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1.
PLoS Genet ; 17(9): e1009777, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34587162

RESUMO

Perturbation of the excitation/inhibition (E/I) balance leads to neurodevelopmental diseases including to autism spectrum disorders, intellectual disability, and epilepsy. Loss-of-function mutations in the DYRK1A gene, located on human chromosome 21 (Hsa21,) lead to an intellectual disability syndrome associated with microcephaly, epilepsy, and autistic troubles. Overexpression of DYRK1A, on the other hand, has been linked with learning and memory defects observed in people with Down syndrome (DS). Dyrk1a is expressed in both glutamatergic and GABAergic neurons, but its impact on each neuronal population has not yet been elucidated. Here we investigated the impact of Dyrk1a gene copy number variation in glutamatergic neurons using a conditional knockout allele of Dyrk1a crossed with the Tg(Camk2-Cre)4Gsc transgenic mouse. We explored this genetic modification in homozygotes, heterozygotes and combined with the Dp(16Lipi-Zbtb21)1Yey trisomic mouse model to unravel the consequence of Dyrk1a dosage from 0 to 3, to understand its role in normal physiology, and in MRD7 and DS. Overall, Dyrk1a dosage in postnatal glutamatergic neurons did not impact locomotor activity, working memory or epileptic susceptibility, but revealed that Dyrk1a is involved in long-term explicit memory. Molecular analyses pointed at a deregulation of transcriptional activity through immediate early genes and a role of DYRK1A at the glutamatergic post-synapse by deregulating and interacting with key post-synaptic proteins implicated in mechanism leading to long-term enhanced synaptic plasticity. Altogether, our work gives important information to understand the action of DYRK1A inhibitors and have a better therapeutic approach.


Assuntos
Transtorno Autístico/genética , Transtornos Cognitivos/genética , Síndrome de Down/genética , Dosagem de Genes , Ácido Glutâmico/metabolismo , Deficiência Intelectual/genética , Neurônios/metabolismo , Distúrbios da Fala/genética , Animais , Encéfalo/patologia , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Transtornos Cognitivos/complicações , Modelos Animais de Doenças , Síndrome de Down/complicações , Regulação da Expressão Gênica , Humanos , Camundongos , Camundongos Transgênicos , Proteômica/métodos , Transmissão Sináptica/genética , Transcrição Gênica
2.
Methods ; 191: 95-106, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-32721466

RESUMO

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.


Assuntos
Reação em Cadeia da Polimerase em Tempo Real , Animais , DNA/genética , Camundongos , RNA/genética , Ratos
3.
Methods ; 191: 107-119, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33838271

RESUMO

Gene targeting and additive (random) transgenesis have proven to be powerful technologies with which to decipher the mammalian genome. With the advent of CRISPR/Cas9 genome editing, the ability to inactivate or modify the function of a gene has become even more accessible. However, the impact of each generated modification may be different from what was initially desired. Minimal validation of mutant alleles from genetically altered (GA) rodents remains essential to guarantee the interpretation of experimental results. The protocol described here combines design strategies for genomic and functional validation of genetically modified alleles with droplet digital PCR (ddPCR) or quantitative PCR (qPCR) for target DNA or mRNA quantification. In-depth analysis of the results obtained with GA models through the analysis of target DNA and mRNA quantification is also provided, to evaluate which pitfalls can be detected using these two methods, and we propose recommendations for the characterization of different type of mutant allele (knock-out, knock-in, conditional knock-out, FLEx, IKMC model or transgenic). Our results also highlight the possibility that mRNA expression of any mutated allele can be different from what might be expected in theory or according to common assumptions. For example, mRNA analyses on knock-out lines showed that nonsense-mediated mRNA decay is generally not achieved with a critical-exon approach. Likewise, comparison of multiple conditional lines crossed with the same CreERT2 deleter showed that the inactivation outcome was very different for each conditional model. DNA quantification by ddPCR of G0 to G2 generations of transgenic rodents generated by pronuclear injection showed an unexpected variability, demonstrating that G1 generation rodents cannot be considered as established lines.


