked previous phenotypic annotation. The function of the majority of genes in the mouse and human genomes remains unknown. The mouse embryonic stem cell knockout resource provides a basis for the characterization of relationships between genes and phenotypes. The EUMODIC consortium developed and validated robust methodologies for the broad-based phenotyping of knockouts through a pipeline comprising 20 disease-oriented platforms. We developed new statistical methods for pipeline design and data analysis aimed at detecting reproducible phenotypes with high power. We acquired phenotype data from 449 mutant alleles, representing 320 unique genes, of which half had no previous functional annotation. We captured data from over 27,000 mice, finding that 83% of the mutant lines are phenodeviant, with 65% demonstrating pleiotropy. Surprisingly, we found significant differences in phenotype annotation according to zygosity. New phenotypes were uncovered for many genes with previously unknown function, providing a powerful basis for hypothesis generation and further investigation in diverse systems.

Background: Methods: Results: Interpretation: