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1.
World J Microbiol Biotechnol ; 35(7): 111, 2019 Jul 06.
Artigo em Inglês | MEDLINE | ID: mdl-31280424

RESUMO

Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) immune systems in bacteria have been used as tools for genome engineering. Thus far, the CRISPR-Cas system has been used in various yeast, bacterial, and mammalian cells. Saccharomyces cerevisiae is a nonpathogenic yeast, classified under "generally recognized as safe", and has long been used to produce consumables such as alcohol or bread. Additionally, recombinant cells of S. cerevisiae have been constructed and used to produce various bio-based chemicals. Some types of CRISPR-Cas system for genetic manipulation have been constructed during the early developmental stages of the CRISPR-Cas system and have been mainly used for gene knock-in and knock-out manipulations. Thereafter, these systems have been used for various novel purposes such as metabolic engineering and tolerance engineering. In this review, we have summarized different aspects of the CRISPR-Cas in the yeast S. cerevisiae, from its basic principles to various applications. This review describes the CRISPR system in S. cerevisiae based on the differences in its origin and efficiency followed by its basic applications; for example, its involvement in gene knock-in and knock-out has been outlined. Finally, advanced applications of the CRISPR system in the bioproduction of useful chemicals have been summarized.


Assuntos
Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Engenharia Metabólica/métodos , Saccharomyces cerevisiae/metabolismo , Edição de Genes/métodos , Regulação Fúngica da Expressão Gênica , Técnicas de Introdução de Genes/métodos , Técnicas de Inativação de Genes/métodos , Saccharomyces cerevisiae/genética
2.
Rinsho Ketsueki ; 60(6): 680-690, 2019.
Artigo em Japonês | MEDLINE | ID: mdl-31281161

RESUMO

Recent progress in whole genome sequencing has identified recurrent somatic mutations in the additional sex combs like 1 (ASXL1) gene in a variety of hematological disorders and even in premalignant conditions. However, the molecular mechanisms regarding the contribution of ASXL1 mutation to the pathogenesis of premalignant conditions remain largely unelucidated. Thus, we investigated the biological effects of mutant Asxl1 using newly-generated knock-in (KI) mice. Heterozygous mutant KI mice developed phenotypes resembling human low-risk myelodysplastic syndromes (MDS), and some of them developed an MDS/myeloproliferative neoplasm-like disease after a long latency. The H2AK119ub1 level around the promoter region of p16Ink4a was significantly decreased in KI hematopoietic stem cells (HSCs), suggesting perturbation of Bmi1-driven H2AK119ub1 histone modification by mutant Asxl1. The mutant Asxl1 failed to interact with Bmi1, although wild type ASXL1 protein did not. When p16Ink4a expression was depleted in Asxl1 KI mice, the HSC pool was restored, and apoptosis was ameliorated in HSCs. These findings demonstrate that the loss of protein interaction between mutant Asxl1 and Bmi1 affected the activity of Prc1. The subsequent derepression of p16Ink4a by aberrant histone ubiquitination could induce cellular senescence, resulting in low-risk MDS-like phenotypes in heterozygous Asxl1 KI mice.


Assuntos
Mutação , Síndromes Mielodisplásicas/genética , Proteínas Repressoras/genética , Animais , Modelos Animais de Doenças , Técnicas de Introdução de Genes , Células-Tronco Hematopoéticas , Histonas/metabolismo , Camundongos , Fenótipo , Complexo Repressor Polycomb 1/genética , Proteínas Proto-Oncogênicas/genética , Ubiquitinação
3.
Nat Commun ; 10(1): 2394, 2019 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-31160584

RESUMO

To understand the molecular processes that link Aß amyloidosis, tauopathy and neurodegeneration, we screened for tau-interacting proteins by immunoprecipitation/LC-MS. We identified the carboxy-terminal PDZ ligand of nNOS (CAPON) as a novel tau-binding protein. CAPON is an adaptor protein of neuronal nitric oxide synthase (nNOS), and activated by the N-methyl-D-aspartate receptor. We observed accumulation of CAPON in the hippocampal pyramidal cell layer in the AppNL-G-F -knock-in (KI) brain. To investigate the effect of CAPON accumulation on Alzheimer's disease (AD) pathogenesis, CAPON was overexpressed in the brain of AppNL-G-F mice crossbred with MAPT (human tau)-KI mice. This produced significant hippocampal atrophy and caspase3-dependent neuronal cell death in the CAPON-expressing hippocampus, suggesting that CAPON accumulation increases neurodegeneration. CAPON expression also induced significantly higher levels of phosphorylated, oligomerized and insoluble tau. In contrast, CAPON deficiency ameliorated the AD-related pathological phenotypes in tauopathy model. These findings suggest that CAPON could be a druggable AD target.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Doença de Alzheimer/metabolismo , Hipocampo/metabolismo , Agregação Patológica de Proteínas/metabolismo , Células Piramidais/metabolismo , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Precursor de Proteína beta-Amiloide/genética , Animais , Atrofia , Caspase 3/metabolismo , Morte Celular , Cromatografia Líquida , Modelos Animais de Doenças , Técnicas de Introdução de Genes , Hipocampo/patologia , Humanos , Imunoprecipitação , Espectrometria de Massas , Camundongos , Neurônios/metabolismo , Neurônios/patologia , Agregação Patológica de Proteínas/patologia , Células Piramidais/patologia , Tauopatias , Proteínas tau/metabolismo
4.
Nat Commun ; 10(1): 2434, 2019 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-31164647

