RESUMEN
Droplet digital PCR (ddPCR) is a recent method developed for the quantification of nucleic acids sequences. It is an evolution of PCR methodology incorporating two principal differences: a PCR reaction is performed in thousands of water-oil emulsion droplets and fluorescence is measured at the end of PCR amplification. It leads to the precise and reproducible quantification of DNA and RNA sequences. Here, we present quantitative methods for DNA and RNA analysis using Bio-Rad QX100 or QX200 systems, respectively. The aim of these methods is to provide useful molecular tools for validating genetically altered animal models such as those subject to CRISPR/Cas9 genome editing, as well for expression or CNV studies. A standard procedure for simultaneous DNA and RNA extraction adapted for mouse organs is also described. These methods were initially designed for mouse studies but also work for samples from other species like rat or human. In our lab, thousands of samples and hundreds of target genes from genetically altered lines were examined using these methods. This large dataset was analyzed to evaluate technical optimizations and limitations. Finally, we propose additional recommendations to be included in dMIQE (Minimum information for publication of quantitative digital PCR experiments) guidelines when using ddPCR instruments.
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Reacción en Cadena en Tiempo Real de la Polimerasa , Animales , ADN/genética , Ratones , ARN/genética , RatasRESUMEN
The genetic landscape of diseases associated with changes in bone mineral density (BMD), such as osteoporosis, is only partially understood. Here, we explored data from 3,823 mutant mouse strains for BMD, a measure that is frequently altered in a range of bone pathologies, including osteoporosis. A total of 200 genes were found to significantly affect BMD. This pool of BMD genes comprised 141 genes with previously unknown functions in bone biology and was complementary to pools derived from recent human studies. Nineteen of the 141 genes also caused skeletal abnormalities. Examination of the BMD genes in osteoclasts and osteoblasts underscored BMD pathways, including vesicle transport, in these cells and together with in silico bone turnover studies resulted in the prioritization of candidate genes for further investigation. Overall, the results add novel pathophysiological and molecular insight into bone health and disease.
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Densidad Ósea/genética , Regulación de la Expresión Génica/genética , Osteoblastos/metabolismo , Osteoclastos/metabolismo , Osteoporosis/genética , Animales , Femenino , Ontología de Genes , Pleiotropía Genética , Estudio de Asociación del Genoma Completo , Genotipo , Masculino , Ratones , Ratones Transgénicos , Mutación , Osteoblastos/patología , Osteoclastos/patología , Osteoporosis/metabolismo , Fenotipo , Regiones Promotoras Genéticas , Mapas de Interacción de Proteínas , Caracteres Sexuales , TranscriptomaRESUMEN
Genotyping consists of searching for a DNA sequence variation localized at a well-defined locus in the genome. It is an essential step in animal research because it allows the identification of animals that will be bred to generate and maintain a colony, euthanized to control the available space in the animal facility, or used in experiment protocols. Here we describe polymerase chain reaction (PCR) genotyping protocols for fast, sensitive, easy, and cost-effective characterization of mouse genotype. We discuss optimization of parameters to improve the reliability of each assay and propose recommendations for enhancing reproducibility and reducing the occurrence of inconclusive genotyping. All steps required for efficient genotyping are presented: tissue collection; sample verification and direct DNA lysis; establishment of a robust genotyping strategy with reliable, rapid, and cost-effective assays; and finally, transition to high-throughput automatized PCR, including mix miniaturization and automation. © 2019 The Authors. Basic Protocol 1: Tissue sampling methods and procedure Basic Protocol 2: Sample verification and DNA lysis Basic Protocol 3: Design of a genotyping strategy Basic Protocol 4: Moving to high-throughput genotyping.