Assuntos
Sistemas CRISPR-Cas , Alelos , Animais , Animais Geneticamente Modificados , Sistemas CRISPR-Cas/genética , DNA , Genômica , RNA Mensageiro , Reação em Cadeia da Polimerase em Tempo Real , Roedores/genética
4.
Mol Ther ; 28(6): 1422-1431, 2020 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-32243835

RESUMO

Genome editing tools have already revolutionized biomedical research and are also expected to have an important impact in the clinic. However, their extensive use in research has revealed much unpredictability, both off and on target, in the outcome of their application. We discuss the challenges associated with this unpredictability, both for research and in the clinic. For the former, an extensive validation of the model is essential. For the latter, potential unpredicted activity does not preclude the use of these tools but requires that molecular evidence to underpin the relevant risk:benefit evaluation is available. Safe and successful clinical application will also depend on the mode of delivery and the cellular context.


Assuntos
Edição de Genes/métodos , Edição de Genes/normas , Experimentação Animal , Animais , Sistemas CRISPR-Cas , Estudos Clínicos como Assunto , Expressão Gênica , Técnicas de Transferência de Genes , Terapia Genética/efeitos adversos , Terapia Genética/métodos , Terapia Genética/normas , Humanos , Especificidade de Órgãos , Reprodutibilidade dos Testes , Medição de Risco , Pesquisa Translacional Biomédica/métodos , Pesquisa Translacional Biomédica/normas
5.
Hum Mol Genet ; 27(12): 2138-2153, 2018 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-29659809

RESUMO

The aristaless-related homeobox (ARX) transcription factor is involved in the development of GABAergic and cholinergic neurons in the forebrain. ARX mutations have been associated with a wide spectrum of neurodevelopmental disorders in humans, among which the most frequent, a 24 bp duplication in the polyalanine tract 2 (c.428_451dup24), gives rise to intellectual disability, fine motor defects with or without epilepsy. To understand the functional consequences of this mutation, we generated a partially humanized mouse model carrying the c.428_451dup24 duplication (Arxdup24/0) that we characterized at the behavior, neurological and molecular level. Arxdup24/0 males presented with hyperactivity, enhanced stereotypies and altered contextual fear memory. In addition, Arxdup24/0 males had fine motor defects with alteration of reaching and grasping abilities. Transcriptome analysis of Arxdup24/0 forebrains at E15.5 showed a down-regulation of genes specific to interneurons and an up-regulation of genes normally not expressed in this cell type, suggesting abnormal interneuron development. Accordingly, interneuron migration was altered in the cortex and striatum between E15.5 and P0 with consequences in adults, illustrated by the defect in the inhibitory/excitatory balance in Arxdup24/0 basolateral amygdala. Altogether, we showed that the c.428_451dup24 mutation disrupts Arx function with a direct consequence on interneuron development, leading to hyperactivity and defects in precise motor movement control and associative memory. Interestingly, we highlighted striking similarities between the mouse phenotype and a cohort of 33 male patients with ARX c.428_451dup24, suggesting that this new mutant mouse line is a good model for understanding the pathophysiology and evaluation of treatment.


Assuntos
Epilepsia/genética , Proteínas de Homeodomínio/genética , Transtornos do Neurodesenvolvimento/genética , Fatores de Transcrição/genética , Adolescente , Adulto , Animais , Criança , Pré-Escolar , Neurônios Colinérgicos/metabolismo , Neurônios Colinérgicos/patologia , Contratura , Modelos Animais de Doenças , Epilepsia/fisiopatologia , Neurônios GABAérgicos/metabolismo , Neurônios GABAérgicos/patologia , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Lactente , Deficiência Intelectual , Masculino , Camundongos , Mutação , Transtornos do Neurodesenvolvimento/fisiopatologia , Peptídeos/genética , Prosencéfalo/fisiopatologia , Paraplegia Espástica Hereditária , Transcriptoma/genética , Adulto Jovem
6.
Nature ; 504(7479): 277-281, 2013 Dec 12.
Artigo em Inglês | MEDLINE | ID: mdl-24336287