RESUMO

To date, genome-wide association studies have implicated at least 35 loci in osteoarthritis but, due to linkage disequilibrium, the specific variants underlying these associations and the mechanisms by which they contribute to disease risk have yet to be pinpointed. Here, we functionally test 1,605 single nucleotide variants associated with osteoarthritis for regulatory activity using a massively parallel reporter assay. We identify six single nucleotide polymorphisms (SNPs) with differential regulatory activity between the major and minor alleles. We show that the most significant SNP, rs4730222, exhibits differential nuclear protein binding in electrophoretic mobility shift assays and drives increased expression of an alternative isoform of HBP1 in a heterozygote chondrosarcoma cell line, in a CRISPR-edited osteosarcoma cell line, and in chondrocytes derived from osteoarthritis patients. This study provides a framework for prioritization of GWAS variants and highlights a role of HBP1 and Wnt signaling in osteoarthritis pathogenesis.


Assuntos
Condrócitos/metabolismo , Redes Reguladoras de Genes , Proteínas de Grupo de Alta Mobilidade/genética , Osteoartrite/genética , Proteínas Repressoras/genética , Alelos , Cartilagem Articular/citologia , Linhagem Celular Tumoral , Ensaio de Desvio de Mobilidade Eletroforética , Técnicas de Introdução de Genes , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Haplótipos , Proteínas de Grupo de Alta Mobilidade/metabolismo , Humanos , Desequilíbrio de Ligação , Proteínas Nucleares/metabolismo , Polimorfismo de Nucleotídeo Único , Ligação Proteica , Isoformas de Proteínas , Proteínas Repressoras/metabolismo , Via de Sinalização Wnt
5.
Nat Commun ; 10(1): 2141, 2019 05 20.
Artigo em Inglês | MEDLINE | ID: mdl-31105267

RESUMO

Costimulation of T cell responses with monoclonal antibody agonists (mAb-AG) targeting 4-1BB showed robust anti-tumor activity in preclinical models, but their clinical development was hampered by low efficacy (Utomilumab) or severe liver toxicity (Urelumab). Here we show that isotype and intrinsic agonistic strength co-determine the efficacy and toxicity of anti-4-1BB mAb-AG. While intrinsically strong agonistic anti-4-1BB can activate 4-1BB in the absence of FcγRs, weak agonistic antibodies rely on FcγRs to activate 4-1BB. All FcγRs can crosslink anti-41BB antibodies to strengthen co-stimulation, but activating FcγR-induced antibody-dependent cell-mediated cytotoxicity compromises anti-tumor immunity by deleting 4-1BB+ cells. This suggests balancing agonistic activity with the strength of FcγR interaction as a strategy to engineer 4-1BB mAb-AG with optimal therapeutic performance. As a proof of this concept, we have developed LVGN6051, a humanized 4-1BB mAb-AG that shows high anti-tumor efficacy in the absence of liver toxicity in a mouse model of cancer immunotherapy.


Assuntos
Anticorpos Monoclonais Humanizados/farmacologia , Imunoterapia/métodos , Melanoma Experimental/terapia , Receptores de IgG/metabolismo , Neoplasias Cutâneas/terapia , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/antagonistas & inibidores , Células 3T3 , Animais , Anticorpos Monoclonais Humanizados/imunologia , Anticorpos Monoclonais Humanizados/uso terapêutico , Linhagem Celular Tumoral/transplante , Ensaios de Seleção de Medicamentos Antitumorais , Técnicas de Introdução de Genes , Humanos , Fígado/efeitos dos fármacos , Melanoma Experimental/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de IgG/genética , Neoplasias Cutâneas/imunologia , Linfócitos T/imunologia , Linfócitos T/metabolismo , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/genética , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/imunologia
6.
Nat Commun ; 10(1): 2134, 2019 05 13.
Artigo em Inglês | MEDLINE | ID: mdl-31086183