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Técnicas de Genotipaje/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Mutación , Reacción en Cadena de la Polimerasa/métodos , Animales , Técnicas de Genotipaje/instrumentación , Secuenciación de Nucleótidos de Alto Rendimiento/instrumentación , Ratones , Reproducibilidad de los ResultadosRESUMEN
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 , Genotipo , Control de Calidad , Animales , RatonesRESUMEN
To facilitate the use of the new mutant resource developed in the mouse, we have generated Cre and FlpO deleter mice on a pure inbred C57BL/6N background. The new transgenic constructs were designed to drive either the Cre or FlpO recombinase, fused to a specific fluorescent marker, respectively the eGFP or the eYFP, and were inserted by homologous recombination in the neutral Rosa26 locus. They allow a rapid, cost-effective, and efficient identification of the carrier individuals through the coexpression of the fluorescent marker. The recombination efficiency of the two deleter lines, Gt(ROSA)26S or < tm1(ACTB-cre,-EGFP)Ics> and Gt(ROSA) 26S or < tm2(CAG-flpo, EYFP)Ics>, was carefully evaluated using five loxP-flanked or four FRT-flanked alleles located at different positions in the mouse genome. For each tested locus, we observed a 100% excision rate. The transgenic mice are easily distinguishable from wild type animals by their bright fluorescence that remains easily detectable until 10 days after birth. In the adult, fluorescence can still be detected in the unpigmented paws. Furthermore, they both display accumulation of the specific recombinase during oogenesis. These fluorescent 'Cre- and Flp- deleter' transgenic lines are valuable tools for the scientific community by their high and stable recombination efficiency, the simplicity of genotype identification and the maintenance of a pure genetic background when used to remove specific selection cassette or to induce complete loss-of-function allele.
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ADN Nucleotidiltransferasas/genética , Integrasas/genética , Ratones Transgénicos , Proteínas/genética , Animales , Proteínas Bacterianas/genética , Efecto Fundador , Marcación de Gen , Ingeniería Genética , Genotipo , Proteínas Fluorescentes Verdes/genética , Recombinación Homóloga , Proteínas Luminiscentes/genética , Ratones , Ratones Endogámicos C57BL , ARN no Traducido , Eliminación de SecuenciaRESUMEN
The Jak2(V617F) mutation is found in most classical BCR/ABL-negative myeloproliferative neoplasms (MPNs). Usually, heterozygosity of the mutation is associated with essential thrombocythemia (ET) and homozygosity with polycythemia vera (PV). Retrovirally transduced or transgenic animal models have shown that the mutation is sufficient for MPN development but that the level of expression is crucial for MPN phenotypes. Therefore we investigated the effect of an endogenous heterozygous expression of Jak2(V617F) in knock-in (KI) mice. These animals displayed constitutive JAK2 activation and autonomous erythroid progenitor cell growth. Mice suffered from marked polycythemia, granulocytosis and thrombocytosis. Spleens and marrows displayed myeloid trilineage hyperplasia. Most animals survived to develop advanced fibrosis in these organs at around 9 months of age. In conclusion, constitutive heterozygous expression of JAK2(V617F) in mice is not embryo-lethal but results in severe PV-like disease with secondary myelofibrosis and not in ET-like disease as expected from patient study.
