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1.
PLoS Genet ; 20(3): e1011187, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38457464

RESUMO

BACKGROUND: Recent developments in CRISPR/Cas9 genome-editing tools have facilitated the introduction of precise alleles, including genetic intervals spanning several kilobases, directly into the embryo. However, the introduction of donor templates, via homology directed repair, can be erroneous or incomplete and these techniques often produce mosaic founder animals. Thus, newly generated alleles must be verified at the sequence level across the targeted locus. Screening for the presence of the desired mutant allele using traditional sequencing methods can be challenging due to the size of the interval to be sequenced, together with the mosaic nature of founders. METHODOLOGY/PRINCIPAL FINDINGS: In order to help disentangle the genetic complexity of these animals, we tested the application of Oxford Nanopore Technologies long-read sequencing at the targeted locus and found that the achievable depth of sequencing is sufficient to offset the sequencing error rate associated with the technology used to validate targeted regions of interest. We have assembled an analysis workflow that facilitates interrogating the entire length of a targeted segment in a single read, to confirm that the intended mutant sequence is present in both heterozygous animals and mosaic founders. We used this workflow to compare the output of PCR-based and Cas9 capture-based targeted sequencing for validation of edited alleles. CONCLUSION: Targeted long-read sequencing supports in-depth characterisation of all experimental models that aim to produce knock-in or conditional alleles, including those that contain a mix of genome-edited alleles. PCR- or Cas9 capture-based modalities bring different advantages to the analysis.


Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Animais , Sistemas CRISPR-Cas/genética , Alelos , Edição de Genes/métodos , Reparo de DNA por Recombinação , Reação em Cadeia da Polimerase
2.
PLoS Genet ; 18(1): e1009937, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-35100259

RESUMO

Mammalian hearing involves the mechanoelectrical transduction (MET) of sound-induced fluid waves in the cochlea. Essential to this process are the specialised sensory cochlear cells, the inner (IHCs) and outer hair cells (OHCs). While genetic hearing loss is highly heterogeneous, understanding the requirement of each gene will lead to a better understanding of the molecular basis of hearing and also to therapeutic opportunities for deafness. The Neuroplastin (Nptn) gene, which encodes two protein isoforms Np55 and Np65, is required for hearing, and homozygous loss-of-function mutations that affect both isoforms lead to profound deafness in mice. Here we have utilised several distinct mouse models to elaborate upon the spatial, temporal, and functional requirement of Nptn for hearing. While we demonstrate that both Np55 and Np65 are present in cochlear cells, characterisation of a Np65-specific mouse knockout shows normal hearing thresholds indicating that Np65 is functionally redundant for hearing. In contrast, we find that Nptn-knockout mice have significantly reduced maximal MET currents and MET channel open probabilities in mature OHCs, with both OHCs and IHCs also failing to develop fully mature basolateral currents. Furthermore, comparing the hearing thresholds and IHC synapse structure of Nptn-knockout mice with those of mice that lack Nptn only in IHCs and OHCs shows that the majority of the auditory deficit is explained by hair cell dysfunction, with abnormal afferent synapses contributing only a small proportion of the hearing loss. Finally, we show that continued expression of Neuroplastin in OHCs of adult mice is required for membrane localisation of Plasma Membrane Ca2+ ATPase 2 (PMCA2), which is essential for hearing function. Moreover, Nptn haploinsufficiency phenocopies Atp2b2 (encodes PMCA2) mutations, with heterozygous Nptn-knockout mice exhibiting hearing loss through genetic interaction with the Cdh23ahl allele. Together, our findings provide further insight to the functional requirement of Neuroplastin for mammalian hearing.


Assuntos
Caderinas/genética , Células Ciliadas Auditivas Internas/fisiologia , Audição/genética , Glicoproteínas de Membrana/genética , Isoformas de Proteínas/genética , Animais , Mutação com Perda de Função , Camundongos , Camundongos Knockout , ATPases Transportadoras de Cálcio da Membrana Plasmática/metabolismo
3.
Neurobiol Dis ; 190: 106363, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-37996040

