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Deleção de Genes , Estudos de Associação Genética , Genoma , Genótipo , Animais , Disseminação de Informação , Cooperação Internacional , Internet , Camundongos , Camundongos Knockout , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/metabolismo , Mutagênese , FenótipoRESUMO
Ploidy represents the number of chromosome sets in a cell. Although gametes have a haploid genome (n), most mammalian cells have diploid genomes (2n). The diploid status of most cells correlates with the number of probable alleles for each autosomal gene and makes it difficult to target these genes via mutagenesis techniques. Here, we describe a 7-week protocol for the derivation of mouse haploid embryonic stem cells (hESCs) from female gametes that also outlines how to maintain the cells once derived. We detail additional procedures that can be used with cell lines obtained from the mouse Haplobank, a biobank of >100,000 individual mouse hESC lines with targeted mutations in 16,970 genes. hESCs can spontaneously diploidize and can be maintained in both haploid and diploid states. Mouse hESCs are genomically and karyotypically stable, are innately immortal and isogenic, and can be derived in an array of differentiated cell types; they are thus highly amenable to genetic screens and to defining molecular connectivity pathways.
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Técnicas de Cultura de Células/métodos , Haploidia , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/fisiologia , Animais , Blastocisto/citologia , Linhagem Celular , Separação Celular/métodos , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos , Fluxo de TrabalhoRESUMO
FBXO7 encodes an F box containing protein that interacts with multiple partners to facilitate numerous cellular processes and has a canonical role as part of an SCF E3 ubiquitin ligase complex. Mutation of FBXO7 is responsible for an early onset Parkinsonian pyramidal syndrome and genome-wide association studies have linked variants in FBXO7 to erythroid traits. A putative orthologue in Drosophila, nutcracker, has been shown to regulate the proteasome, and deficiency of nutcracker results in male infertility. Therefore, we reasoned that modulating Fbxo7 levels in a murine model could provide insights into the role of this protein in mammals. We used a targeted gene trap model which retained 4-16% residual gene expression and assessed the sensitivity of phenotypic traits to gene dosage. Fbxo7 hypomorphs showed regenerative anaemia associated with a shorter erythrocyte half-life, and male mice were infertile. Alterations to T cell phenotypes were also observed, which intriguingly were both T cell intrinsic and extrinsic. Hypomorphic mice were also sensitive to infection with Salmonella, succumbing to a normally sublethal challenge. Despite these phenotypes, Fbxo7 hypomorphs were produced at a normal Mendelian ratio with a normal lifespan and no evidence of neurological symptoms. These data suggest that erythrocyte survival, T cell development and spermatogenesis are particularly sensitive to Fbxo7 gene dosage.
Assuntos
Alelos , Proteínas F-Box , Dosagem de Genes , Regulação da Expressão Gênica , Infertilidade Masculina , Característica Quantitativa Herdável , Animais , Proteínas F-Box/biossíntese , Proteínas F-Box/genética , Infertilidade Masculina/genética , Infertilidade Masculina/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Salmonella , Infecções por Salmonella/genética , Espermatogênese/genéticaRESUMO
CRISPR-Cas9 technologies have transformed genome-editing of experimental organisms and have immense therapeutic potential. Despite significant advances in our understanding of the CRISPR-Cas9 system, concerns remain over the potential for off-target effects. Recent studies have addressed these concerns using whole-genome sequencing (WGS) of gene-edited embryos or animals to search for de novo mutations (DNMs), which may represent candidate changes introduced by poor editing fidelity. Critically, these studies used strain-matched, but not pedigree-matched controls and thus were unable to reliably distinguish generational or colony-related differences from true DNMs. Here we used a trio design and whole genome sequenced 8 parents and 19 embryos, where 10 of the embryos were mutagenised with well-characterised gRNAs targeting the coat colour Tyrosinase (Tyr) locus. Detailed analyses of these whole genome data allowed us to conclude that if CRISPR mutagenesis were causing SNV or indel off-target mutations in treated embryos, then the number of these mutations is not statistically distinguishable from the background rate of DNMs occurring due to other processes.
Assuntos
Sistemas CRISPR-Cas/genética , Edição de Genes/métodos , Monofenol Mono-Oxigenase/genética , Mutagênese/genética , Sequenciamento Completo do Genoma/métodos , Animais , Variação Biológica da População/genética , Análise Mutacional de DNA/métodos , Feminino , Genoma/genética , Cor de Cabelo/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Linhagem , RNA Guia de Cinetoplastídeos/genética , Projetos de PesquisaRESUMO
A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.