RESUMO

Fibroblasts are the major mesenchymal cell type in connective tissue and deposit the collagen and elastic fibres of the extracellular matrix (ECM). Even within a single tissue, fibroblasts exhibit considerable functional diversity, but it is not known whether this reflects the existence of a differentiation hierarchy or is a response to different environmental factors. Here we show, using transplantation assays and lineage tracing in mice, that the fibroblasts of skin connective tissue arise from two distinct lineages. One forms the upper dermis, including the dermal papilla that regulates hair growth and the arrector pili muscle, which controls piloerection. The other forms the lower dermis, including the reticular fibroblasts that synthesize the bulk of the fibrillar ECM, and the preadipocytes and adipocytes of the hypodermis. The upper lineage is required for hair follicle formation. In wounded adult skin, the initial wave of dermal repair is mediated by the lower lineage and upper dermal fibroblasts are recruited only during re-epithelialization. Epidermal ß-catenin activation stimulates the expansion of the upper dermal lineage, rendering wounds permissive for hair follicle formation. Our findings explain why wounding is linked to formation of ECM-rich scar tissue that lacks hair follicles. They also form a platform for discovering fibroblast lineages in other tissues and for examining fibroblast changes in ageing and disease.


Assuntos
Linhagem da Célula , Fibroblastos/citologia , Pele/citologia , Pele/crescimento & desenvolvimento , Cicatrização/fisiologia , Adipócitos/citologia , Adipócitos/metabolismo , Animais , Derme/anatomia & histologia , Derme/citologia , Derme/embriologia , Derme/crescimento & desenvolvimento , Feminino , Fibroblastos/transplante , Folículo Piloso/citologia , Folículo Piloso/metabolismo , Técnicas In Vitro , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Camundongos Transgênicos , Músculo Liso/citologia , Músculo Liso/metabolismo , Pele/anatomia & histologia , Pele/embriologia , beta Catenina/metabolismo
7.
Proc Natl Acad Sci U S A ; 113(39): 11004-9, 2016 09 27.
Artigo em Inglês | MEDLINE | ID: mdl-27621431

RESUMO

The multifunctional protein E4 transcription factor 1 (E4F1) is an essential regulator of epidermal stem cell (ESC) maintenance. Here, we found that E4F1 transcriptionally regulates a metabolic program involved in pyruvate metabolism that is required to maintain skin homeostasis. E4F1 deficiency in basal keratinocytes resulted in deregulated expression of dihydrolipoamide acetyltransferase (Dlat), a gene encoding the E2 subunit of the mitochondrial pyruvate dehydrogenase (PDH) complex. Accordingly, E4f1 knock-out (KO) keratinocytes exhibited impaired PDH activity and a redirection of the glycolytic flux toward lactate production. The metabolic reprogramming of E4f1 KO keratinocytes associated with remodeling of their microenvironment and alterations of the basement membrane, led to ESC mislocalization and exhaustion of the ESC pool. ShRNA-mediated depletion of Dlat in primary keratinocytes recapitulated defects observed upon E4f1 inactivation, including increased lactate secretion, enhanced activity of extracellular matrix remodeling enzymes, and impaired clonogenic potential. Altogether, our data reveal a central role for Dlat in the metabolic program regulated by E4F1 in basal keratinocytes and illustrate the importance of PDH activity in skin homeostasis.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Di-Hidrolipoil-Lisina-Resíduo Acetiltransferase/metabolismo , Homeostase , Proteínas Mitocondriais/metabolismo , Pele/metabolismo , Fatores de Transcrição/metabolismo , Animais , Animais Recém-Nascidos , Membrana Basal/metabolismo , Adesão Celular , Células Cultivadas , Microambiente Celular , Proteínas de Ligação a DNA/deficiência , Di-Hidrolipoil-Lisina-Resíduo Acetiltransferase/genética , Células Epidérmicas , Epiderme/metabolismo , Regulação da Expressão Gênica , Queratinócitos/citologia , Queratinócitos/metabolismo , Camundongos Knockout , Proteínas Mitocondriais/genética , Transportadores de Ácidos Monocarboxílicos/metabolismo , Proteínas Musculares/metabolismo , Piruvatos/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Repressoras , Células-Tronco/metabolismo , Fatores de Transcrição/deficiência , Ubiquitina-Proteína Ligases
9.
Am J Physiol Endocrinol Metab ; 315(3): E386-E393, 2018 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-29870677