RESUMO

Sporadic Creutzfeldt-Jakob disease (sCJD), the most common human prion disease, is thought to occur when the cellular prion protein (PrPC) spontaneously misfolds and assembles into prion fibrils, culminating in fatal neurodegeneration. In a genome-wide association study of sCJD, we recently identified risk variants in and around the gene STX6, with evidence to suggest a causal increase of STX6 expression in disease-relevant brain regions. STX6 encodes syntaxin-6, a SNARE protein primarily involved in early endosome to trans-Golgi network retrograde transport. Here we developed and characterised a mouse model with genetic depletion of Stx6 and investigated a causal role of Stx6 expression in mouse prion disease through a classical prion transmission study, assessing the impact of homozygous and heterozygous syntaxin-6 knockout on disease incubation periods and prion-related neuropathology. Following inoculation with RML prions, incubation periods in Stx6-/- and Stx6+/- mice differed by 12 days relative to wildtype. Similarly, in Stx6-/- mice, disease incubation periods following inoculation with ME7 prions also differed by 12 days. Histopathological analysis revealed a modest increase in astrogliosis in ME7-inoculated Stx6-/- animals and a variable effect of Stx6 expression on microglia activation, however no differences in neuronal loss, spongiform change or PrP deposition were observed at endpoint. Importantly, Stx6-/- mice are viable and fertile with no gross impairments on a range of neurological, biochemical, histological and skeletal structure tests. Our results provide some support for a pathological role of Stx6 expression in prion disease, which warrants further investigation in the context of prion disease but also other neurodegenerative diseases considering syntaxin-6 appears to have pleiotropic risk effects in progressive supranuclear palsy and Alzheimer's disease.


Assuntos
Síndrome de Creutzfeldt-Jakob , Doenças Priônicas , Príons , Camundongos , Humanos , Animais , Síndrome de Creutzfeldt-Jakob/genética , Síndrome de Creutzfeldt-Jakob/patologia , Príons/genética , Príons/metabolismo , Estudo de Associação Genômica Ampla , Camundongos Transgênicos , Encéfalo/metabolismo , Doenças Priônicas/genética , Doenças Priônicas/patologia , Proteínas Qa-SNARE/genética , Proteínas Qa-SNARE/metabolismo
4.
Methods ; 191: 59-67, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-32599056

RESUMO

The widespread availability of recombineered vectors and gene targeted embryonic stem cells from large-scale repositories facilitates the generation of mouse models for functional genetic studies. Southern blotting validates the structure of these targeted alleles produced by homologous recombination, as well as indicating any additional integrations of the vector into the genome. Traditionally this technique employs radioactively-labelled probes; however, there are many laboratories that are restricted in their use of radioactivity. Here, we present a widely applicable protocol for Southern blot analysis using cold probes and alternative procedures employing radioactive probes. Furthermore, the probes are designed to recognise standardised regions of gene-targeting cassettes and so represent universally applicable reagents for assessing allelic integrity.


Assuntos
Radioatividade , Alelos , Animais , Southern Blotting , Marcação de Genes , Vetores Genéticos , Recombinação Homóloga , Camundongos
5.
Nucleic Acids Res ; 47(14): 7402-7417, 2019 08 22.
Artigo em Inglês | MEDLINE | ID: mdl-31127293

RESUMO

The CRISPR system is widely used in genome editing for biomedical research. Here, using either dual paired Cas9D10A nickases or paired Cas9 nuclease we characterize unintended larger deletions at on-target sites that frequently evade common genotyping practices. We found that unintended larger deletions are prevalent at multiple distinct loci on different chromosomes, in cultured cells and mouse embryos alike. We observed a high frequency of microhomologies at larger deletion breakpoint junctions, suggesting the involvement of microhomology-mediated end joining in their generation. In populations of edited cells, the distribution of larger deletion sizes is dependent on proximity to sgRNAs and cannot be predicted by microhomology sequences alone.


Assuntos
Sistemas CRISPR-Cas , Deleção Cromossômica , Cromossomos de Mamíferos/genética , Edição de Genes/métodos , Deleção de Sequência , Animais , Linhagem Celular , Pontos de Quebra do Cromossomo , Cromossomos de Mamíferos/metabolismo , Reparo do DNA por Junção de Extremidades , Desoxirribonuclease I/genética , Desoxirribonuclease I/metabolismo , Endonucleases/genética , Endonucleases/metabolismo , Camundongos , Modelos Genéticos , RNA Guia de Cinetoplastídeos/genética , RNA Guia de Cinetoplastídeos/metabolismo
6.
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
7.
Methods ; 121-122: 68-76, 2017 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-28363792

RESUMO

The application of CRISPR/Cas9 technology has revolutionised genetics by greatly enhancing the efficacy of genome editing in the early embryo. Furthermore, the system has enabled the generation of allele types previously incompatible with in vivo mutagenesis. Despite its versatility and ease of implementation, CRISPR/Cas9 editing outcome is unpredictable and can generate mosaic founders. Therefore, careful genotyping and characterisation of new mutants is proving essential. The literature presents a wide range of protocols for molecular characterisation, each representing different levels of investment. We present strategies and protocols for designing, producing and screening CRISPR/Cas9 edited founders and genotyping their offspring according to desired allele type (indel, point mutation and deletion).