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Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) assisted generation of mutant animals has become the method of choice for the elucidation of gene function in development and disease due to the shortened timelines for generation of a desired mutant, the ease of producing materials in comparison to other methodologies (such as embryonic stem cells, ESCs) and the ability to simultaneously target multiple genes in one injection session. Here we describe a step by step protocol, from preparation of materials through to injection and validation of a cytoplasmic injection, which can be used to generate CRISPR mutants. This can be accomplished from start of injection to completion within 2-4 h with high survival and developmental rates of injected zygotes and offers significant advantages over pronuclear and other previously described methodologies for microinjection.
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This corrects the article DOI: 10.1038/nature19356.
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Modelling human diseases caused by large genomic rearrangements has become more accessible since the utilization of CRISPR/Cas9 in mammalian systems. In a previous study, we showed that genomic rearrangements of up to one million base pairs can be generated by direct injection of CRISPR/Cas9 reagents into mouse zygotes. Although these rearrangements are ascertained by junction PCR, we describe here a variety of anticipated structural changes often involving reintegration of the region demarcated by the gRNAs in the vicinity of the edited locus. We illustrate here some of this diversity detected by high-resolution fibre-FISH and conclude that extensive molecular analysis is required to fully understand the structure of engineered chromosomes generated by Cas9.
Assuntos
Proteína 9 Associada à CRISPR/metabolismo , Rearranjo Gênico/genética , Genoma , Animais , Feminino , Duplicação Gênica , Hibridização in Situ Fluorescente , Camundongos Endogâmicos C57BL , NADPH Oxidase 4/metabolismo , Deleção de Sequência , Inversão de SequênciaRESUMO
PAXX was identified recently as a novel nonhomologous end-joining DNA repair factor in human cells. To characterize its physiological roles, we generated Paxx-deficient mice. Like Xlf-/- mice, Paxx-/- mice are viable, grow normally, and are fertile but show mild radiosensitivity. Strikingly, while Paxx loss is epistatic with Ku80, Lig4, and Atm deficiency, Paxx/Xlf double-knockout mice display embryonic lethality associated with genomic instability, cell death in the central nervous system, and an almost complete block in lymphogenesis, phenotypes that closely resemble those of Xrcc4-/- and Lig4-/- mice. Thus, combined loss of Paxx and Xlf is synthetic-lethal in mammals.
Assuntos
Proteínas de Ligação a DNA/genética , Desenvolvimento Embrionário/genética , Mutações Sintéticas Letais/genética , Trissacarídeos/genética , Animais , Apoptose/genética , Proteínas de Ligação a DNA/metabolismo , Epistasia Genética , Instabilidade Genômica/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Tolerância a Radiação/genética , Trissacarídeos/metabolismoRESUMO
Approximately one-third of all mammalian genes are essential for life. Phenotypes resulting from knockouts of these genes in mice have provided tremendous insight into gene function and congenital disorders. As part of the International Mouse Phenotyping Consortium effort to generate and phenotypically characterize 5,000 knockout mouse lines, here we identify 410 lethal genes during the production of the first 1,751 unique gene knockouts. Using a standardized phenotyping platform that incorporates high-resolution 3D imaging, we identify phenotypes at multiple time points for previously uncharacterized genes and additional phenotypes for genes with previously reported mutant phenotypes. Unexpectedly, our analysis reveals that incomplete penetrance and variable expressivity are common even on a defined genetic background. In addition, we show that human disease genes are enriched for essential genes, thus providing a dataset that facilitates the prioritization and validation of mutations identified in clinical sequencing efforts.
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Embrião de Mamíferos/embriologia , Embrião de Mamíferos/metabolismo , Genes Essenciais/genética , Genes Letais/genética , Mutação/genética , Fenótipo , Animais , Sequência Conservada/genética , Doença , Estudo de Associação Genômica Ampla , Ensaios de Triagem em Larga Escala , Humanos , Imageamento Tridimensional , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Penetrância , Polimorfismo de Nucleotídeo Único/genética , Homologia de SequênciaRESUMO
Single-nucleotide variations in C13orf31 (LACC1) that encode p.C284R and p.I254V in a protein of unknown function (called 'FAMIN' here) are associated with increased risk for systemic juvenile idiopathic arthritis, leprosy and Crohn's disease. Here we set out to identify the biological mechanism affected by these coding variations. FAMIN formed a complex with fatty acid synthase (FASN) on peroxisomes and promoted flux through de novo lipogenesis to concomitantly drive high levels of fatty-acid oxidation (FAO) and glycolysis and, consequently, ATP regeneration. FAMIN-dependent FAO controlled inflammasome activation, mitochondrial and NADPH-oxidase-dependent production of reactive oxygen species (ROS), and the bactericidal activity of macrophages. As p.I254V and p.C284R resulted in diminished function and loss of function, respectively, FAMIN determined resilience to endotoxin shock. Thus, we have identified a central regulator of the metabolic function and bioenergetic state of macrophages that is under evolutionary selection and determines the risk of inflammatory and infectious disease.
