ABSTRACT
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.
Subject(s)
Genome/genetics , Animals , Behavior, Animal , Disease Resistance/immunology , Eye/pathology , Female , Femur/diagnostic imaging , Hypersensitivity/immunology , INDEL Mutation/genetics , Killer Cells, Natural/immunology , Listeriosis/immunology , Listeriosis/microbiology , Male , Maze Learning , Mice, Inbred C57BL , Phenotype , Polymorphism, Single Nucleotide/genetics , Spleen/immunology , X-Ray MicrotomographyABSTRACT
Vertebrate organs show consistent left-right (L-R) asymmetry in placement and patterning. To identify genes involved in this process we performed an ENU-based genetic screen. Of 135 lines analyzed 11 showed clear single gene defects affecting L-R patterning, including 3 new alleles of known L-R genes and mutants in novel L-R loci. We identified six lines (termed "gasping") that, in addition to abnormal L-R patterning and associated cardiovascular defects, had complex phenotypes including pulmonary agenesis, exencephaly, polydactyly, ocular and craniofacial malformations. These complex abnormalities are present in certain human disease syndromes (e.g., HYLS, SRPS, VACTERL). Gasping embryos also show defects in ciliogenesis, suggesting a role for cilia in these human congenital malformation syndromes. Our results indicate that genes controlling ciliogenesis and left-right asymmetry have, in addition to their known roles in cardiac patterning, major and unexpected roles in pulmonary, craniofacial, ocular and limb development with implications for human congenital malformation syndromes.
Subject(s)
Body Patterning/genetics , Extremities/embryology , Eye/embryology , Facial Bones/embryology , Mutagenesis/genetics , Respiratory System/embryology , Amino Acid Sequence , Animals , Embryo, Mammalian/embryology , Embryo, Mammalian/metabolism , Eye/metabolism , Facial Bones/metabolism , Gene Expression Regulation, Developmental/genetics , Humans , Mice , Microscopy, Electron, Scanning , Molecular Sequence Data , Mutation/genetics , Phenotype , Respiratory System/metabolism , Sequence AlignmentABSTRACT
By screening N-ethyl-N-nitrosourea-mutagenized animals for alterations in rhythms of wheel-running activity, we identified a mouse mutation, after hours (Afh). The mutation, a Cys(358)Ser substitution in Fbxl3, an F-box protein with leucine-rich repeats, results in long free-running rhythms of about 27 hours in homozygotes. Circadian transcriptional and translational oscillations are attenuated in Afh mice. The Afh allele significantly affected Per2 expression and delayed the rate of Cry protein degradation in Per2::Luciferase tissue slices. Our in vivo and in vitro studies reveal a central role for Fbxl3 in mammalian circadian timekeeping.
Subject(s)
Circadian Rhythm , F-Box Proteins/genetics , F-Box Proteins/physiology , Point Mutation , ARNTL Transcription Factors , Amino Acid Sequence , Amino Acid Substitution , Animals , Basic Helix-Loop-Helix Transcription Factors/genetics , Basic Helix-Loop-Helix Transcription Factors/metabolism , CLOCK Proteins , COS Cells , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Chlorocebus aethiops , Circadian Rhythm/genetics , Crosses, Genetic , Cryptochromes , Female , Flavoproteins/genetics , Flavoproteins/metabolism , Gene Expression Regulation , Liver/metabolism , Lung/metabolism , Male , Mice , Mice, Inbred BALB C , Mice, Inbred C3H , Molecular Sequence Data , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Period Circadian Proteins , Suprachiasmatic Nucleus/metabolism , Trans-Activators/genetics , Trans-Activators/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism , Transcription, GeneticABSTRACT
Otitis media (OM), inflammation of the middle ear, remains the most common cause of hearing impairment in children. It is also the most common cause of surgery in children in the developed world. There is evidence from studies of the human population and mouse models that there is a significant genetic component predisposing to OM, yet nothing is known about the underlying genetic pathways involved in humans. We identified an N-ethyl-N-nitrosourea-induced dominant mouse mutant Junbo with hearing loss due to chronic suppurative OM and otorrhea. This develops from acute OM that arises spontaneously in the postnatal period, with the age of onset and early severity dependent on the microbiological status of the mice and their air quality. We have identified the causal mutation, a missense change in the C-terminal zinc finger region of the transcription factor Evi1. This protein is expressed in middle ear basal epithelial cells, fibroblasts, and neutrophil leukocytes at postnatal day 13 and 21 when inflammatory changes are underway. The identification and characterization of the Junbo mutant elaborates a novel role for Evi1 in mammalian disease and implicates a new pathway in genetic predisposition to OM.
Subject(s)
DNA-Binding Proteins/genetics , Genetic Predisposition to Disease/genetics , Mutation/genetics , Otitis Media/genetics , Proto-Oncogenes/genetics , Transcription Factors/genetics , Amino Acid Sequence , Animals , Chromosome Mapping , Cloning, Molecular , DNA Mutational Analysis , DNA-Binding Proteins/chemistry , Ear, Middle/cytology , Ear, Middle/pathology , Flow Cytometry , Granulocytes/immunology , Lung/cytology , Lung/pathology , MDS1 and EVI1 Complex Locus Protein , Male , Mice , Mice, Inbred BALB C , Mice, Mutant Strains , Molecular Sequence Data , Nose/cytology , Nose/pathology , Otitis Media/immunology , Phenotype , Specific Pathogen-Free Organisms , Transcription Factors/chemistryABSTRACT
The semi-dominantly inherited mouse mutation pardon (Pdo) was isolated due to the lack of a Preyer reflex (ear flick) in response to sound from a large-scale N -ethyl- N -nitrosourea (ENU) mutagenesis programme. Dissection of the middle ear revealed malformations in all three ossicles, rendering the ossicular chain incomplete. Hair cell counts in the apical turn of the organ of Corti revealed a significant 22.7% increase in the number of outer hair cells. Raised compound action potential thresholds in Pdo/+ mutants suggested a combined sensorineural/conductive hearing loss. We show that a missense mutation in the homeobox gene Emx2 is responsible for these defects, identifying a new function for this gene in the development of specific structures in the ear.