Assuntos
Proteínas de Bactérias/genética , Sistemas CRISPR-Cas , Endonucleases/genética , Edição de Genes/métodos , Técnicas de Inativação de Genes , Técnicas de Transferência de Genes , RNA Guia de Cinetoplastídeos/genética , Alelos , Animais , Animais Recém-Nascidos , Proteínas de Bactérias/metabolismo , Proteína 9 Associada à CRISPR , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades , Embrião de Mamíferos , Endonucleases/metabolismo , Marcação de Genes/métodos , Genoma , Técnicas de Genotipagem , Mutação INDEL , Camundongos , Camundongos Transgênicos , Microinjeções , Mutação Puntual , Controle de Qualidade , RNA Guia de Cinetoplastídeos/metabolismo , Reparo de DNA por Recombinação , Zigoto/citologia , Zigoto/metabolismo
8.
BMC Cell Biol ; 17(1): 30, 2016 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-27496052

RESUMO

BACKGROUND: Karyotypic integrity is essential for the successful germline transmission of alleles mutated in embryonic stem (ES) cells. Classical methods for the identification of aneuploidy involve cytological analyses that are both time consuming and require rare expertise to identify mouse chromosomes. RESULTS: As part of the International Mouse Phenotyping Consortium, we gathered data from over 1,500 ES cell clones and found that the germline transmission (GLT) efficiency of clones is compromised when over 50 % of cells harbour chromosome number abnormalities. In JM8 cells, chromosomes 1, 8, 11 or Y displayed copy number variation most frequently, whilst the remainder generally remain unchanged. We developed protocols employing droplet digital polymerase chain reaction (ddPCR) to accurately quantify the copy number of these four chromosomes, allowing efficient triage of ES clones prior to microinjection. We verified that assessments of aneuploidy, and thus decisions regarding the suitability of clones for microinjection, were concordant between classical cytological and ddPCR-based methods. Finally, we improved the method to include assay multiplexing so that two unstable chromosomes are counted simultaneously (and independently) in one reaction, to enhance throughput and further reduce the cost. CONCLUSION: We validated a PCR-based method as an alternative to classical karyotype analysis. This technique enables laboratories that are non-specialist, or work with large numbers of clones, to precisely screen ES cells for the most common aneuploidies prior to microinjection to ensure the highest level of germline transmission potential. The application of this method allows early exclusion of aneuploid ES cell clones in the ES cell to mouse conversion process, thus improving the chances of obtaining germline transmission and reducing the number of animals used in failed microinjection attempts. This method can be applied to any other experiments that require accurate analysis of the genome for copy number variation (CNV).


Assuntos
Aneuploidia , Cariotipagem/métodos , Metáfase , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/metabolismo , Reação em Cadeia da Polimerase/métodos , Animais , Células Cultivadas , Cromossomos de Mamíferos/metabolismo , Variações do Número de Cópias de DNA , Células Germinativas , Camundongos , Camundongos Endogâmicos C57BL
9.
Proc Natl Acad Sci U S A ; 110(23): 9547-52, 2013 Jun 04.
Artigo em Inglês | MEDLINE | ID: mdl-23690615