Assuntos
Artrite Juvenil/genética , Doença de Crohn/genética , Infecções/genética , Hanseníase/genética , Macrófagos/imunologia , Proteínas/genética , Choque Séptico/genética , Trifosfato de Adenosina/metabolismo , Animais , Bacteriólise , Células Cultivadas , Metabolismo Energético , Ácido Graxo Sintase Tipo I/metabolismo , Predisposição Genética para Doença , Humanos , Inflamassomos/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Metabolismo dos Lipídeos/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , NADPH Oxidases/metabolismo , Oxirredução , Polimorfismo de Nucleotídeo Único , RiscoRESUMO
Deletions, duplications, and inversions of large genomic regions covering several genes are an important class of disease causing variants in humans. Modeling these structural variants in mice requires multistep processes in ES cells, which has limited their availability. Mutant mice containing small insertions, deletions, and single nucleotide polymorphisms can be reliably generated using CRISPR/Cas9 directly in mouse zygotes. Large structural variants can be generated using CRISPR/Cas9 in ES cells, but it has not been possible to generate these directly in zygotes. We now demonstrate the direct generation of deletions, duplications and inversions of up to one million base pairs by zygote injection.
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Sistemas CRISPR-Cas , Cromossomos , Engenharia Genética/métodos , Animais , Sequência de Bases , Duplicação Cromossômica , Inversão Cromossômica , DNA , Estudos de Viabilidade , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência MolecularRESUMO
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.
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Estudos de Associação Genética , Animais , Feminino , Heterozigoto , Homozigoto , Humanos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Anotação de Sequência Molecular , Mutação , FenótipoRESUMO
With the advent of modern developmental biology and molecular genetics, the scientific community has generated thousands of newly genetically altered strains of laboratory mice with the aim of elucidating gene function. To this end, a large group of Institutions which form the International Mouse Phenotyping Consortium is generating and phenotyping a knockout mouse strain for each of the ~20,000 protein-coding genes using the mutant ES cell resource produced by the International Knockout Mouse Consortium. These strains are made available to the research community via public repositories, mostly as cryopreserved sperm or embryos. To ensure the quality of this frozen resource there is a requirement that for each strain the frozen sperm/embryos are proven able to produce viable mutant progeny, before the live animal resource is removed from cages. Given the current requirement to generate live pups to demonstrate their mutant genotype, this quality control check necessitates the use and generation of many animals and requires considerable time, cage space, technical and economic resources. Here, we describe a simple and efficient method of genotyping pre-implantation stage blastocysts with significant ethical and economic advantages especially beneficial for current and future large-scale mouse mutagenesis projects.
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Blastocisto/metabolismo , Genótipo , Controle de Qualidade , Animais , CamundongosRESUMO
Fertilization occurs when sperm and egg recognize each other and fuse to form a new, genetically distinct organism. The molecular basis of sperm-egg recognition is unknown, but is likely to require interactions between receptor proteins displayed on their surface. Izumo1 is an essential sperm cell-surface protein, but its receptor on the egg has not been described. Here we identify folate receptor 4 (Folr4) as the receptor for Izumo1 on the mouse egg, and propose to rename it Juno. We show that the Izumo1-Juno interaction is conserved within several mammalian species, including humans. Female mice lacking Juno are infertile and Juno-deficient eggs do not fuse with normal sperm. Rapid shedding of Juno from the oolemma after fertilization suggests a mechanism for the membrane block to polyspermy, ensuring eggs normally fuse with just a single sperm. Our discovery of an essential receptor pair at the nexus of conception provides opportunities for the rational development of new fertility treatments and contraceptives.
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Fertilização/fisiologia , Imunoglobulinas/metabolismo , Proteínas de Membrana/metabolismo , Óvulo/metabolismo , Receptores de Superfície Celular/metabolismo , Espermatozoides/metabolismo , Animais , Sequência Conservada , Evolução Molecular , Feminino , Fertilidade/genética , Fertilização/genética , Genes Essenciais , Glicosilfosfatidilinositóis/metabolismo , Humanos , Infertilidade Feminina/genética , Masculino , Mamíferos , Camundongos , Oócitos/citologia , Oócitos/metabolismo , Óvulo/citologia , Partenogênese , Receptores de Superfície Celular/deficiência , Receptores de Superfície Celular/genética , Injeções de Esperma Intracitoplásmicas , Fatores de TempoRESUMO
We describe here use of a cell-permeable Cre to efficiently convert the EUCOMM/KOMP-CSD tm1a allele to the tm1b form in preimplantation mouse embryos in a high-throughput manner, consistent with the requirements of the International Mouse Phenotyping Consortium-affiliated NIH KOMP2 project. This method results in rapid allele conversion and minimizes the use of experimental animals when compared to conventional Cre transgenic mouse breeding, resulting in a significant reduction in costs and time with increased welfare benefits.
