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Targeted knockout and lacZ reporter expression of the mouse Tmhs deafness gene and characterization of the hscy-2J mutation.

ABSTRACT: The Tmhs gene codes for a tetraspan transmembrane protein that is expressed in hair cell stereocilia. We previously showed that a spontaneous missense mutation of Tmhs underlies deafness and vestibular dysfunction in the hurry-scurry (hscy) mouse. Subsequently, mutations in the human TMHS gene were shown to be responsible for DFNB67, an autosomal recessive nonsyndromic deafness locus. Here we describe a genetically engineered null mutation of the mouse Tmhs gene (Tmhs ( tm1Kjn )) and show that its phenotype is identical to that of the hscy missense mutation, confirming the deleterious nature of the hscy cysteine-to-phenylalanine substitution. In the targeted null allele, the Tmhs promoter drives expression of a lacZ reporter gene. Visualization of beta-galactosidase activity in Tmhs ( tm1Kjn ) heterozygous mice indicates that Tmhs is highly expressed in the cochlear and vestibular hair cells of the inner ear. Expression is first detectable at E15.5, peaks around P0, decreases slightly at P6, and is absent by P15, a duration that supports the involvement of Tmhs in stereocilia development. Tmhs reporter gene expression also was detected in several cranial and cervical sensory ganglia, but not in the vestibular or spiral ganglia. We also describe a new nontargeted mutation of the Tmhs gene, hscy-2J, that causes abnormal splicing from a cryptic splice site within exon 2 and is predicted to produce a functionally null protein lacking 51 amino acids of the wild-type sequence.

SUBMITTER: Longo-Guess CM

PROVIDER: S-EPMC2613174 | biostudies-literature | 2007 Sep

REPOSITORIES: biostudies-literature

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Targeted knockout and lacZ reporter expression of the mouse Tmhs deafness gene and characterization of the hscy-2J mutation.

Longo-Guess Chantal M CM Gagnon Leona H LH Fritzsch Bernd B Johnson Kenneth R KR

Mammalian genome : official journal of the International Mammalian Genome Society 20070918 9

The Tmhs gene codes for a tetraspan transmembrane protein that is expressed in hair cell stereocilia. We previously showed that a spontaneous missense mutation of Tmhs underlies deafness and vestibular dysfunction in the hurry-scurry (hscy) mouse. Subsequently, mutations in the human TMHS gene were shown to be responsible for DFNB67, an autosomal recessive nonsyndromic deafness locus. Here we describe a genetically engineered null mutation of the mouse Tmhs gene (Tmhs ( tm1Kjn )) and show that i ...[more]

PMID: 17876667

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Project description:BackgroundMice carrying targeted mutations are important for investigating gene function and the role of genes in disease, but off-target mutagenic effects associated with the processes of generating targeted alleles, for instance using Crispr, and culturing embryonic stem cells, offer opportunities for spontaneous mutations to arise. Identifying spontaneous mutations relies on the detection of phenotypes segregating independently of targeted alleles, and having a broad estimate of the level of mutations generated by intensive breeding programmes is difficult given that many phenotypes are easy to miss if not specifically looked for. Here we present data from a large, targeted knockout programme in which mice were analysed through a phenotyping pipeline. Such spontaneous mutations segregating within mutant lines may confound phenotypic analyses, highlighting the importance of record-keeping and maintaining correct pedigrees.ResultsTwenty-five lines out of 1311 displayed different deafness phenotypes that did not segregate with the targeted allele. We observed a variety of phenotypes by Auditory Brainstem Response (ABR) and behavioural assessment and isolated eight lines showing early-onset severe progressive hearing loss, later-onset progressive hearing loss, low frequency hearing loss, or complete deafness, with vestibular dysfunction. The causative mutations identified include deletions, insertions, and point mutations, some of which involve new genes not previously associated with deafness while others are new alleles of genes known to underlie hearing loss. Two of the latter show a phenotype much reduced in severity compared to other mutant alleles of the same gene. We investigated the ES cells from which these lines were derived and determined that only one of the 8 mutations could have arisen in the ES cell, and in that case, only after targeting. Instead, most of the non-segregating mutations appear to have occurred during breeding of mutant mice. In one case, the mutation arose within the wildtype colony used for expanding mutant lines.ConclusionsOur data show that spontaneous mutations with observable effects on phenotype are a common side effect of intensive breeding programmes, including those underlying targeted mutation programmes. Such spontaneous mutations segregating within mutant lines may confound phenotypic analyses, highlighting the importance of record-keeping and maintaining correct pedigrees.

Project description:BackgroundDystrophin is a rod-shaped cytoplasmic protein that provides sarcolemmal stability as a structural link between the cytoskeleton and the extracellular matrix via the dystrophin-associated protein complex (DAPC). Mutations in the dystrophin-encoding DMD gene cause X-linked dystrophinopathies with variable phenotypes, the most severe being Duchenne muscular dystrophy (DMD) characterized by progressive muscle wasting and fibrosis. However, dystrophin deficiency does not only impair the function of skeletal and heart muscle but may also affect other organ systems such as the brain, eye, and gastrointestinal tract. The generation of a dystrophin reporter mouse would facilitate research into dystrophin muscular and extramuscular pathophysiology without the need for immunostaining.ResultsWe generated a Dmd (EGFP) reporter mouse through the in-frame insertion of the EGFP coding sequence behind the last Dmd exon 79, which is known to be expressed in all major dystrophin isoforms. We analyzed EGFP and dystrophin expression in various tissues and at the single muscle fiber level. Immunostaining of various members of the DAPC was done to confirm the correct subsarcolemmal location of dystrophin-binding partners. We found strong natural EGFP fluorescence at all expected sites of dystrophin expression in the skeletal and smooth muscle, heart, brain, and retina. EGFP fluorescence exactly colocalized with dystrophin immunostaining. In the skeletal muscle, dystrophin and other proteins of the DAPC were expressed at their correct sarcolemmal/subsarcolemmal localization. Skeletal muscle maintained normal tissue architecture, suggesting the correct function of the dystrophin-EGFP fusion protein. EGFP expression could be easily verified in isolated myofibers as well as in satellite cell-derived myotubes.ConclusionsThe novel dystrophin reporter mouse provides a valuable tool for direct visualization of dystrophin expression and will allow the study of dystrophin expression in vivo and in vitro in various tissues by live cell imaging.

Dataset Information

Targeted knockout and lacZ reporter expression of the mouse Tmhs deafness gene and characterization of the hscy-2J mutation.

Publications

Targeted knockout and lacZ reporter expression of the mouse Tmhs deafness gene and characterization of the hscy-2J mutation.

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