RESUMO

The suprachiasmatic nucleus (SCN) coordinates circadian rhythms that adapt the individual to solar time. SCN pacemaking revolves around feedback loops in which expression of Period (Per) and Cryptochrome (Cry) genes is periodically suppressed by their protein products. Specifically, PER/CRY complexes act at E-box sequences in Per and Cry to inhibit their transactivation by CLOCK/BMAL1 heterodimers. To function effectively, these closed intracellular loops need to be synchronized between SCN cells and to the light/dark cycle. For Per expression, this is mediated by neuropeptidergic and glutamatergic extracellular cues acting via cAMP/calcium-responsive elements (CREs) in Per genes. Cry genes, however, carry no CREs, and how CRY-dependent SCN pacemaking is synchronized remains unclear. Furthermore, whereas reporter lines are available to explore Per circadian expression in real time, no Cry equivalent exists. We therefore created a mouse, B6.Cg-Tg(Cry1-luc)01Ld, carrying a transgene (mCry1-luc) consisting of mCry1 elements containing an E-box and E'-box driving firefly luciferase. mCry1-luc organotypic SCN slices exhibited stable circadian bioluminescence rhythms with appropriate phase, period, profile, and spatial organization. In SCN lacking vasoactive intestinal peptide or its receptor, mCry1 expression was damped and desynchronized between cells. Despite the absence of CREs, mCry1-luc expression was nevertheless (indirectly) sensitive to manipulation of cAMP-dependent signaling. In mPer1/2-null SCN, mCry1-luc bioluminescence was arrhythmic and no longer suppressed by elevation of cAMP. Finally, an SCN graft procedure showed that PER-independent as well as PER-dependent mechanisms could sustain circadian expression of mCry1. The mCry1-luc mouse therefore reports circadian mCry1 expression and its interactions with vasoactive intestinal peptide, cAMP, and PER at the heart of the SCN pacemaker.


Assuntos
Ritmo Circadiano/fisiologia , Criptocromos/metabolismo , Retroalimentação Fisiológica/fisiologia , Proteínas Circadianas Period/metabolismo , Núcleo Supraquiasmático/fisiologia , Animais , AMP Cíclico/metabolismo , Primers do DNA/genética , Luciferases , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos
10.
J Mol Endocrinol ; 73(1)2024 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-38381448

RESUMO

Humans with the mutation Y509C in transducin beta like 1 X-linked (TBL1X HGNC ID HGNC:11585) have been reported to present with the combination of central congenital hypothyroidism and impaired hearing. TBL1X belongs to the WD40 repeat-containing protein family, is part of NCoR and SMRT corepressor complexes, and thereby involved in thyroid hormone signaling. In order to investigate the effects of the Y509C mutation in TBL1X on cellular thyroid hormone action, we aimed to generate a hemizygous male mouse cohort carrying the Tbl1x Y459C mutation which is equivalent to the human TBL1X Y509C mutation using CRISPR/Cas9 technology. Hemizygous male mice were small at birth and inactive. Their life span (median life span 93 days) was very short compared with heterozygous female mice (survived to >200 days with no welfare issues). About 52% of mice did not survive to weaning (133 mice). Of the remaining 118 mice, only 8 were hemizygous males who were unable to mate whereby it was impossible to generate homozygous female mice. In conclusion, the Tbl1x Y459C mutation in male mice has a marked negative effect on birth weight, survival, and fertility of male mice. The present findings are unexpected as they are in contrast to the mild phenotype in human males carrying the equivalent TBL1X Y509C mutation.


Assuntos
Longevidade , Mutação , Transducina , Animais , Feminino , Humanos , Masculino , Camundongos , Hemizigoto , Longevidade/genética , Mutação/genética , Fenótipo , Transducina/genética , Transducina/metabolismo
11.
Methods Mol Biol ; 2631: 103-134, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36995665

RESUMO

Targeted nucleases allow the production of many types of genetic mutations directly in the early embryo. However, the outcome of their activity is a repair event of unpredictable nature, and the founder animals that are produced are generally of a mosaic nature. Here, we present the molecular assays and genotyping strategies that will support the screening of the first generation for potential founders and the validation of positive animals in the subsequent generation, according to the type of mutation generated.


Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Animais , Genótipo , Mutação , Genoma
12.
Immunogenetics ; 64(6): 435-45, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22454046

RESUMO

Six major histocompatibility complex (MHC) classical class I genes have been identified in cattle, and up to three of these are expressed in variable combinations on different haplotypes. The origin and functional significance of this genetic complexity is unknown. However, an improved assembly of the cattle genome, an expanded database of full-length cDNA sequences and high-resolution frequency data concerning expressed class I genes in an economically important cattle breed combine to provide a new opportunity to study the significance of cattle MHC class I diversity. Analysis of these new data supports assignment of alleles to six discrete genes and further shows that all these classical genes share a common ancestor with a single non-classical gene, NC1. While haplotype structure is variable, with thirteen gene configurations identified, there are nevertheless clear constraints relating to both the number and combination of genes. Haplotypes expressing two classical genes are most frequently observed, and the classical class I gene 2 is almost invariably present. The frequency data support the dominance of gene 2, showing that close to 100 % of individuals carry at least one copy. This indicates a hierarchy in the functional importance of particular genes and haplotype structures. Haplotype frequency in cattle populations is therefore likely to impact on differential disease susceptibilities. This knowledge will be important for development of informed breeding strategies aimed at increasing the ability of cattle to survive in the face of future unpredictable pathogen exposure.


