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1.
Methods ; 191: 15-22, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-32721467

RESUMO

Aberrant microsatellite repeat-expansions at specific loci within the human genome cause several distinct, heritable, and predominantly neurological, disorders. Creating models for these diseases poses a challenge, due to the instability of such repeats in bacterial vectors, especially with large repeat expansions. Designing constructs for more precise genome engineering projects, such as engineering knock-in mice, proves a greater challenge still, since these unstable repeats require numerous cloning steps in order to introduce homology arms or selection cassettes. Here, we report our efforts to clone a large hexanucleotide repeat in the C9orf72 gene, originating from within a BAC construct, derived from a C9orf72-ALS patient. We provide detailed methods for efficient repeat sizing and growth conditions in bacteria to facilitate repeat retention during growth and sub-culturing. We report that sub-cloning into a linear vector dramatically improves stability, but is dependent on the relative orientation of DNA replication through the repeat, consistent with previous studies. We envisage the findings presented here provide a relatively straightforward route to maintaining large-range microsatellite repeat-expansions, for efficient cloning into vectors.

2.
BMC Genomics ; 21(1): 754, 2020 Nov 02.
Artigo em Inglês | MEDLINE | ID: mdl-33138777

RESUMO

BACKGROUND: Efforts to elucidate the function of enhancers in vivo are underway but their vast numbers alongside differing enhancer architectures make it difficult to determine their impact on gene activity. By systematically annotating multiple mouse tissues with super- and typical-enhancers, we have explored their relationship with gene function and phenotype. RESULTS: Though super-enhancers drive high total- and tissue-specific expression of their associated genes, we find that typical-enhancers also contribute heavily to the tissue-specific expression landscape on account of their large numbers in the genome. Unexpectedly, we demonstrate that both enhancer types are preferentially associated with relevant 'tissue-type' phenotypes and exhibit no difference in phenotype effect size or pleiotropy. Modelling regulatory data alongside molecular data, we built a predictive model to infer gene-phenotype associations and use this model to predict potentially novel disease-associated genes. CONCLUSION: Overall our findings reveal that differing enhancer architectures have a similar impact on mammalian phenotypes whilst harbouring differing cellular and expression effects. Together, our results systematically characterise enhancers with predicted phenotypic traits endorsing the role for both types of enhancers in human disease and disorders.


Assuntos
Elementos Facilitadores Genéticos , Animais , Elementos Facilitadores Genéticos/genética , Humanos , Camundongos , Fenótipo
3.
Curr Protoc Mouse Biol ; 9(3): e64, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31532925

RESUMO

Genetically modified mice are an essential tool for modeling disease-causing mechanisms and discovering gene function. SNP genotyping was traditionally used to associate candidate regions with traits in the mouse, but failed to reveal novel variants without further targeted sequencing. Using a robust set of computational protocols, we present a platform to enable scientists to detect variants arising from whole-genome and exome sequencing experiments. This article guides researchers on aligning reads to the mouse genome, quality-assurance strategies, mutation discovery, comparing mutations to previously discovered mouse SNPs, and the annotation of novel variants, in order to predict mutation consequences on the protein level. Challenges unique to the mouse are discussed, and two protocols use self-contained containers to maintain version control and allow users to adapt our approach to new techniques by upgrading container versions. Our protocols are suited for servers or office workstations and are usable by non-bioinformatics specialists. © 2019 by John Wiley & Sons, Inc.


Assuntos
Biologia Computacional/métodos , Genômica/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Camundongos/genética , Mutação , Animais , Análise de Sequência de DNA , Sequenciamento Completo do Genoma
4.
EMBO Mol Med ; 11(9): e10288, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31448880

RESUMO

Hearing relies on mechanically gated ion channels present in the actin-rich stereocilia bundles at the apical surface of cochlear hair cells. Our knowledge of the mechanisms underlying the formation and maintenance of the sound-receptive structure is limited. Utilizing a large-scale forward genetic screen in mice, genome mapping and gene complementation tests, we identified Clrn2 as a new deafness gene. The Clrn2clarinet/clarinet mice (p.Trp4* mutation) exhibit a progressive, early-onset hearing loss, with no overt retinal deficits. Utilizing data from the UK Biobank study, we could show that CLRN2 is involved in human non-syndromic progressive hearing loss. Our in-depth morphological, molecular and functional investigations establish that while it is not required for initial formation of cochlear sensory hair cell stereocilia bundles, clarin-2 is critical for maintaining normal bundle integrity and functioning. In the differentiating hair bundles, lack of clarin-2 leads to loss of mechano-electrical transduction, followed by selective progressive loss of the transducing stereocilia. Together, our findings demonstrate a key role for clarin-2 in mammalian hearing, providing insights into the interplay between mechano-electrical transduction and stereocilia maintenance.


