ENU-induced Mouse Mutant Research Articles

Loss of function in ROBO1 is associated with tetralogy of Fallot and septal defects

BackgroundCongenital heart disease (CHD) is a common birth defect affecting approximately 1% of newborns. Great progress has been made in elucidating the genetic aetiology of CHD with advances in genomic...

A Novel ENU-Induced Mutation in Myo6 Causes Vestibular Dysfunction and Deafness.

Mouse N-ethyl-N-nitrosourea (ENU) mutagenesis has generated many useful animal models for human diseases. Here we describe the identification of a novel ENU-induced mouse mutant strain Turner (Tur) that displays circling and headtossing behavior and progressive hearing loss. Tur/Tur homozygous animals lack Preyer and righting reflexes and display severe headtossing and reaching response defect. We mapped the Tur mutation to a critical region of 11 cM on chromosome 9 that includes myosin VI. Direct sequence analysis revealed a c.820A>T substitution in exon 8 of the Myo6 gene that changes amino acid Asn200 to Ile (p.N200I) in the motor domain. Analysis of inner ear hair cells by immunohistochemistry, scanning electron microscopy and histology revealed degeneration of hair cells in the inner ear and structural malformation of the stereocilia in the cochlea of Turner homozygous mutant mice. Our data indicate that this novel mouse strain provides a useful model for future studies on the function of myosin VI in mammalian auditory and non-auditory systems and in human syndromes.

Characterization of Highper, an ENU-induced mouse mutant with abnormal psychostimulant and stress responses

RationaleChemical mutagenesis in the mouse is a forward genetics approach that introduces random mutations into the genome, thereby providing an opportunity to annotate gene function and characterize phenotypes that have not been previously linked to a given gene.ObjectivesWe report on the behavioral characterization of Highper, an N-ethyl-N-nitrosourea (ENU)-induced mutant mouse line.Methods Highper and B6 control mice were assessed for locomotor activity in the open field and home cage environments. Basal and acute restraint stress-induced corticosterone levels were measured. Mice were tested for locomotor response to cocaine (5, 20, 30, and 45 mg/kg), methylphenidate (30 mg/kg), and ethanol (0.75, 1.25, and 1.75 g/kg). The rewarding and reinforcing effects of cocaine were assessed using conditioned place preference and self-administration paradigms.Results Highper mice are hyperactive during behavioral tests but show normal home cage locomotor behavior. Highper mice also exhibit a twofold increase in locomotor response to cocaine, methylphenidate, and ethanol and prolonged activation of the hypothalamic–pituitary–adrenal axis in response to acute stress. Highper mice are more sensitive to the rewarding and reinforcing effects of cocaine, although place preference in Highper mice appears to be significantly influenced by the environment in which the drug is administered.ConclusionsAltogether, our findings indicate that Highper mice may provide important insights into the genetic, molecular, and biological mechanisms underlying stress and the drug reward pathway.Electronic supplementary materialThe online version of this article (doi:10.1007/s00213-012-2827-5) contains supplementary material, which is available to authorized users.

An ENU-induced mouse mutant of SHIP1 reveals a critical role of the stem cell isoform for suppression of macrophage activation

AbstractIn a recessive ENU mutagenesis screen for embryonic lethality, we identified a mouse pedigree with a missense mutation of SHIP1 (SHIP1el20) leading to an amino acid substitution I641T in the inositol-5′-phosphatase domain that represses phosphatidylinositol-3-kinase signaling. Despite detectable expression of functional SHIP1 protein, the phenotype of homozygous SHIP1el20/el20 mice was more severe than gene-targeted SHIP1-null (SHIP1−/−) mice. Compared with age-matched SHIP1−/− mice, 5-week-old SHIP1el20/el20 mice had increased myeloid cells, serum IL-6 levels, marked reductions in lymphoid cells, and died by 7 weeks of age with infiltration of the lungs by activated macrophages. Bone marrow transplantation demonstrated that these defects were hematopoietic-cell-autonomous. We show that the el20 mutation reduces expression in SHIP1el20/el20 macrophages of both SHIP1 and s-SHIP, an isoform of SHIP1 generated by an internal promoter. In contrast, SHIP1−/− macrophages express normal levels of s-SHIP. Compound heterozygous mice (SHIP1−/el20) had the same phenotype as SHIP1−/− mice, thus providing genetic proof that the more severe phenotype of SHIP1el20/el20 mice is probably the result of concomitant loss of SHIP1 and s-SHIP. Our results suggest that s-SHIP synergizes with SHIP1 for suppression of macrophage activation, thus providing the first evidence for a role of s-SHIP in adult hematopoiesis.

