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1.
Nat Commun ; 10(1): 2905, 2019 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-31266953

RESUMO

Delivery into mammalian cells remains a significant challenge for many applications of proteins as research tools and therapeutics. We recently reported that the fusion of cargo proteins to a supernegatively charged (-30)GFP enhances encapsulation by cationic lipids and delivery into mammalian cells. To discover polyanionic proteins with optimal delivery properties, we evaluate negatively charged natural human proteins for their ability to deliver proteins into cultured mammalian cells and human primary fibroblasts. Here we discover that ProTα, a small, widely expressed, intrinsically disordered human protein, enables up to ~10-fold more efficient cationic lipid-mediated protein delivery compared to (-30)GFP. ProTα enables efficient delivery at low- to mid-nM concentrations of two unrelated genome editing proteins, Cre recombinase and zinc-finger nucleases, under conditions in which (-30)GFP fusion or cationic lipid alone does not result in substantial activity. ProTα may enable mammalian cell protein delivery applications when delivery potency is limiting.


Assuntos
Edição de Genes/métodos , Lipossomos/química , Proteínas/química , Edição de Genes/instrumentação , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Integrases/química , Integrases/genética , Integrases/metabolismo , Lipossomos/metabolismo , Transporte Proteico , Proteínas/genética , Proteínas/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Nucleases de Dedos de Zinco/química , Nucleases de Dedos de Zinco/genética , Nucleases de Dedos de Zinco/metabolismo
2.
Nature ; 571(7764): 219-225, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31189177

RESUMO

Conventional CRISPR-Cas systems maintain genomic integrity by leveraging guide RNAs for the nuclease-dependent degradation of mobile genetic elements, including plasmids and viruses. Here we describe a notable inversion of this paradigm, in which bacterial Tn7-like transposons have co-opted nuclease-deficient CRISPR-Cas systems to catalyse RNA-guided integration of mobile genetic elements into the genome. Programmable transposition of Vibrio cholerae Tn6677 in Escherichia coli requires CRISPR- and transposon-associated molecular machineries, including a co-complex between the DNA-targeting complex Cascade and the transposition protein TniQ. Integration of donor DNA occurs in one of two possible orientations at a fixed distance downstream of target DNA sequences, and can accommodate variable length genetic payloads. Deep-sequencing experiments reveal highly specific, genome-wide DNA insertion across dozens of unique target sites. This discovery of a fully programmable, RNA-guided integrase lays the foundation for genomic manipulations that obviate the requirements for double-strand breaks and homology-directed repair.


Assuntos
Sistemas CRISPR-Cas/genética , Elementos de DNA Transponíveis/genética , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Edição de Genes/métodos , Mutagênese Insercional/métodos , RNA Bacteriano/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Proteínas Associadas a CRISPR/genética , Proteínas Associadas a CRISPR/metabolismo , Endodesoxirribonucleases/genética , Endodesoxirribonucleases/metabolismo , Escherichia coli/genética , Genoma Bacteriano/genética , Integrases/genética , Integrases/metabolismo , Mutagênese Sítio-Dirigida/métodos , RNA Guia/genética , Especificidade por Substrato , Vibrio cholerae/genética
3.
Nat Commun ; 10(1): 2870, 2019 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-31253773

RESUMO

An important channel of cell-to-cell communication is direct contact. The immune synapse is a paradigmatic example of such type of interaction: it forms upon engagement of antigen receptors in lymphocytes by antigen-presenting cells and allows the local exchange of molecules and information. Although mechanics has been shown to play an important role in this process, how forces organize and impact on synapse function is unknown. We find that mechanical forces are spatio-temporally patterned at the immune synapse: global pulsatile myosin II-driven tangential forces are observed at the synapse periphery while localised forces generated by invadosome-like F-actin protrusions are detected at its centre. Noticeably, we observe that these force-producing actin protrusions constitute the main site of antigen extraction and endocytosis and require myosin II contractility to form. The interplay between global and local forces dictated by the organization of the actomyosin cytoskeleton therefore controls endocytosis at the immune synapse.


Assuntos
Citoesqueleto de Actina/fisiologia , Actomiosina/metabolismo , Linfócitos B/fisiologia , Endocitose/fisiologia , Miosina Tipo II/metabolismo , Actomiosina/genética , Animais , Comunicação Celular , Cruzamentos Genéticos , Integrases/genética , Integrases/metabolismo , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Miosina Tipo II/genética , Receptores de Complemento 3d
4.
Methods Mol Biol ; 1947: 361-376, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30969428