Assuntos
Bovinos/imunologia , Haplótipos , Antígenos de Histocompatibilidade Classe I/genética , Animais , Frequência do Gene , Variação Genética , Polimorfismo Genético
13.
Lab Anim ; 56(1): 69-82, 2022 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-34192966

RESUMO

The emergence of an array of genome-editing tools in recent years has facilitated the introduction of genetic modifications directly into the embryo, increasing the ease, efficiency and catalogue of alleles accessible to researchers across a range of species. Bypassing the requirement for a selection cassette and resulting in a broad range of outcomes besides the desired allele, genome editing has altered the allele validation process both temporally and technically. Whereas traditional gene targeting relies upon selection and allows allele validation at the embryonic stem cell modification stage, screening for the presence of the intended allele now occurs in the (frequently mosaic) founder animals. Final confirmation of the edited allele can only take place at the subsequent G1 generation and the validation strategy must differentiate the desired allele from a range of unintended outcomes. Here we present some of the challenges posed by gene editing, strategies for validation and considerations for animal colony management.


Assuntos
Edição de Genes , Testes Genéticos , Alelos , Animais , Embrião de Mamíferos , Edição de Genes/métodos , Edição de Genes/normas , Reprodutibilidade dos Testes
15.
iScience ; 24(12): 103463, 2021 Dec 17.
Artigo em Inglês | MEDLINE | ID: mdl-34988393

RESUMO

Amyotrophic lateral sclerosis/frontotemporal dementia (ALS/FTD) is a fatal neurodegenerative disorder, and continued innovation is needed for improved understanding and for developing therapeutics. We have created next-generation genomically humanized knockin mouse models, by replacing the mouse genomic region of Sod1, Tardbp (TDP-43), and Fus, with their human orthologs, preserving human protein biochemistry and splicing with exons and introns intact. We establish a new standard of large knockin allele quality control, demonstrating the utility of indirect capture for enrichment of a genomic region of interest followed by Oxford Nanopore sequencing. Extensive analysis shows that homozygous humanized animals only express human protein at endogenous levels. Characterization of humanized FUS animals showed that they are phenotypically normal throughout their lifespan. These humanized strains are vital for preclinical assessment of interventions and serve as templates for the addition of coding or non-coding human ALS/FTD mutations to dissect disease pathomechanisms, in a physiological context.

16.
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
17.
Nat Genet ; 49(8): 1231-1238, 2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-28650483

RESUMO

Although next-generation sequencing has revolutionized the ability to associate variants with human diseases, diagnostic rates and development of new therapies are still limited by a lack of knowledge of the functions and pathobiological mechanisms of most genes. To address this challenge, the International Mouse Phenotyping Consortium is creating a genome- and phenome-wide catalog of gene function by characterizing new knockout-mouse strains across diverse biological systems through a broad set of standardized phenotyping tests. All mice will be readily available to the biomedical community. Analyzing the first 3,328 genes identified models for 360 diseases, including the first models, to our knowledge, for type C Bernard-Soulier, Bardet-Biedl-5 and Gordon Holmes syndromes. 90% of our phenotype annotations were novel, providing functional evidence for 1,092 genes and candidates in genetically uncharacterized diseases including arrhythmogenic right ventricular dysplasia 3. Finally, we describe our role in variant functional validation with The 100,000 Genomes Project and others.


Assuntos
Modelos Animais de Doenças , Técnicas de Inativação de Genes , Animais , Feminino , Doenças Genéticas Inatas , Predisposição Genética para Doença , Humanos , Masculino , Camundongos , Camundongos Knockout , Fenótipo
19.
Nat Genet ; 47(9): 969-978, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26214591

RESUMO

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.


Assuntos
Estudos de Associação Genética , Animais , Feminino , Heterozigoto , Homozigoto , Humanos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Anotação de Sequência Molecular , Mutação , Fenótipo
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