Discovery of Candidate Disease Genes in ENU–Induced Mouse Mutants by Large-Scale Sequencing, Including a Splice-Site Mutation in Nucleoredoxin

An accurate and precisely annotated genome assembly is a fundamental requirement for functional genomic analysis. Here, the complete DNA sequence and gene annotation of mouse Chromosome 11 was used to test the efficacy of large-scale sequencing for mutation identification. We re-sequenced the 14,000 annotated exons and boundaries from over 900 genes in 41 recessive mutant mouse lines that were isolated in an N-ethyl-N-nitrosourea (ENU) mutation screen targeted to mouse Chromosome 11. Fifty-nine sequence variants were identified in 55 genes from 31 mutant lines. 39% of the lesions lie in coding sequences and create primarily missense mutations. The other 61% lie in noncoding regions, many of them in highly conserved sequences. A lesion in the perinatal lethal line l11Jus13 alters a consensus splice site of nucleoredoxin (Nxn), inserting 10 amino acids into the resulting protein. We conclude that point mutations can be accurately and sensitively recovered by large-scale sequencing, and that conserved noncoding regions should be included for disease mutation identification. Only seven of the candidate genes we report have been previously targeted by mutation in mice or rats, showing that despite ongoing efforts to functionally annotate genes in the mammalian genome, an enormous gap remains between phenotype and function. Our data show that the classical positional mapping approach of disease mutation identification can be extended to large target regions using high-throughput sequencing.

The Novel Mouse Mutation Oblivion Inactivates the PMCA2 Pump and Causes Progressive Hearing Loss

Progressive hearing loss is common in the human population, but we have few clues to the molecular basis. Mouse mutants with progressive hearing loss offer valuable insights, and ENU (N-ethyl-N-nitrosourea) mutagenesis is a useful way of generating models. We have characterised a new ENU-induced mouse mutant, Oblivion (allele symbol Obl), showing semi-dominant inheritance of hearing impairment. Obl/+ mutants showed increasing hearing impairment from post-natal day (P)20 to P90, and loss of auditory function was followed by a corresponding base to apex progression of hair cell degeneration. Obl/Obl mutants were small, showed severe vestibular dysfunction by 2 weeks of age, and were completely deaf from birth; sensory hair cells were completely degenerate in the basal turn of the cochlea, although hair cells appeared normal in the apex. We mapped the mutation to Chromosome 6. Mutation analysis of Atp2b2 showed a missense mutation (2630C→T) in exon 15, causing a serine to phenylalanine substitution (S877F) in transmembrane domain 6 of the PMCA2 pump, the resident Ca2+ pump of hair cell stereocilia. Transmembrane domain mutations in these pumps generally are believed to be incompatible with normal targeting of the protein to the plasma membrane. However, analyses of hair cells in cultured utricular maculae of Obl/Obl mice and of the mutant Obl pump in model cells showed that the protein was correctly targeted to the plasma membrane. Biochemical and biophysical characterisation showed that the pump had lost a significant portion of its non-stimulated Ca2+ exporting ability. These findings can explain the progressive loss of auditory function, and indicate the limits in our ability to predict mechanism from sequence alone.