Assuntos
Perda Auditiva/metabolismo , Estereocílios/metabolismo , Adulto , Idoso , Animais , Estudos de Coortes , Feminino , Células Ciliadas Auditivas/metabolismo , Audição , Perda Auditiva/genética , Perda Auditiva/fisiopatologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Estereocílios/genética
5.
J Bone Miner Res ; 34(7): 1324-1335, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-30830987

RESUMO

Nephrolithiasis (NL) and nephrocalcinosis (NC), which comprise renal calcification of the collecting system and parenchyma, respectively, have a multifactorial etiology with environmental and genetic determinants and affect ∼10% of adults by age 70 years. Studies of families with hereditary NL and NC have identified >30 causative genes that have increased our understanding of extracellular calcium homeostasis and renal tubular transport of calcium. However, these account for <20% of the likely genes that are involved, and to identify novel genes for renal calcification disorders, we investigated 1745 12-month-old progeny from a male mouse that had been treated with the chemical mutagen N-ethyl-N-nitrosourea (ENU) for radiological renal opacities. This identified a male mouse with renal calcification that was inherited as an autosomal dominant trait with >80% penetrance in 152 progeny. The calcification consisted of calcium phosphate deposits in the renal papillae and was associated with the presence of the urinary macromolecules osteopontin and Tamm-Horsfall protein, which are features found in Randall's plaques of patients with NC. Genome-wide mapping located the disease locus to a ∼30 Mbp region on chromosome 17A3.3-B3 and whole-exome sequence analysis identified a heterozygous mutation, resulting in a missense substitution (Met149Thr, M149T), in the bromodomain-containing protein 4 (BRD4). The mutant heterozygous (Brd4+/M149T ) mice, when compared with wild-type (Brd4+/+ ) mice, were normocalcemic and normophosphatemic, with normal urinary excretions of calcium and phosphate, and had normal bone turnover markers. BRD4 plays a critical role in histone modification and gene transcription, and cDNA expression profiling, using kidneys from Brd4+/M149T and Brd4+/+ mice, revealed differential expression of genes involved in vitamin D metabolism, cell differentiation, and apoptosis. Kidneys from Brd4+/M149T mice also had increased apoptosis at sites of calcification within the renal papillae. Thus, our studies have established a mouse model, due to a Brd4 Met149Thr mutation, for inherited NC. © 2019 American Society for Bone and Mineral Research.


Assuntos
Mutação de Sentido Incorreto/genética , Nefrocalcinose/genética , Proteínas Nucleares/genética , Fatores de Transcrição/genética , Sequência de Aminoácidos , Animais , Apoptose/genética , Segregação de Cromossomos/genética , Cromossomos de Mamíferos/genética , Modelos Animais de Doenças , Feminino , Loci Gênicos , Rim/patologia , Masculino , Camundongos , Nefrocalcinose/urina , Proteínas Nucleares/química , Fenótipo , Fatores de Transcrição/química , Transcrição Genética , Sequenciamento Completo do Exoma
6.
J Bone Miner Res ; 34(3): 497-507, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30395686