RESUMO

The transient receptor potential vanilloid 1 (TRPV1) ion channel is a prototypical molecular sensor for noxious heat in mammals. Its role in sustained heat response remains poorly understood, because rapid heat-induced desensitization (Dh) follows tightly heat-induced activation (Ah). To understand the physiological role and structural basis of Dh, we carried out a comparative study of TRPV1 channels in mouse (mV1) and those in platypus (pV1), which naturally lacks Dh. Here we show that a temperature-sensitive interaction between the N- and C-terminal domains of mV1 but not pV1 drives a conformational rearrangement in the pore leading to Dh. We further show that knock-in mice expressing pV1 sensed heat normally but suffered scald damages in a hot environment. Our findings suggest that Dh evolved late during evolution as a protective mechanism and a delicate balance between Ah and Dh is crucial for mammals to sense and respond to noxious heat.


Assuntos
Aclimatação/fisiologia , Temperatura Alta/efeitos adversos , Domínios Proteicos/fisiologia , Canais de Cátion TRPV/metabolismo , Animais , Evolução Biológica , Temperatura Corporal/fisiologia , Dermatite , Feminino , Técnicas de Introdução de Genes , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Filogenia , Ornitorrinco/fisiologia , Canais de Cátion TRPV/genética
7.
Nat Commun ; 10(1): 2129, 2019 05 13.
Artigo em Inglês | MEDLINE | ID: mdl-31086189

RESUMO

De novo heterozygous missense variants in the γ-tubulin gene TUBG1 have been linked to human malformations of cortical development associated with intellectual disability and epilepsy. Here, we investigated through in-utero electroporation and in-vivo studies, how four of these variants affect cortical development. We show that TUBG1 mutants affect neuronal positioning, disrupting the locomotion of new-born neurons but without affecting progenitors' proliferation. We further demonstrate that pathogenic TUBG1 variants are linked to reduced microtubule dynamics but without major structural nor functional centrosome defects in subject-derived fibroblasts. Additionally, we developed a knock-in Tubg1Y92C/+ mouse model and assessed consequences of the mutation. Although centrosomal positioning in bipolar neurons is correct, they fail to initiate locomotion. Furthermore, Tubg1Y92C/+ animals show neuroanatomical and behavioral defects and increased epileptic cortical activity. We show that Tubg1Y92C/+ mice partially mimic the human phenotype and therefore represent a relevant model for further investigations of the physiopathology of cortical malformations.


Assuntos
Malformações do Desenvolvimento Cortical/genética , Microtúbulos/metabolismo , Neurogênese/genética , Neurônios/fisiologia , Tubulina (Proteína)/genética , Animais , Comportamento Animal , Movimento Celular/genética , Centrossomo/metabolismo , Córtex Cerebral/anormalidades , Córtex Cerebral/citologia , Córtex Cerebral/diagnóstico por imagem , Modelos Animais de Doenças , Embrião de Mamíferos , Epilepsia/genética , Feminino , Fibroblastos/citologia , Fibroblastos/metabolismo , Fibroblastos/ultraestrutura , Técnicas de Introdução de Genes , Predisposição Genética para Doença , Células HeLa , Humanos , Microscopia Intravital , Masculino , Camundongos , Camundongos Transgênicos , Microscopia Confocal , Microscopia Eletrônica , Microtúbulos/genética , Mutação de Sentido Incorreto
8.
Dev Growth Differ ; 61(4): 265-275, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31037730

RESUMO

The CRISPR-Cas9 technology has been a powerful means to manipulate the genome in a wide range of organisms. A series of GFP knocked-in (GFPKI ) Drosophila strains have been generated through CRISPR-Cas9-induced double strand breaks coupled with homology-directed repairs in the presence of donor plasmids. They visualized specific cell types or intracellular structures in both fixed and live specimen. We provide a rapid and efficient strategy to identify KI lines. This method requires neither co-integration of a selection marker nor prior establishment of sgRNA-expressing transgenic lines. The injection of the mixture of a sgRNA/Cas9 expression plasmid and a donor plasmid into cleavage stage embryos efficiently generated multiple independent KI lines. A PCR-based selection allows to identify KI fly lines at the F1 generation (approximately 4 weeks after injection). These GFPKI strains have been deposited in the Kyoto Drosophila stock center, and made freely available to researchers at non-profit organizations. Thus, they will be useful resources for Drosophila research.