RESUMO

Fructose consumption in humans and animals has been linked to enhanced de novo lipogenesis, dyslipidemia, and insulin resistance. Hereditary deficiency of ketohexokinase (KHK), the first enzymatic step in fructose metabolism, leads to essential fructosuria in humans, characterized by elevated levels of blood and urinary fructose following fructose ingestion but is otherwise clinically benign. To address whether KHK deficiency is associated with altered glucose and lipid metabolism, a Khk knockout (KO) mouse line was generated and characterized. NMR spectroscopic analysis of plasma following ingestion of [6-13C] fructose revealed striking differences in biomarkers of fructose metabolism. Significantly elevated urine and plasma 13C-fructose levels were observed in Khk KO vs. wild-type (WT) control mice, as was reduced conversion of 13C-fructose into plasma 13C-glucose and 13C-lactate. In addition, the observation of significant levels of fructose-6-phosphate in skeletal muscle tissue of Khk KO, but not WT, mice suggests a potential mechanism, whereby fructose is metabolized via muscle hexokinase in the absence of KHK. Khk KO mice on a standard chow diet displayed no metabolic abnormalities with respect to ambient glucose, glucose tolerance, body weight, food intake, and circulating trigylcerides, ß-hydroxybutyrate, and lactate. When placed on a high-fat and high-fructose (HF/HFruc) diet, Khk KO mice had markedly reduced liver weight, triglyceride levels, and insulin levels. Together, these results suggest that Khk KO mice may serve as a good model for essential fructosuria in humans and that inhibition of KHK offers the potential to protect from diet-induced hepatic steatosis and insulin resistance.


Assuntos
Dieta , Frutoquinases/deficiência , Erros Inatos do Metabolismo da Frutose/genética , Doenças Metabólicas/genética , Doenças Metabólicas/metabolismo , Animais , Glicemia/metabolismo , Peso Corporal/genética , Dieta Hiperlipídica , Ingestão de Alimentos/genética , Frutoquinases/genética , Frutoquinases/metabolismo , Erros Inatos do Metabolismo da Frutose/metabolismo , Frutosefosfatos/sangue , Intolerância à Glucose/genética , Resistência à Insulina , Metabolismo dos Lipídeos/genética , Camundongos Knockout
10.
J Biol Chem ; 291(45): 23428-23439, 2016 Nov 04.
Artigo em Inglês | MEDLINE | ID: mdl-27621313