RESUMO

Renal calcification (RCALC) resulting in nephrolithiasis and nephrocalcinosis, which affects ∼10% of adults by 70 years of age, involves environmental and genetic etiologies. Thus, nephrolithiasis and nephrocalcinosis occurs as an inherited disorder in ∼65% of patients, and may be associated with endocrine and metabolic disorders including: primary hyperparathyroidism, hypercalciuria, renal tubular acidosis, cystinuria, and hyperoxaluria. Investigations of families with nephrolithiasis and nephrocalcinosis have identified some causative genes, but further progress is limited as large families are unavailable for genetic studies. We therefore embarked on establishing mouse models for hereditary nephrolithiasis and nephrocalcinosis by performing abdominal X-rays to identify renal opacities in N-ethyl-N-nitrosourea (ENU)-mutagenized mice. This identified a mouse with RCALC inherited as an autosomal dominant trait, designated RCALC type 2 (RCALC2). Genomewide mapping located the Rcalc2 locus to a ∼16-Mbp region on chromosome 11D-E2 and whole-exome sequence analysis identified a heterozygous mutation in the DNA polymerase gamma-2, accessory subunit (Polg2) resulting in a nonsense mutation, Tyr265Stop (Y265X), which co-segregated with RCALC2. Kidneys of mutant mice (Polg2+ / Y265X ) had lower POLG2 mRNA and protein expression, compared to wild-type littermates (Polg2+/+ ). The Polg2+/Y265X and Polg2+ / + mice had similar plasma concentrations of sodium, potassium, calcium, phosphate, chloride, urea, creatinine, glucose, and alkaline phosphatase activity; and similar urinary fractional excretion of calcium, phosphate, oxalate, and protein. Polg2 encodes the minor subunit of the mitochondrial DNA (mtDNA) polymerase and the mtDNA content in Polg2+ / Y265X kidneys was reduced compared to Polg2+/+ mice, and cDNA expression profiling revealed differential expression of 26 genes involved in several biological processes including mitochondrial DNA function, apoptosis, and ubiquitination, the complement pathway, and inflammatory pathways. In addition, plasma of Polg2+ / Y265X mice, compared to Polg2+ / + littermates had higher levels of reactive oxygen species. Thus, our studies have identified a mutant mouse model for inherited renal calcification associated with a Polg2 nonsense mutation. © 2018 The Authors. Journal of Bone and Mineral Research Published by Wiley Periodicals, Inc.


Assuntos
Calcinose , Códon de Terminação , DNA Polimerase gama , Etilnitrosoureia/toxicidade , Nefropatias , Rim , Animais , Calcinose/genética , Calcinose/metabolismo , Calcinose/patologia , DNA Polimerase gama/genética , DNA Polimerase gama/metabolismo , Rim/metabolismo , Rim/patologia , Nefropatias/genética , Nefropatias/metabolismo , Nefropatias/patologia , Camundongos , Camundongos Mutantes
7.
Dis Model Mech ; 11(12)2018 12 18.
Artigo em Inglês | MEDLINE | ID: mdl-30478029

RESUMO

Isocitrate dehydrogenase (IDH) is an enzyme required for the production of α-ketoglutarate from isocitrate. IDH3 generates the NADH used in the mitochondria for ATP production, and is a tetramer made up of two α, one ß and one γ subunit. Loss-of-function and missense mutations in both IDH3A and IDH3B have previously been implicated in families exhibiting retinal degeneration. Using mouse models, we investigated the role of IDH3 in retinal disease and mitochondrial function. We identified mice with late-onset retinal degeneration in a screen of ageing mice carrying an ENU-induced mutation, E229K, in Idh3a Mice homozygous for this mutation exhibit signs of retinal stress, indicated by GFAP staining, as early as 3 months, but no other tissues appear to be affected. We produced a knockout of Idh3a and found that homozygous mice do not survive past early embryogenesis. Idh3a-/E229K compound heterozygous mutants exhibit a more severe retinal degeneration compared with Idh3aE229K/E229K homozygous mutants. Analysis of mitochondrial function in mutant cell lines highlighted a reduction in mitochondrial maximal respiration and reserve capacity levels in both Idh3aE229K/E229K and Idh3a-/E229K cells. Loss-of-function Idh3b mutants do not exhibit the same retinal degeneration phenotype, with no signs of retinal stress or reduction in mitochondrial respiration. It has previously been reported that the retina operates with a limited mitochondrial reserve capacity and we suggest that this, in combination with the reduced reserve capacity in mutants, explains the degenerative phenotype observed in Idh3a mutant mice.This article has an associated First Person interview with the first author of the paper.