Assuntos
Sistemas CRISPR-Cas/genética , Drosophila/genética , Edição de Genes/métodos , Técnicas de Introdução de Genes/métodos , Proteínas de Fluorescência Verde/genética , Animais , Fatores de Tempo
9.
Nat Commun ; 10(1): 1718, 2019 04 12.
Artigo em Inglês | MEDLINE | ID: mdl-30979869

RESUMO

Hypothalamic neurons expressing the anorectic peptide Pro-opiomelanocortin (Pomc) regulate food intake and body weight. Here, we show that Steroid Receptor Coactivator-1 (SRC-1) interacts with a target of leptin receptor activation, phosphorylated STAT3, to potentiate Pomc transcription. Deletion of SRC-1 in Pomc neurons in mice attenuates their depolarization by leptin, decreases Pomc expression and increases food intake leading to high-fat diet-induced obesity. In humans, fifteen rare heterozygous variants in SRC-1 found in severely obese individuals impair leptin-mediated Pomc reporter activity in cells, whilst four variants found in non-obese controls do not. In a knock-in mouse model of a loss of function human variant (SRC-1L1376P), leptin-induced depolarization of Pomc neurons and Pomc expression are significantly reduced, and food intake and body weight are increased. In summary, we demonstrate that SRC-1 modulates the function of hypothalamic Pomc neurons, and suggest that targeting SRC-1 may represent a useful therapeutic strategy for weight loss.


Assuntos
Hipotálamo/metabolismo , Neurônios/metabolismo , Coativador 1 de Receptor Nuclear/genética , Coativador 1 de Receptor Nuclear/metabolismo , Obesidade/genética , Alelos , Animais , Peso Corporal , Linhagem Celular Tumoral , Cruzamentos Genéticos , Deleção de Genes , Técnicas de Introdução de Genes , Variação Genética , Células HEK293 , Heterozigoto , Homeostase , Humanos , Leptina/metabolismo , Masculino , Potenciais da Membrana , Camundongos , Camundongos Transgênicos , Mutação de Sentido Incorreto , Obesidade/metabolismo , Fenótipo
10.
Science ; 364(6437): 283-285, 2019 Apr 19.
Artigo em Inglês | MEDLINE | ID: mdl-31000662

RESUMO

Malignancies arising from mutation of tumor suppressors have unexplained tissue proclivity. For example, BAP1 encodes a widely expressed deubiquitinase for histone H2A, but germline mutations are predominantly associated with uveal melanomas and mesotheliomas. We show that BAP1 inactivation causes apoptosis in mouse embryonic stem cells, fibroblasts, liver, and pancreatic tissue but not in melanocytes and mesothelial cells. Ubiquitin ligase RNF2, which silences genes by monoubiquitinating H2A, promoted apoptosis in BAP1-deficient cells by suppressing expression of the prosurvival genes Bcl2 and Mcl1. In contrast, BAP1 loss in melanocytes had little impact on expression of prosurvival genes, instead inducing Mitf Thus, BAP1 appears to modulate gene expression by countering H2A ubiquitination, but its loss only promotes tumorigenesis in cells that do not engage an RNF2-dependent apoptotic program.


Assuntos
Apoptose/genética , Carcinogênese/genética , Regulação Neoplásica da Expressão Gênica , Melanoma/genética , Complexo Repressor Polycomb 1/metabolismo , Proteínas Supressoras de Tumor/genética , Ubiquitina Tiolesterase/genética , Ubiquitina-Proteína Ligases/metabolismo , Neoplasias Uveais/genética , Animais , Técnicas de Introdução de Genes , Mutação em Linhagem Germinativa , Histonas , Humanos , Melanócitos/metabolismo , Melanócitos/patologia , Melanoma/patologia , Mesotelioma/genética , Mesotelioma/patologia , Camundongos , Camundongos Mutantes , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Ubiquitinação , Neoplasias Uveais/patologia
11.
Bioengineered ; 10(1): 98-107, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31023186

RESUMO

Progranulin has multiple functions in several physiological and pathological processes, including embryonic development, wound repair, tumorigenesis, inflammation and neurodegeneration. To investigate the transcriptional regulation of the PGRN gene, a luciferase knock-in reporter system was established in HEK293 cells by integrating luciferase gene in the genome controlled by the endogenous PGRN promoter using CRISPR/Cas9. PCR results demonstrated the site-specific integration of the exogenous luciferase gene into the genome. To validate the novel luciferase knock-in system, a CRISPR/Cas9 transcription activation/repression system for the PGRN gene was constructed and applied to the knock-in system. In addition, phorbol ester (phorbol 12-myristate, 13-acetate), previously reported as activating the expression of PGRN, was applied to the system. The results indicated that luciferase activity was directly correlated with the activity of the PGRN endogenous promoter. This novel system will be a useful tool for investigating the transcriptional regulation of PGRN, and it has great potential in screening the drugs targeting PGRN.