RESUMO

Mutations of the AMP-activated kinase gamma 2 subunit (AMPKγ2), N488I (AMPKγ2NI) and R531G (AMPKγ2RG), are associated with Wolff-Parkinson-White (WPW) syndrome, a cardiac disorder characterized by ventricular pre-excitation in humans. Cardiac-specific transgenic overexpression of human AMPKγ2NI or AMPKγ2RG leads to constitutive AMPK activation and the WPW phenotype in mice. However, overexpression of these mutant proteins also caused profound, non-physiological increase in cardiac glycogen, which might abnormally alter the true phenotype. To investigate whether physiological levels of AMPKγ2NI or AMPKγ2RG mutation cause WPW syndrome and metabolic changes in other organs, we generated two knock-in mouse lines on the C57BL/6N background harboring mutations of human AMPKγ2NI and AMPKγ2RG, respectively. Similar to the reported phenotypes of mice overexpressing AMPKγ2NI or AMPKγ2RG in the heart, both lines developed WPW syndrome and cardiac hypertrophy; however, these effects were independent of cardiac glycogen accumulation. Compared with AMPKγ2WT mice, AMPKγ2NI and AMPKγ2RG mice exhibited reduced body weight, fat mass, and liver steatosis when fed with a high fat diet (HFD). Surprisingly, AMPKγ2RG but not AMPKγ2NI mice fed with an HFD exhibited severe kidney injury characterized by glycogen accumulation, inflammation, apoptosis, cyst formation, and impaired renal function. These results demonstrate that expression of AMPKγ2NI and AMPKγ2RG mutations at physiological levels can induce beneficial metabolic effects but that this is accompanied by WPW syndrome. Our data also reveal an unexpected effect of AMPKγ2RG in the kidney, linking lifelong constitutive activation of AMPK to a potential risk for kidney dysfunction in the context of an HFD.


Assuntos
Proteínas Quinases Ativadas por AMP/genética , Mutação , Insuficiência Renal/genética , Síndrome de Wolff-Parkinson-White/genética , Animais , Apoptose , Modelos Animais de Doenças , Técnicas de Introdução de Genes , Inflamação/genética , Inflamação/patologia , Rim/metabolismo , Rim/patologia , Masculino , Camundongos Endogâmicos C57BL , Insuficiência Renal/patologia , Síndrome de Wolff-Parkinson-White/patologia
11.
Mamm Genome ; 28(7-8): 291-301, 2017 08.
Artigo em Inglês | MEDLINE | ID: mdl-28677007

RESUMO

Modeling human disease has proven to be a challenge for the scientific community. For years, generating an animal model was complicated and restricted to very few species. With the rise of CRISPR/Cas9, it is now possible to generate more or less any animal model. In this review, we will show how this technology is and will change our way to obtain relevant disease animal models and how it should impact human health.


Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Estudos de Associação Genética/métodos , Predisposição Genética para Doença , Modelos Biológicos , Animais , Animais Geneticamente Modificados , Modelos Animais de Doenças , Terapia Genética/métodos , Variação Genética , Genômica/métodos , Humanos , Camundongos , Herança Multifatorial , Fases de Leitura Aberta , Medicina de Precisão , RNA não Traduzido , Sequências Reguladoras de Ácido Ribonucleico
12.
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
14.
Nat Neurosci ; 27(7): 1285-1298, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-38849523

RESUMO

Fibrotic scar tissue formation occurs in humans and mice. The fibrotic scar impairs tissue regeneration and functional recovery. However, the origin of scar-forming fibroblasts is unclear. Here, we show that stromal fibroblasts forming the fibrotic scar derive from two populations of perivascular cells after spinal cord injury (SCI) in adult mice of both sexes. We anatomically and transcriptionally identify the two cell populations as pericytes and perivascular fibroblasts. Fibroblasts and pericytes are enriched in the white and gray matter regions of the spinal cord, respectively. Both cell populations are recruited in response to SCI and inflammation. However, their contribution to fibrotic scar tissue depends on the location of the lesion. Upon injury, pericytes and perivascular fibroblasts become activated and transcriptionally converge on the generation of stromal myofibroblasts. Our results show that pericytes and perivascular fibroblasts contribute to the fibrotic scar in a region-dependent manner.