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Desenvolvimento Embrionário/genética , Integrases/genética , Alelos , Animais , Cruzamento , Embrião de Mamíferos , Células-Tronco Embrionárias , Camundongos , Camundongos TransgênicosRESUMO
In 2007, the International Knockout Mouse Consortium (IKMC) made the ambitious promise to generate mutations in virtually every protein-coding gene of the mouse genome in a concerted worldwide action. Now, 5 years later, the IKMC members have developed high-throughput gene trapping and, in particular, gene-targeting pipelines and generated more than 17,400 mutant murine embryonic stem (ES) cell clones and more than 1,700 mutant mouse strains, most of them conditional. A common IKMC web portal (www.knockoutmouse.org) has been established, allowing easy access to this unparalleled biological resource. The IKMC materials considerably enhance functional gene annotation of the mammalian genome and will have a major impact on future biomedical research.
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Camundongos Knockout/genética , Animais , Internacionalidade , Internet , CamundongosRESUMO
Creating transgenic mammals is currently a very inefficient process. In addition to problems with transgene integration and unpredictable expression patterns of the inserted gene, embryo loss occurs at various developmental stages. In the present study, we demonstrate that this loss is due to chromosomal damage. We examined the integrity of chromosomes in embryos produced by microinjection of pronuclei, intracytoplasmic sperm injection (ICSI), and in vitro fertilization (IVF)-mediated transgenesis, and correlated these findings with the abilities of embryos to develop in vitro and yield transgenic morulas/blastocysts. Chromosomal analysis was performed after microinjection of the pronuclei in zygotes, as well as in parthenogenetic and androgenetic embryos. In all the pronuclei injection groups, significant oocyte arrest and increased incidence of chromosome breaks were observed after both transgenic DNA injection and sham injection. This indicates that the DNA damage is a transgene-independent effect. In ICSI-mediated transgenesis, there was no significant oocyte arrest. The observed chromosomal damage was lower than that after pronuclei microinjection in zygotes and was dependent upon the presence of exogenous DNA. The occurrence of DNA breaks, as measured by comet assay performed on the sperm prior to ICSI, showed that DNA damage was present in the sperm before fertilization. Embryonic development in vitro and transgene expression at the morula/blastocyst stage were higher in ICSI-mediated transgenesis than after microinjection of pronuclei into zygotes. Sperm-mediated gene transfer via IVF did not affect chromosome integrity, allowed good embryo development, but did not yield any transgenic embryos. The present study demonstrates that DNA damage occurs after both the microinjection of pronuclei and ICSI-mediated transgenesis, albeit through different mechanisms.
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Quebra Cromossômica , Cromossomos/ultraestrutura , Técnicas de Transferência de Genes , Camundongos Transgênicos/genética , Animais , Núcleo Celular/ultraestrutura , Montagem e Desmontagem da Cromatina , Ensaio Cometa , Dano ao DNA , Desenvolvimento Embrionário/genética , Feminino , Genoma/genética , Masculino , Camundongos , Oócitos/citologia , Oócitos/metabolismo , Injeções de Esperma Intracitoplásmicas , Espermatozoides/metabolismo , Espermatozoides/transplante , Espermatozoides/ultraestrutura , Zigoto/ultraestruturaRESUMO
Pax6 is a key transcriptional regulator in eye, olfactory system, forebrain, pituitary cerebellum, spinal cord and pancreas development. Alternative splicing, promoter usage and multiple enhancers regulate the complex Pax6 spatio-temporal expression pattern. Chromosomal rearrangements which abolish PAX6 gene expression have been characterised downstream of the coding region. Through evolutionary sequence comparison and transgenic reporter studies, we have identified a new Pax6 3' cis-regulatory region. This region, C1170 Box 123, contains three distinct modules of human-mouse sequence conservation, while only Box 1 is conserved to Fugu. Both the human and the orthologous Fugu sequence direct similar reporter gene expression in the developing pretectum, neural retina and olfactory region, indicating evolutionary conservation of Pax6 regulatory mechanisms despite the low level of overall sequence conservation.