Assuntos
Proteína 9 Associada à CRISPR/genética , Sistemas CRISPR-Cas , Efeito Fundador , Técnicas de Introdução de Genes/métodos , Luciferases/genética , Progranulinas/genética , Sequência de Bases , Proteína 9 Associada à CRISPR/metabolismo , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Edição de Genes , Regulação da Expressão Gênica/efeitos dos fármacos , Genes Reporter , Genoma Humano , Células HEK293 , Humanos , Luciferases/metabolismo , Progranulinas/agonistas , Progranulinas/metabolismo , Regiões Promotoras Genéticas/efeitos dos fármacos , Transdução de Sinais , Acetato de Tetradecanoilforbol/farmacologia , Transcrição Genética/efeitos dos fármacos
12.
Nat Commun ; 10(1): 1476, 2019 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-30931940

RESUMO

Early detection and accurate monitoring of chronic kidney disease (CKD) could improve care and retard progression to end-stage renal disease. Here, using untargeted metabolomics in 2155 participants including patients with stage 1-5 CKD and healthy controls, we identify five metabolites, including 5-methoxytryptophan (5-MTP), whose levels strongly correlate with clinical markers of kidney disease. 5-MTP levels decrease with progression of CKD, and in mouse kidneys after unilateral ureteral obstruction (UUO). Treatment with 5-MTP ameliorates renal interstitial fibrosis, inhibits IκB/NF-κB signaling, and enhances Keap1/Nrf2 signaling in mice with UUO or ischemia/reperfusion injury, as well as in cultured human kidney cells. Overexpression of tryptophan hydroxylase-1 (TPH-1), an enzyme involved in 5-MTP synthesis, reduces renal injury by attenuating renal inflammation and fibrosis, whereas TPH-1 deficiency exacerbates renal injury and fibrosis by activating NF-κB and inhibiting Nrf2 pathways. Together, our results suggest that TPH-1 may serve as a target in the treatment of CKD.


Assuntos
Fibrose/metabolismo , Proteínas I-kappa B/metabolismo , NF-kappa B/metabolismo , Insuficiência Renal Crônica/metabolismo , Triptofano Hidroxilase/genética , Triptofano/análogos & derivados , Acetilcarnitina/metabolismo , Animais , Canavanina/análogos & derivados , Canavanina/metabolismo , Carnitina/análogos & derivados , Carnitina/metabolismo , Estudos de Casos e Controles , Modelos Animais de Doenças , Progressão da Doença , Técnicas de Introdução de Genes , Técnicas de Silenciamento de Genes , Humanos , Inflamação/metabolismo , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Rim/citologia , Rim/efeitos dos fármacos , Rim/patologia , Metabolômica , Camundongos , Fator 2 Relacionado a NF-E2/metabolismo , Índice de Gravidade de Doença , Transdução de Sinais , Taurina/metabolismo , Triptofano/metabolismo , Triptofano/farmacologia , Obstrução Ureteral
13.
Cell Mol Life Sci ; 76(18): 3657-3665, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-30976840

RESUMO

D190N, a missense mutation in rhodopsin, causes photoreceptor degeneration in patients with autosomal dominant retinitis pigmentosa (adRP). Two competing hypotheses have been developed to explain why D190N rod photoreceptors degenerate: (a) defective rhodopsin trafficking prevents proteins from correctly exiting the endoplasmic reticulum, leading to their accumulation, with deleterious effects or (b) elevated mutant rhodopsin expression and unabated signaling causes excitotoxicity. A knock-in D190N mouse model was engineered to delineate the mechanism of pathogenesis. Wild type (wt) and mutant rhodopsin appeared correctly localized in rod outer segments of D190N heterozygotes. Moreover, the rhodopsin glycosylation state in the mutants appeared similar to that in wt mice. Thus, it seems plausible that the injurious effect of the heterozygous mutation is not related to mistrafficking of the protein, but rather from constitutive rhodopsin activity and a greater propensity for chromophore isomerization even in the absence of light.