Assuntos
Cicatriz , Fibroblastos , Fibrose , Pericitos , Traumatismos da Medula Espinal , Animais , Fibroblastos/patologia , Fibroblastos/metabolismo , Fibrose/patologia , Traumatismos da Medula Espinal/patologia , Camundongos , Pericitos/patologia , Pericitos/metabolismo , Masculino , Feminino , Cicatriz/patologia , Camundongos Endogâmicos C57BL , Células Estromais/patologia
15.
Nat Commun ; 15(1): 5574, 2024 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-38956430

RESUMO

The biomedical research community addresses reproducibility challenges in animal studies through standardized nomenclature, improved experimental design, transparent reporting, data sharing, and centralized repositories. The ARRIVE guidelines outline documentation standards for laboratory animals in experiments, but genetic information is often incomplete. To remedy this, we propose the Laboratory Animal Genetic Reporting (LAG-R) framework. LAG-R aims to document animals' genetic makeup in scientific publications, providing essential details for replication and appropriate model use. While verifying complete genetic compositions may be impractical, better reporting and validation efforts enhance reliability of research. LAG-R standardization will bolster reproducibility, peer review, and overall scientific rigor.


Assuntos
Animais de Laboratório , Guias como Assunto , Animais , Animais de Laboratório/genética , Reprodutibilidade dos Testes , Projetos de Pesquisa , Experimentação Animal/normas , Pesquisa Biomédica/normas
16.
J Neurosci ; 32(6): 1962-8, 2012 Feb 08.
Artigo em Inglês | MEDLINE | ID: mdl-22323709

RESUMO

Vesicular (v)- and target (t)-SNARE proteins assemble in SNARE complex to mediate membrane fusion. Tetanus neurotoxin-insensitive vesicular-associated membrane protein (TI-VAMP/VAMP7), a vesicular SNARE expressed in several cell types including neurons, was previously shown to play a major role in exocytosis involved in neurite growth in cultured neurons. Here we generated a complete constitutive knock-out by deleting the exon 3 of Vamp7. Loss of TI-VAMP expression did not lead to any striking developmental or neurological defect. Knock-out mice displayed decreased brain weight and increased third ventricle volume. Axon growth appeared normal in cultured knock-out neurons. Behavioral characterization unraveled that TI-VAMP knock-out was associated with increased anxiety. Our results thus suggest compensatory mechanisms allowing the TI-VAMP knock-out mice to fulfill major developmental processes. The phenotypic traits unraveled here further indicate an unexpected role of TI-VAMP-mediated vesicular traffic in anxiety and suggest a role for TI-VAMP in higher brain functions.


Assuntos
Ansiedade/genética , Metaloendopeptidases , Proteínas R-SNARE/deficiência , Proteínas R-SNARE/genética , Toxina Tetânica , Animais , Ansiedade/etiologia , Ansiedade/psicologia , Células COS , Células Cultivadas , Chlorocebus aethiops , Masculino , Metaloendopeptidases/administração & dosagem , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Coelhos , Toxina Tetânica/administração & dosagem
17.
Methods Mol Biol ; 2631: 277-297, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36995673

RESUMO

CRISPR/Cas9 technology is a versatile tool for engineering biology that has dramatically transformed our ability to manipulate genomes. In this protocol, we use its capacity to generate two double-strand breaks simultaneously, at precise positions in the genome, to generate mouse or rat lines with deletion, inversion, and duplication of a specific genomic segment. The technic is called CRISMERE for CRISpr-MEdiated REarrangement. This protocol describes the different steps to generate and validate the different chromosomal rearrangements that can be obtained with the technology. These new genetic configurations can be useful to model rare diseases with copy number variation, understand the genomic organization, or provide genetic tools (like balancer chromosome) to keep lethal mutations.