Assuntos
Isocitrato Desidrogenase/genética , Mitocôndrias/patologia , Mutação/genética , Degeneração Retiniana/genética , Degeneração Retiniana/fisiopatologia , Animais , Fibroblastos/metabolismo , Genótipo , Isocitrato Desidrogenase/metabolismo , Mutação com Perda de Função/genética , Camundongos , Mutação de Sentido Incorreto/genética , Fenótipo , Células Fotorreceptoras de Vertebrados/metabolismo , Células Fotorreceptoras de Vertebrados/patologia , Retina/patologia , Retina/fisiopatologia
8.
JBMR Plus ; 2(3): 154-163, 2018 May.
Artigo em Inglês | MEDLINE | ID: mdl-30283900

RESUMO

Kyphosis and scoliosis are common spinal disorders that occur as part of complex syndromes or as nonsyndromic, idiopathic diseases. Familial and twin studies implicate genetic involvement, although the causative genes for idiopathic kyphoscoliosis remain to be identified. To facilitate these studies, we investigated progeny of mice treated with the chemical mutagen N-ethyl-N-nitrosourea (ENU) and assessed them for morphological and radiographic abnormalities. This identified a mouse with kyphoscoliosis due to fused lumbar vertebrae, which was inherited as an autosomal dominant trait; the phenotype was designated as hereditary vertebral fusion (HVF) and the locus as Hvf. Micro-computed tomography (µCT) analysis confirmed the occurrence of nonsyndromic kyphoscoliosis due to fusion of lumbar vertebrae in HVF mice, consistent with a pattern of blocked vertebrae due to failure of segmentation. µCT scans also showed the lumbar vertebral column of HVF mice to have generalized disc narrowing, displacement with compression of the neural spine, and distorted transverse processes. Histology of lumbar vertebrae revealed HVF mice to have irregularly shaped vertebral bodies and displacement of intervertebral discs and ossification centers. Genetic mapping using a panel of single nucleotide polymorphic (SNP) loci arranged in chromosome sets and DNA samples from 23 HVF (eight males and 15 females) mice, localized Hvf to chromosome 4A3 and within a 5-megabase (Mb) region containing nine protein coding genes, two processed transcripts, three microRNAs, five small nuclear RNAs, three large intergenic noncoding RNAs, and 24 pseudogenes. However, genome sequence analysis in this interval did not identify any abnormalities in the coding exons, or exon-intron boundaries of any of these genes. Thus, our studies have established a mouse model for a monogenic form of nonsyndromic kyphoscoliosis due to fusion of lumbar vertebrae, and further identification of the underlying genetic defect will help elucidate the molecular mechanisms involved in kyphoscoliosis. © 2018 The Authors. JBMR Plus is published by Wiley Periodicals, Inc. on behalf of the American Society for Bone and Mineral Research.

10.
BMC Biol ; 16(1): 70, 2018 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-29925374

RESUMO

BACKGROUND: Recent advances in clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) genome editing have led to the use of long single-stranded DNA (lssDNA) molecules for generating conditional mutations. However, there is still limited available data on the efficiency and reliability of this method. RESULTS: We generated conditional mouse alleles using lssDNA donor templates and performed extensive characterization of the resulting mutations. We observed that the use of lssDNA molecules as donors efficiently yielded founders bearing the conditional allele, with seven out of nine projects giving rise to modified alleles. However, rearranged alleles including nucleotide changes, indels, local rearrangements and additional integrations were also frequently generated by this method. Specifically, we found that alleles containing unexpected point mutations were found in three of the nine projects analyzed. Alleles originating from illegitimate repairs or partial integration of the donor were detected in eight projects. Furthermore, additional integrations of donor molecules were identified in four out of the seven projects analyzed by copy counting. This highlighted the requirement for a thorough allele validation by polymerase chain reaction, sequencing and copy counting of the mice generated through this method. We also demonstrated the feasibility of using lssDNA donors to generate thus far problematic point mutations distant from active CRISPR cutting sites by targeting two distinct genes (Gckr and Rims1). We propose a strategy to perform extensive quality control and validation of both types of mouse models generated using lssDNA donors. CONCLUSION: lssDNA donors reproducibly generate conditional alleles and can be used to introduce point mutations away from CRISPR/Cas9 cutting sites in mice. However, our work demonstrates that thorough quality control of new models is essential prior to reliably experimenting with mice generated by this method. These advances in genome editing techniques shift the challenge of mutagenesis from generation to the validation of new mutant models.