An ENU-induced mouse mutant with ADHD-related behavioral abnormality carries a missense mutation in the intracellular C0 domain of NR1

An ENU-induced mouse mutant with ADHD-related behavioral abnormality carries a missense mutation in the intracellular C0 domain of NR1

Enamelin ( Enam ) is essential for amelogenesis: ENU-induced mouse mutants as models for different clinical subtypes of human amelogenesis imperfecta (AI)

Amelogenesis imperfecta (AI) is a group of commonly inherited defects of dental enamel formation, which exhibits marked genetic and clinical heterogeneity. The genetic basis of this heterogeneity is still poorly understood. Enamelin, the affected gene product in one form of AI (AIH2), is an extracellular matrix protein that is one of the components of enamel. We isolated three ENU-induced dominant mouse mutations, M100395, M100514 and M100521, which caused AI-like phenotypes in the incisors and molars of the affected individuals. Linkage analyses mapped each of the three mutations to a region of chromosome 5 that contained the genes encoding enamelin (Enam) and ameloblastin (Ambn). Sequence analysis revealed that each mutation was a single-base substitution in Enam. M100395 (Enam(Rgsc395)) and M100514 (Enam(Rgsc514)) were putative missense mutations that caused S to I and E to G substitutions at positions 55 and 57 of the translated protein, respectively. Enam(Rgsc395) and Enam(Rgsc514) heterozygotes showed severe breakage of the enamel surface, a phenotype that resembled local hypoplastic AI. The M100521 mutation (Enam(Rgsc521)) was a T to A substitution at the splicing donor site in intron 4. This mutation resulted in a frameshift that gave rise to a premature stop codon. The transcript of the Enam(Rgsc521) mutant allele was degraded, indicating that Enam(Rgsc521) is a loss-of-function mutation. Enam(Rgsc521) heterozygotes showed a hypomaturation-type AI phenotype in the incisors, possibly due to haploinsufficiency of Enam. Enam(Rgsc521) homozygotes showed complete loss of enamel on the incisors and the molars. Thus, we report here that the Enam gene is essential for amelogenesis, and that mice with different point mutations at Enam may provide good animal models to study the different clinical subtypes of AI.

Assessment of a systematic expression profiling approach in ENU-induced mouse mutant lines

Comparative genomewide expression profiling is a powerful tool in the effort to annotate the mouse genome with biological function. The systematic analysis of RNA expression data of mouse lines from the Munich ENU mutagenesis screen might support the understanding of the molecular biology of such mutants and provide new insights into mammalian gene function. In a direct comparison of DNA microarray experiments of individual versus pooled RNA samples of organs from ENU-induced mouse mutants, we provide evidence that individual RNA samples may outperform pools in some aspects. Genes with high biological variability in their expression levels (noisy genes) are identified as false positives in pooled samples. Evidence suggests that highly stringent housing conditions and standardized procedures for the isolation of organs significantly reduce biological variability in gene expression profiling experiments. Data on wild-type individuals demonstrate the positive effect of controlling variables such as social status, food intake before organ sampling, and stress with regard to reproducibility of gene expression patterns. Analyses of several organs from various ENU-induced mutant lines in general show low numbers of differentially expressed genes. We demonstrate the feasibility to detect transcriptionally affected organs employing RNA expression profiling as a tool for molecular phenotyping.

Hypercholesterolemia in ENU-induced mouse mutants

Hypercholesterolemia is caused by multiple environmental factors and genetic predispositions, and plays an important role in the development and pathogenesis of various human diseases. In this study, we aimed to establish randomly mutant mouse lines showing hypercholesterolemia for their further use in the detection of novel causative alleles. In the Munich ENU Mouse Mutagenesis Project, clinical chemistry blood analysis was performed on more than 15,000 G1 mice and 230 G3 pedigrees of chemically mutagenized mice to detect dominant and recessive mutations leading to an increased plasma total cholesterol level. Using inbred C3HeB/FeJ mice we identified more than 100 animals consistently showing hypercholesterolemia. Transmission of the altered phenotype to the subsequent generations led to the production of nine hypercholesterolemic lines. A single line showed further obvious deviations in the analysis of additional clinical chemistry blood parameters. Thus, the lines produced will contribute to the search for alleles that selectively cause primary hypercholesterolemia.