Assuntos
Variações do Número de Cópias de DNA , Genoma , Camundongos , Ratos , Animais , Genômica , Mutação , Cromossomos , Sistemas CRISPR-Cas/genética , Engenharia Genética
18.
Genes (Basel) ; 14(2)2023 02 03.
Artigo em Inglês | MEDLINE | ID: mdl-36833328

RESUMO

The French mouse clinic (Institut Clinique de la Souris; ICS) has produced more than 2000 targeting vectors for 'à la carte' mutagenesis in C57BL/6N mice. Although most of the vectors were used successfully for homologous recombination in murine embryonic stem cells (ESCs), a few have failed to target a specific locus after several attempts. We show here that co-electroporation of a CRISPR plasmid with the same targeting construct as the one that failed previously allows the systematic achievement of positive clones. A careful validation of these clones is, however, necessary as a significant number of clones (but not all) show a concatemerization of the targeting plasmid at the locus. A detailed Southern blot analysis permitted characterization of the nature of these events as standard long-range 5' and 3' PCRs were not able to distinguish between correct and incorrect alleles. We show that a simple and inexpensive PCR performed prior to ESC amplification allows detection and elimination of those clones with concatemers. Finally, although we only tested murine ESCs, our results highlight the risk of mis-validation of any genetically modified cell line (such as established lines, induced pluripotent stem cells or those used for ex vivo gene therapy) that combines the use of CRISPR/Cas9 and a circular double-stranded donor. We strongly advise the CRISPR community to perform a Southern blot with internal probes when using CRISPR to enhance homologous recombination in any cell type, including fertilized oocytes.


Assuntos
Sistemas CRISPR-Cas , Células-Tronco Embrionárias , Camundongos , Animais , Camundongos Endogâmicos C57BL , Células-Tronco Embrionárias/metabolismo , Recombinação Homóloga , Mutagênese
19.
Lab Anim Res ; 39(1): 14, 2023 Jun 12.
Artigo em Inglês | MEDLINE | ID: mdl-37308929

RESUMO

BACKGROUND: Animal models are essential to understand the physiopathology of human diseases but also to evaluate new therapies. However, for several diseases there is no appropriate animal model, which complicates the development of effective therapies. HPV infections, responsible for carcinoma cancers, are among these. So far, the lack of relevant animal models has hampered the development of therapeutic vaccines. In this study, we used a candidate therapeutic vaccine named C216, similar to the ProCervix candidate therapeutic vaccine, to validate new mouse and dog HPV preclinical models. ProCervix has shown promising results with classical subcutaneous murine TC-1 cell tumor isografts but has failed in a phase II study. RESULTS: We first generated E7/HPV16 syngeneic transgenic mice in which the expression of the E7 antigen could be switched on through the use of Cre-lox recombination. Non-integrative LentiFlash® viral particles were used to locally deliver Cre mRNA, resulting in E7/HPV16 expression and GFP reporter fluorescence. The expression of E7/HPV16 was monitored by in vivo fluorescence using Cellvizio imaging and by local mRNA expression quantification. In the experimental conditions used, we observed no differences in E7 expression between C216 vaccinated and control groups. To mimic the MHC diversity of humans, E7/HPV16 transgenes were locally delivered by injection of lentiviral particles in the muscle of dogs. Vaccination with C216, tested with two different adjuvants, induced a strong immune response in dogs. However, we detected no relationship between the level of cellular response against E7/HPV16 and the elimination of E7-expressing cells, either by fluorescence or by RT-ddPCR analysis. CONCLUSIONS: In this study, we have developed two animal models, with a genetic design that is easily transposable to different antigens, to validate the efficacy of candidate vaccines. Our results indicate that, despite being immunogenic, the C216 candidate vaccine did not induce a sufficiently strong immune response to eliminate infected cells. Our results are in line with the failure of the ProCervix vaccine that was observed at the end of the phase II clinical trial, reinforcing the relevance of appropriate animal models.

20.
Genesis ; 50(6): 482-9, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22121025

RESUMO

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.


Assuntos
DNA Nucleotidiltransferases/genética , Integrases/genética , Camundongos Transgênicos , Proteínas/genética , Animais , Proteínas de Bactérias/genética , Efeito Fundador , Marcação de Genes , Engenharia Genética , Genótipo , Proteínas de Fluorescência Verde/genética , Recombinação Homóloga , Proteínas Luminescentes/genética , Camundongos , Camundongos Endogâmicos C57BL , RNA não Traduzido , Deleção de Sequência
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