Assuntos
Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Retinite Pigmentosa/patologia , Rodopsina/genética , Sequência de Aminoácidos , Animais , Modelos Animais de Doenças , Eletrorretinografia , Técnicas de Introdução de Genes , Glicosilação , Camundongos , Camundongos Endogâmicos C57BL , Mutação de Sentido Incorreto , Estrutura Terciária de Proteína , Retina/metabolismo , Retina/patologia , Retinite Pigmentosa/metabolismo , Rodopsina/química , Rodopsina/metabolismo , Alinhamento de Sequência
14.
Int J Mol Sci ; 20(6)2019 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-30875922

RESUMO

Spinal bulbar muscular atrophy (SBMA) is a slowly progressive, androgen-dependent neuromuscular disease in men that is characterized by both muscle and synaptic dysfunction. Because gene expression in muscle is heterogeneous, with synaptic myonuclei expressing genes that regulate synaptic function and extrasynaptic myonuclei expressing genes to regulate contractile function, we used quantitative PCR to compare gene expression in these two domains of muscle from three different mouse models of SBMA: the "97Q" model that ubiquitously expresses mutant human androgen receptor (AR), the 113Q knock-in (KI) model that expresses humanized mouse AR with an expanded glutamine tract, and the "myogenic" model that overexpresses wild-type rat AR only in skeletal muscle. We were particularly interested in neurotrophic factors because of their role in maintaining neuromuscular function via effects on both muscle and synaptic function, and their implicated role in SBMA. We confirmed previous reports of the enriched expression of select genes (e.g., the acetylcholine receptor) in the synaptic region of muscle, and are the first to report the synaptic enrichment of others (e.g., glial cell line-derived neurotrophic factor). Interestingly, all three models displayed comparably dysregulated expression of most genes examined in both the synaptic and extrasynaptic domains of muscle, with only modest differences between regions and models. These findings of comprehensive gene dysregulation in muscle support the emerging view that skeletal muscle may be a prime therapeutic target for restoring function of both muscles and motoneurons in SBMA.


Assuntos
Fator Neurotrófico Derivado do Encéfalo/genética , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Músculo Esquelético/metabolismo , Atrofia Muscular Espinal/genética , Receptores Androgênicos/genética , Animais , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Técnicas de Introdução de Genes , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Atrofia Muscular Espinal/metabolismo , Ratos , Receptores Androgênicos/metabolismo
15.
Methods Mol Biol ; 1953: 105-119, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30912018

RESUMO

Small molecule-induced targeted protein degradation is a powerful approach for drug target validation given its selectivity, high kinetic resolution, dose dependency, and reversibility. Out of the several methods that have been reported so far, the 12 kDa degradation tag (dTAG) system has the advantage of hijacking a degradation machinery that is ubiquitously expressed in all human tissues. Therefore it is independent of expressing additional, exogenous factors and additionally permits target validation in vivo. Here, we describe the protocol for generation and validation of clones harboring knock-in of a selectable dTAG cassette at the endogenous locus of proteins of interest using the near-haploid cell line KBM7.


Assuntos
Descoberta de Drogas/métodos , Técnicas de Introdução de Genes/métodos , Proteólise/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia , Linhagem Celular , Clonagem Molecular/métodos , Edição de Genes/métodos , Vetores Genéticos/genética , Haploidia , Humanos , Plasmídeos/genética , Proteínas/genética , Proteínas/metabolismo
16.
Methods Mol Biol ; 1953: 213-230, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30912024

RESUMO

The use of CRISPR/Cas9 to modify the mouse genome has gained immense interest in the past few years since it allows the direct modification of embryos, bypassing the need of labor-intensive procedures for the manipulation of embryonic stem cells. By shortening the overall timelines and reducing the costs for the generation of new genetically modified mouse lines (Li et al., Nat Biotechnol 31: 681-683, 2013), this technology has rapidly become a major tool for in vivo drug discovery applications.


Assuntos
Sistemas CRISPR-Cas , Edição de Genes/métodos , Técnicas de Introdução de Genes/métodos , Técnicas de Inativação de Genes/métodos , Camundongos/genética , Alelos , Animais , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Feminino , Genoma , Técnicas de Genotipagem/métodos , Humanos , Masculino , Camundongos/embriologia , Camundongos Endogâmicos C57BL , RNA Guia/genética
17.
EBioMedicine ; 41: 649-658, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30819512