Assuntos
DNA de Cadeia Simples , Edição de Genes/métodos , Marcação de Genes , Camundongos/genética , Alelos , Animais , Sistemas CRISPR-Cas , Mutação , Reprodutibilidade dos Testes
11.
Nat Genet ; 50(4): 572-580, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29632379

RESUMO

Individual risk of type 2 diabetes (T2D) is modified by perturbations to the mass, distribution and function of adipose tissue. To investigate the mechanisms underlying these associations, we explored the molecular, cellular and whole-body effects of T2D-associated alleles near KLF14. We show that KLF14 diabetes-risk alleles act in adipose tissue to reduce KLF14 expression and modulate, in trans, the expression of 385 genes. We demonstrate, in human cellular studies, that reduced KLF14 expression increases pre-adipocyte proliferation but disrupts lipogenesis, and in mice, that adipose tissue-specific deletion of Klf14 partially recapitulates the human phenotype of insulin resistance, dyslipidemia and T2D. We show that carriers of the KLF14 T2D risk allele shift body fat from gynoid stores to abdominal stores and display a marked increase in adipocyte cell size, and that these effects on fat distribution, and the T2D association, are female specific. The metabolic risk associated with variation at this imprinted locus depends on the sex both of the subject and of the parent from whom the risk allele derives.


Assuntos
Adipócitos/patologia , Composição Corporal/genética , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/patologia , Fatores de Transcrição Sp/genética , Alelos , Animais , Distribuição da Gordura Corporal , Tamanho Celular , Elementos Facilitadores Genéticos , Feminino , Expressão Gênica , Estudo de Associação Genômica Ampla , Impressão Genômica , Humanos , Fatores de Transcrição Kruppel-Like/deficiência , Fatores de Transcrição Kruppel-Like/genética , Lipogênese/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fenótipo , Fatores de Risco , Caracteres Sexuais
12.
Nat Commun ; 8(1): 886, 2017 10 12.
Artigo em Inglês | MEDLINE | ID: mdl-29026089

RESUMO

The developmental and physiological complexity of the auditory system is likely reflected in the underlying set of genes involved in auditory function. In humans, over 150 non-syndromic loci have been identified, and there are more than 400 human genetic syndromes with a hearing loss component. Over 100 non-syndromic hearing loss genes have been identified in mouse and human, but we remain ignorant of the full extent of the genetic landscape involved in auditory dysfunction. As part of the International Mouse Phenotyping Consortium, we undertook a hearing loss screen in a cohort of 3006 mouse knockout strains. In total, we identify 67 candidate hearing loss genes. We detect known hearing loss genes, but the vast majority, 52, of the candidate genes were novel. Our analysis reveals a large and unexplored genetic landscape involved with auditory function.The full extent of the genetic basis for hearing impairment is unknown. Here, as part of the International Mouse Phenotyping Consortium, the authors perform a hearing loss screen in 3006 mouse knockout strains and identify 52 new candidate genes for genetic hearing loss.


Assuntos
Perda Auditiva/genética , Mapas de Interação de Proteínas/genética , Animais , Conjuntos de Dados como Assunto , Testes Genéticos , Perda Auditiva/epidemiologia , Testes Auditivos , Camundongos , Camundongos Knockout , Fenótipo
13.
PLoS Genet ; 13(8): e1006969, 2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-28806779

RESUMO

Otitis media (OM), inflammation of the middle ear (ME), is a common cause of conductive hearing impairment. Despite the importance of the disease, the aetiology of chronic and recurrent forms of middle ear inflammatory disease remains poorly understood. Studies of the human population suggest that there is a significant genetic component predisposing to the development of chronic OM, although the underlying genes are largely unknown. Using N-ethyl-N-nitrosourea mutagenesis we identified a recessive mouse mutant, edison, that spontaneously develops a conductive hearing loss due to chronic OM. The causal mutation was identified as a missense change, L972P, in the Nischarin (NISCH) gene. edison mice develop a serous or granulocytic effusion, increasingly macrophage and neutrophil rich with age, along with a thickened, inflamed mucoperiosteum. We also identified a second hypomorphic allele, V33A, with only modest increases in auditory thresholds and reduced incidence of OM. NISCH interacts with several proteins, including ITGA5 that is thought to have a role in modulating VEGF-induced angiogenesis and vascularization. We identified a significant genetic interaction between Nisch and Itga5; mice heterozygous for Itga5-null and homozygous for edison mutations display a significantly increased penetrance and severity of chronic OM. In order to understand the pathological mechanisms underlying the OM phenotype, we studied interacting partners to NISCH along with downstream signalling molecules in the middle ear epithelia of edison mouse. Our analysis implicates PAK1 and RAC1, and downstream signalling in LIMK1 and NF-κB pathways in the development of chronic OM.


Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/genética , Quinases Lim/metabolismo , Mutação de Sentido Incorreto , NF-kappa B/metabolismo , Otite Média/genética , Alelos , Animais , Mapeamento Cromossômico , Doença Crônica , Modelos Animais de Doenças , Orelha Média/metabolismo , Etilnitrosoureia/toxicidade , Feminino , Técnicas de Genotipagem , Heterozigoto , Homozigoto , Humanos , Receptores de Imidazolinas , Inflamação/genética , Integrina alfa6/genética , Integrina alfa6/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Quinases Lim/genética , Masculino , Camundongos , Camundongos Knockout , NF-kappa B/genética , Neuropeptídeos/genética , Neuropeptídeos/metabolismo , Otite Média/metabolismo , Penetrância , Análise de Sequência de DNA , Regulação para Cima , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo , Quinases Ativadas por p21/genética , Quinases Ativadas por p21/metabolismo , Proteínas rac1 de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/metabolismo
14.
Nat Commun ; 7: 12444, 2016 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-27534441

RESUMO

Determining the genetic bases of age-related disease remains a major challenge requiring a spectrum of approaches from human and clinical genetics to the utilization of model organism studies. Here we report a large-scale genetic screen in mice employing a phenotype-driven discovery platform to identify mutations resulting in age-related disease, both late-onset and progressive. We have utilized N-ethyl-N-nitrosourea mutagenesis to generate pedigrees of mutagenized mice that were subject to recurrent screens for mutant phenotypes as the mice aged. In total, we identify 105 distinct mutant lines from 157 pedigrees analysed, out of which 27 are late-onset phenotypes across a range of physiological systems. Using whole-genome sequencing we uncover the underlying genes for 44 of these mutant phenotypes, including 12 late-onset phenotypes. These genes reveal a number of novel pathways involved with age-related disease. We illustrate our findings by the recovery and characterization of a novel mouse model of age-related hearing loss.


Assuntos
Envelhecimento/genética , Testes Genéticos , Mutagênese/genética , Animais , Cóclea/metabolismo , Modelos Animais de Doenças , Epitélio/ultraestrutura , Potenciais Evocados Auditivos do Tronco Encefálico/fisiologia , Feminino , Audição/genética , Masculino , Camundongos Endogâmicos C57BL , Mutação/genética , Linhagem , Fenótipo
15.
Genome Med ; 8(1): 16, 2016 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-26876963

RESUMO

BACKGROUND: Nuclease-based technologies have been developed that enable targeting of specific DNA sequences directly in the zygote. These approaches provide an opportunity to modify the genomes of inbred mice, and allow the removal of strain-specific mutations that confound phenotypic assessment. One such mutation is the Cdh23 (ahl) allele, present in several commonly used inbred mouse strains, which predisposes to age-related progressive hearing loss. RESULTS: We have used targeted CRISPR/Cas9-mediated homology directed repair (HDR) to correct the Cdh23 (ahl) allele directly in C57BL/6NTac zygotes. Employing offset-nicking Cas9 (D10A) nickase with paired RNA guides and a single-stranded oligonucleotide donor template we show that allele repair was successfully achieved. To investigate potential Cas9-mediated 'off-target' mutations in our corrected mouse, we undertook whole-genome sequencing and assessed the 'off-target' sites predicted for the guide RNAs (≤4 nucleotide mis-matches). No induced sequence changes were identified at any of these sites. Correction of the progressive hearing loss phenotype was demonstrated using auditory-evoked brainstem response testing of mice at 24 and 36 weeks of age, and rescue of the progressive loss of sensory hair cell stereocilia bundles was confirmed using scanning electron microscopy of dissected cochleae from 36-week-old mice. CONCLUSIONS: CRISPR/Cas9-mediated HDR has been successfully utilised to efficiently correct the Cdh23 (ahl) allele in C57BL/6NTac mice, and rescue the associated auditory phenotype. The corrected mice described in this report will allow age-related auditory phenotyping studies to be undertaken using C57BL/6NTac-derived models, such as those generated by the International Mouse Phenotyping Consortium (IMPC) programme.