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Janus Quinasa 2/genética , Trastornos Mieloproliferativos/etiología , Policitemia Vera/genética , Sustitución de Aminoácidos , Animales , Médula Ósea/patología , Linaje de la Célula , Cruzamientos Genéticos , Técnicas de Sustitución del Gen , Heterocigoto , Humanos , Hiperplasia , Janus Quinasa 2/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Mutación Missense , Trastornos Mieloproliferativos/enzimología , Trastornos Mieloproliferativos/genética , Mutación Puntual , Policitemia Vera/enzimología , Mielofibrosis Primaria/enzimología , Mielofibrosis Primaria/etiología , Mielofibrosis Primaria/genética , Bazo/patología , Trombocitemia Esencial/enzimología , Trombocitemia Esencial/genéticaRESUMEN
Establishing standard operating procedures (SOPs) as tools for the analysis of behavioral phenotypes is fundamental to mouse functional genomics. It is essential that the tests designed provide reliable measures of the process under investigation but most importantly that these are reproducible across both time and laboratories. For this reason, we devised and tested a set of SOPs to investigate mouse behavior. Five research centers were involved across France, Germany, Italy, and the UK in this study, as part of the EUMORPHIA program. All the procedures underwent a cross-validation experimental study to investigate the robustness of the designed protocols. Four inbred reference strains (C57BL/6J, C3HeB/FeJ, BALB/cByJ, 129S2/SvPas), reflecting their use as common background strains in mutagenesis programs, were analyzed to validate these tests. We demonstrate that the operating procedures employed, which includes open field, SHIRPA, grip-strength, rotarod, Y-maze, prepulse inhibition of acoustic startle response, and tail flick tests, generated reproducible results between laboratories for a number of the test output parameters. However, we also identified several uncontrolled variables that constitute confounding factors in behavioral phenotyping. The EUMORPHIA SOPs described here are an important start-point for the ongoing development of increasingly robust phenotyping platforms and their application in large-scale, multicentre mouse phenotyping programs.
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Conducta Animal/fisiología , Técnicas de Laboratorio Clínico , Cooperación Internacional , Animales , Laboratorios , Masculino , Aprendizaje por Laberinto , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Fenotipo , Reflejo de Sobresalto , Reproducibilidad de los Resultados , Prueba de Desempeño de Rotación con Aceleración ConstanteRESUMEN
Coffin-Lowry Syndrome (CLS) is an X-linked mental retardation condition associated with skeletal abnormalities. The gene mutated in CLS, RSK2, encodes a growth factor-regulated kinase. However, the cellular and molecular bases of the skeletal abnormalities associated with CLS remain unknown. Here, we show that RSK2 is required for osteoblast differentiation and function. We identify the transcription factor ATF4 as a critical substrate of RSK2 that is required for the timely onset of osteoblast differentiation, for terminal differentiation of osteoblasts, and for osteoblast-specific gene expression. Additionally, RSK2 and ATF4 posttranscriptionally regulate the synthesis of Type I collagen, the main constituent of the bone matrix. Accordingly, Atf4-deficiency results in delayed bone formation during embryonic development and low bone mass throughout postnatal life. These findings identify ATF4 as a critical regulator of osteoblast differentiation and function, and indicate that lack of ATF4 phosphorylation by RSK2 may contribute to the skeletal phenotype of CLS.
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Anomalías Múltiples/genética , Regulación Enzimológica de la Expresión Génica , Osteoblastos/metabolismo , Proteínas Quinasas S6 Ribosómicas/genética , Transactivadores/metabolismo , Factores de Transcripción/metabolismo , Anomalías Múltiples/enzimología , Factor de Transcripción Activador 4 , Animales , Diferenciación Celular , Línea Celular , Núcleo Celular/química , Colágeno Tipo I/biosíntesis , Matriz Extracelular/química , Genes Reguladores , Discapacidad Intelectual/genética , Ratones , Ratones Mutantes , Morfogénesis , Osteocalcina/metabolismo , Fosforilación , Procesamiento Proteico-Postraduccional , Proteínas Recombinantes/metabolismo , Especificidad por Sustrato , Síndrome , Transactivadores/deficiencia , Transactivadores/genética , Factores de Transcripción/genética , Activación Transcripcional , Cromosoma XRESUMEN
The Coffin-Lowry syndrome (CLS) is a syndromic form of X-linked mental retardation characterised in male patients by psychomotor and growth retardation, and various skeletal anomalies. CLS is caused by mutations in a gene located in Xp22.2 and encoding RSK2, a growth-factor regulated protein kinase. Mutations are extremely heterogeneous and lead to premature termination of translation and/or to loss of phosphotransferase activity. No correlation between the type and location of mutation and the clinical phenotype is evident. However, in one family (MRX19), a missense mutation was associated solely with mild mental retardation and no other clinical feature. Screening for RSK2 mutations is essential in most cases to confirm the diagnosis as well as for genetic counseling.