RESUMO

BACKGROUND: The most prevalent inherited form of generalized dystonia is caused by a mutation in torsinA (DYT1, ∆GAG) with incomplete penetrance. Rodent models with mutated torsinA do not develop dystonic symptoms, but previous ex vivo studies indicated abnormal excitation of cholinergic interneurons (ChI) and increased striatal acetylcholine. METHODS: We used in vivo optogenetics to exacerbate this endophenotype in order to determine its capacity to trigger dystonic symptoms in freely behaving mice. Tor1a+/Δgag DYT1 mice and wildtype littermates expressing channelrhodopsin2 under the Chat promotor were implanted bilaterally with optical LED cannulae and stimulated with blue light pulses of varied durations. FINDINGS: Six months old DYT1 KI mice but not wildtype controls responded with hyperactivity to blue light specifically at 25 ms pulse duration, 10 Hz frequency. Neuronal activity (c-Fos) in cholinergic interneurons was increased immediately after light stimulation and persisted only in DYT1 KI over 15 min. Substance P was increased specifically in striosome compartments in naïve DYT1 KI mice compared to wildtype. Under optogenetic stimulation substance P increased in wildtype to match levels in Dyt1 KI, and acetylcholinesterase was elevated in the striatum of stimulated DYT1 KI. No signs of dystonic movements were observed under stimulation of up to one hour in both genotypes and age groups, and the sensorimotor deficit previously observed in 6 months old DYT1 KI mice persisted under stimulation. INTERPRETATION: Overall this supports an endophenotype of dysregulated cholinergic activity in DYT1 dystonia, but depolarizing cholinergic interneurons was not sufficient to induce overt dystonia in DYT1 KI mice.


Assuntos
Neurônios Colinérgicos/metabolismo , Chaperonas Moleculares/genética , Optogenética , Acetilcolinesterase/genética , Acetilcolinesterase/metabolismo , Animais , Comportamento Animal/efeitos da radiação , Channelrhodopsins/metabolismo , Endofenótipos , Feminino , Técnicas de Introdução de Genes , Luz , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas Proto-Oncogênicas c-fos/metabolismo , Substância P/genética , Substância P/metabolismo
18.
Genesis ; 57(5): e23292, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30884088

RESUMO

Medullary thyroid carcinoma (MTC) develops from hyperplasia of thyroid C cells and represents one of the major causes of thyroid cancer mortality. Mutations in the cysteine-rich domain (CRD) of the RET gene are the most prevalent genetic cause of MTC. The current consensus holds that such cysteine mutations cause ligand-independent dimerization and constitutive activation of RET. However, given the number of the CRD mutations left uncharacterized, our understanding of the pathogenetic mechanisms by which CRD mutations lead to MTC remains incomplete. We report here that RET(C618F), a mutation identified in MTC patients, displays moderately high basal activity and requires the ligand for its full activation. To assess the biological significance of RET(C618F) in organogenesis, we generated a knock-in mouse line conditionally expressing RET(C618F) cDNA by the Ret promoter. The RET(C618F) allele can be made to be Ret-null and express mCherry by Cre-loxP recombination, which allows the assessment of the biological influence of RET(C618F) in vivo. Mice expressing RET(C618F) display mild C cell hyperplasia and increased numbers of enteric neurons, indicating that RET(C618F) confers gain-of-function phenotypes. This mouse line serves as a novel biological platform for investigating pathogenetic mechanisms involved in MTC and enteric hyperganglionosis.


Assuntos
Carcinoma Neuroendócrino/genética , Proteínas Proto-Oncogênicas c-ret/genética , Neoplasias da Glândula Tireoide/genética , Animais , Carcinoma Neuroendócrino/metabolismo , Linhagem Celular Tumoral , Sistema Nervoso Entérico/metabolismo , Sistema Nervoso Entérico/patologia , Técnicas de Introdução de Genes/métodos , Mutação em Linhagem Germinativa , Humanos , Hiperplasia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutação , Doenças do Sistema Nervoso/genética , Doenças do Sistema Nervoso/metabolismo , Doenças do Sistema Nervoso/patologia , Proteínas Proto-Oncogênicas c-ret/biossíntese , Proteínas Proto-Oncogênicas c-ret/metabolismo , Hiperplasia do Timo/genética , Hiperplasia do Timo/metabolismo , Glândula Tireoide/metabolismo , Glândula Tireoide/patologia , Neoplasias da Glândula Tireoide/metabolismo
19.
BMC Neurosci ; 20(1): 13, 2019 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-30894120