Assuntos
Caderinas/genética , Terapia Genética/métodos , Perda Auditiva/terapia , Reparo de DNA por Recombinação , Animais , Sistemas CRISPR-Cas , Potenciais Evocados Auditivos , Perda Auditiva/fisiopatologia , Camundongos , Camundongos Endogâmicos C57BL , RNA Guia/metabolismo , Estereocílios/fisiologia
16.
Mamm Genome ; 26(9-10): 486-500, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26449678

RESUMO

Mutagenesis-based screens in mice are a powerful discovery platform to identify novel genes or gene functions associated with disease phenotypes. An N-ethyl-N-nitrosourea (ENU) mutagenesis screen induces single nucleotide variants randomly in the mouse genome. Subsequent phenotyping of mutant and wildtype mice enables the identification of mutated pathways resulting in phenotypes associated with a particular ENU lesion. This unbiased approach to gene discovery conducts the phenotyping with no prior knowledge of the functional mutations. Before the advent of affordable next generation sequencing (NGS), ENU variant identification was a limiting step in gene characterization, akin to 'finding a needle in a haystack'. The emergence of a reliable reference genome alongside advances in NGS has propelled ENU mutation discovery from an arduous, time-consuming exercise to an effective and rapid form of mutation discovery. This has permitted large mouse facilities worldwide to use ENU for novel mutation discovery in a high-throughput manner, helping to accelerate basic science at the mechanistic level. Here, we describe three different strategies used to identify ENU variants from NGS data and some of the subsequent steps for mutation characterisation.


Assuntos
Genoma/efeitos dos fármacos , Sequenciamento de Nucleotídeos em Larga Escala , Mutação/efeitos dos fármacos , Animais , Modelos Animais de Doenças , Etilnitrosoureia/toxicidade , Camundongos , Mutagênese/efeitos dos fármacos , Mutagênese/genética , Mutação/genética
17.
Dis Model Mech ; 8(12): 1531-42, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26471094

RESUMO

Otitis media with effusion (OME) is the most common cause of hearing loss in children, and tympanostomy (ear tube insertion) to alleviate the condition remains the commonest surgical intervention in children in the developed world. Chronic and recurrent forms of otitis media (OM) are known to have a very substantial genetic component; however, until recently, little was known of the underlying genes involved. The Jeff mouse mutant carries a mutation in the Fbxo11 gene, a member of the F-box family, and develops deafness due to a chronic proliferative OM. We previously reported that Fbxo11 is involved in the regulation of transforming growth factor beta (TGF-ß) signalling by regulating the levels of phospho-Smad2 in the epithelial cells of palatal shelves, eyelids and airways of the lungs. It has been proposed that FBXO11 regulates the cell's response to TGF-ß through the ubiquitination of CDT2. Additional substrates for FBXO11 have been identified, including p53. Here, we have studied both the genetic and biochemical interactions between FBXO11 and p53 in order to better understand the function of FBXO11 in epithelial development and its potential role in OM. In mice, we show that p53 (also known as Tp53) homozygous mutants and double heterozygous mutants (Jf/+ p53/+) exhibit similar epithelial developmental defects to Fbxo11 homozygotes. FBXO11 and p53 interact in the embryonic lung, and mutation in Fbxo11 prevents the interaction with p53. Both p53 and double mutants show raised levels of pSMAD2, recapitulating that seen in Fbxo11 homozygotes. Overall, our results support the conclusion that FBXO11 regulates the TGF-ß pathway in the embryonic lung via cross-talk with p53.


Assuntos
Embrião de Mamíferos/metabolismo , Epistasia Genética , Proteínas F-Box/genética , Pulmão/embriologia , Otite Média/genética , Proteína Supressora de Tumor p53/genética , Animais , Proteínas Culina/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Modelos Animais de Doenças , Desenvolvimento Embrionário , Proteínas F-Box/química , Proteínas F-Box/metabolismo , Heterozigoto , Homozigoto , Pulmão/patologia , Camundongos Knockout , Modelos Moleculares , Mutação , Otite Média/embriologia , Otite Média/patologia , Fenótipo , Fosforilação , Inativadores de Plasminogênio/metabolismo , Proteína Smad2/metabolismo , Proteína Supressora de Tumor p53/química , Proteína Supressora de Tumor p53/metabolismo
18.
Cell ; 162(3): 607-21, 2015 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-26232227

RESUMO

We identified a dominant missense mutation in the SCN transcription factor Zfhx3, termed short circuit (Zfhx3(Sci)), which accelerates circadian locomotor rhythms in mice. ZFHX3 regulates transcription via direct interaction with predicted AT motifs in target genes. The mutant protein has a decreased ability to activate consensus AT motifs in vitro. Using RNA sequencing, we found minimal effects on core clock genes in Zfhx3(Sci/+) SCN, whereas the expression of neuropeptides critical for SCN intercellular signaling was significantly disturbed. Moreover, mutant ZFHX3 had a decreased ability to activate AT motifs in the promoters of these neuropeptide genes. Lentiviral transduction of SCN slices showed that the ZFHX3-mediated activation of AT motifs is circadian, with decreased amplitude and robustness of these oscillations in Zfhx3(Sci/+) SCN slices. In conclusion, by cloning Zfhx3(Sci), we have uncovered a circadian transcriptional axis that determines the period and robustness of behavioral and SCN molecular rhythms.


Assuntos
Ritmo Circadiano , Regulação da Expressão Gênica , Proteínas de Homeodomínio/metabolismo , Neuropeptídeos/genética , Núcleo Supraquiasmático/metabolismo , Sequência de Aminoácidos , Animais , Regulação para Baixo , Proteínas de Homeodomínio/química , Proteínas de Homeodomínio/genética , Técnicas In Vitro , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Mutação , Motivos de Nucleotídeos , Regiões Promotoras Genéticas , Alinhamento de Sequência , Transcrição Genética
19.
Genome Biol ; 14(7): R82, 2013 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-23902802

RESUMO

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.


Assuntos
Genoma/genética , Animais , Comportamento Animal , Resistência à Doença/imunologia , Olho/patologia , Feminino , Fêmur/diagnóstico por imagem , Hipersensibilidade/imunologia , Mutação INDEL/genética , Células Matadoras Naturais/imunologia , Listeriose/imunologia , Listeriose/microbiologia , Masculino , Aprendizagem em Labirinto , Camundongos Endogâmicos C57BL , Fenótipo , Polimorfismo de Nucleotídeo Único/genética , Baço/imunologia , Microtomografia por Raio-X
20.
PLoS Genet ; 9(1): e1003219, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23382690

RESUMO

Forward genetics screens with N-ethyl-N-nitrosourea (ENU) provide a powerful way to illuminate gene function and generate mouse models of human disease; however, the identification of causative mutations remains a limiting step. Current strategies depend on conventional mapping, so the propagation of affected mice requires non-lethal screens; accurate tracking of phenotypes through pedigrees is complex and uncertain; out-crossing can introduce unexpected modifiers; and Sanger sequencing of candidate genes is inefficient. Here we show how these problems can be efficiently overcome using whole-genome sequencing (WGS) to detect the ENU mutations and then identify regions that are identical by descent (IBD) in multiple affected mice. In this strategy, we use a modification of the Lander-Green algorithm to isolate causative recessive and dominant mutations, even at low coverage, on a pure strain background. Analysis of the IBD regions also allows us to calculate the ENU mutation rate (1.54 mutations per Mb) and to model future strategies for genetic screens in mice. The introduction of this approach will accelerate the discovery of causal variants, permit broader and more informative lethal screens to be used, reduce animal costs, and herald a new era for ENU mutagenesis.


Assuntos
Modelos Animais de Doenças , Etilnitrosoureia/toxicidade , Genoma , Mutação/genética , Análise de Sequência de DNA/métodos , Animais , Mapeamento Cromossômico , Genes Dominantes , Genes Recessivos , Humanos , Camundongos , Mutagênese , Fenótipo
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