RESUMO

BACKGROUND: Knock-in (KI) mouse models of Alzheimer's disease (AD) that endogenously overproduce Aß without non-physiological overexpression of amyloid precursor protein (APP) provide important insights into the pathogenic mechanisms of AD. Previously, we reported that AppNL-G-F mice, which harbor three familial AD mutations (Swedish, Beyreuther/Iberian, and Arctic) exhibited emotional alterations before the onset of definitive cognitive deficits. To determine whether these mice exhibit deficits in learning and memory at more advanced ages, we compared the Morris water maze performance of AppNL-G-F and AppNL mice, which harbor only the Swedish mutation, with that of wild-type (WT) C57BL/6J mice at the age of 24 months. To correlate cognitive deficits and neuroinflammation, we also examined Aß plaque formation and reactive gliosis in these mice. RESULTS: In the Morris water maze, a spatial task, 24-month-old AppNL-G-F/NL-G-F mice exhibited significantly poorer spatial learning than WT mice during the hidden training sessions, but similarly to WT mice during the visible training sessions. Not surprisingly, AppNL-G-F/NL-G-F mice also exhibited spatial memory deficits both 1 and 7 days after the last training session. By contrast, 24-month-old AppNL/NL mice had intact spatial learning and memory relative to WT mice. Immunohistochemical analyses revealed that 24-month-old AppNL-G-F/NL-G-F mice developed massive Aß plaques and reactive gliosis (microgliosis and astrocytosis) throughout the brain, including the cortex and hippocampus. By contrast, we observed no detectable brain pathology in AppNL/NL mice despite overproduction of human Aß40 and Aß42 in their brains. CONCLUSIONS: Aß plaque formation, followed by sustained neuroinflammation, is necessary for the induction of definitive cognitive deficits in App-KI mouse models of AD. Our data also indicate that introduction of the Swedish mutation alone in endogenous APP is not sufficient to produce either AD-related brain pathology or cognitive deficits in mice.


Assuntos
Doença de Alzheimer/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Disfunção Cognitiva/etiologia , Disfunção Cognitiva/metabolismo , Gliose/metabolismo , Placa Amiloide/metabolismo , Doença de Alzheimer/patologia , Doença de Alzheimer/psicologia , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/genética , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Disfunção Cognitiva/patologia , Modelos Animais de Doenças , Técnicas de Introdução de Genes , Gliose/patologia , Gliose/psicologia , Humanos , Inflamação/metabolismo , Inflamação/patologia , Inflamação/psicologia , Masculino , Aprendizagem em Labirinto/fisiologia , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fragmentos de Peptídeos/metabolismo , Placa Amiloide/patologia , Placa Amiloide/psicologia , Memória Espacial/fisiologia
20.
Invest Ophthalmol Vis Sci ; 60(4): 858-867, 2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-30821811

RESUMO

Purpose: Investigate the effects of the absence of 17 amino acids at the C-terminal end of Aquaporin 0 (AQP0) on lens transparency, focusing property, and homeostasis. Methods: A knockin (KI) mouse model (AQP0ΔC/ΔC) was developed to express AQP0 only as the end-cleaved form in the lens. For this, AQP0 was genetically engineered as C-terminally end-cleaved with amino acids 1 to 246, instead of the full length 1 to 263 of the wild type (WT). After verifying the KI integration into the genome and its expression, the mouse model was bred for several generations. AQP0 KI homozygous (AQP0ΔC/ΔC) and heterozygous (AQP0+/ΔC) lenses were imaged and analyzed at different developmental stages for transparency. Correspondingly, aberrations in the lens were characterized using the standard metal grid focusing method. Data were compared with age-matched WT, AQP0 knockout (AQP0-/-), and AQP0 heterozygous (AQP0+/-) lenses. Results: AQP0ΔC/ΔC lenses were transparent throughout the embryonic development and until postnatal day 15 (P15) in contrast to age-matched AQP0-/- lenses, which developed cataract at embryonic stage itself. However, there was distortion aberration in AQP0ΔC/ΔC lens at P5; after P15, cataract began to develop and progressed faster surpassing that of age-matched AQP0-/- lenses. AQP0+/ΔC lenses were transparent even at the age of 1 year in contrast to AQP0+/- lenses; however, there was distortion aberration starting at P15. Conclusions: A specific distribution profile of intact and end-cleaved AQP0 from the outer cortex to the inner nucleus is required in the lens for establishing refractive index gradient to enable proper focusing without aberrations and for maintaining transparency.


Assuntos
Sequência de Aminoácidos/genética , Aquaporinas/genética , Catarata/genética , Proteínas do Olho/genética , Cristalino/patologia , Erros de Refração/genética , Deleção de Sequência/genética , Animais , Western Blotting , Catarata/embriologia , Catarata/fisiopatologia , Células Cultivadas , Modelos Animais de Doenças , Técnicas de Introdução de Genes , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos C57BL , Erros de Refração/embriologia , Erros de Refração/fisiopatologia , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Transfecção
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