RESUMO

Engineered G protein-coupled receptors (DREADDs, designer receptors exclusively activated by designer drugs) are convenient tools for specific activation of GPCR signaling in many cell types. DREADDs have been utilized as research tools to study numerous cellular and physiologic processes, including regulation of neuronal activity, behavior, and metabolism. Mice with random insertion transgenes and adeno-associated viruses have been widely used to express DREADDs in individual cell types. We recently created and characterized ROSA26-GsDREADD knock-in mice to allow Cre recombinase-dependent expression of a Gαs-coupled DREADD (GsD) fused to GFP in distinct cell populations in vivo. These animals also harbor a CREB-activated luciferase reporter gene for analysis of CREB activity by in vivo imaging, ex vivo imaging, or biochemical reporter assays. In this chapter, we provide detailed methods for breeding GsD animals, inducing GsD expression, stimulating GsD activity, and measuring basal and stimulated CREB reporter bioluminescence in tissues in vivo, ex vivo, and in vitro. These animals are available from our laboratory for non-profit research.


Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Genes Reporter , Processamento de Imagem Assistida por Computador/métodos , Medições Luminescentes/métodos , Moduladores de Transporte de Membrana/farmacologia , Receptores Acoplados a Proteínas-G/metabolismo , Animais , Clozapina/análogos & derivados , Clozapina/farmacologia , Integrases/metabolismo , Camundongos , Especificidade de Órgãos , Receptores Acoplados a Proteínas-G/efeitos dos fármacos , Receptores Acoplados a Proteínas-G/genética , Transdução de Sinais
5.
Invest Ophthalmol Vis Sci ; 60(5): 1776-1788, 2019 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-31022732

RESUMO

Purpose: To determine the effects of αvß3 integrin expression and activation on intraocular pressure (IOP). Methods: Cre+/-ß3flox/flox mice were treated with topical tamoxifen eye drops for 5 days to activate Cre and excise the ß3 integrin gene from the anterior segment. IOP was measured weekly for 11 weeks using rebound tonometry. Mice were then killed and changes in expression of the ß3 integrin subunit in Cre+/- ß3flox/flox mice were determined using Western blotting analysis and immunofluorescence microscopy. To determine the effect of αvß3 integrin activation on outflow facility, porcine organ culture anterior segments (POCAS) were perfused with the αvß3 integrin-activating antibody AP5 or an isotype IgG control for 21 hours. The effect of αvß3 integrin activation on IOP was measured over 7 days in C57BL/6J mice intracamerally infused with AP5, AP3, IgG, or PBS. Results: Deletion of the ß3 integrin subunit using the tamoxifen-inducible Cre-loxP system resulted in a decrease in expression of the ß3 integrin subunit in the trabecular meshwork and ciliary muscle. Morphologically no gross changes in the anterior segment were detected. Deletion of the ß3 integrin subunit resulted in a significantly (P < 0.05) lower IOP in mice within 2 weeks following the tamoxifen treatment and persisted for 11 weeks. Activating the αvß3 integrin with the AP5 antibody resulted in a significant (P < 0.05) increase in IOP in C57BL/6J mice and a decrease in outflow facility in 42% of the POCAS. Conclusions: These studies demonstrate a role for αvß3 integrin signaling in the regulation of IOP.


Assuntos
Segmento Anterior do Olho/metabolismo , Regulação da Expressão Gênica/fisiologia , Integrina alfaVbeta3/genética , Pressão Intraocular/fisiologia , Idoso de 80 Anos ou mais , Animais , Segmento Anterior do Olho/efeitos dos fármacos , Western Blotting , Feminino , Humanos , Integrases/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência , Técnicas de Cultura de Órgãos , Reação em Cadeia da Polimerase em Tempo Real , Suínos , Tamoxifeno/farmacologia , Tonometria Ocular
6.
Int J Mol Sci ; 20(7)2019 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-30959855

RESUMO

Cathepsin D is one of the major lysosomal aspartic proteases that is essential for the normal functioning of the autophagy-lysosomal system. In the kidney, cathepsin D is enriched in renal proximal tubular epithelial cells, and its levels increase during acute kidney injury. To investigate how cathepsin D-deficiency impacts renal proximal tubular cells, we employed a conditional knockout CtsDflox/-; Spink3Cre mouse. Immunohistochemical analyses using anti-cathepsin D antibody revealed that cathepsin D was significantly decreased in tubular epithelial cells of the cortico-medullary region, mainly in renal proximal tubular cells of this mouse. Cathepsin D-deficient renal proximal tubular cells showed an increase of microtubule-associated protein light chain 3 (LC3; a marker for autophagosome/autolysosome)-signals and an accumulation of abnormal autophagic structures. Renal ischemia/reperfusion injury resulted in an increase of early kidney injury marker, Kidney injury molecule 1 (Kim-1), in the cathepsin D-deficient renal tubular epithelial cells of the CtsDflox/-; Spink3Cre mouse. Inflammation marker was also increased in the cortico-medullary region of the CtsDflox/-; Spink3Cre mouse. Our results indicated that lack of cathepsin D in the renal tubular epithelial cells led to an increase of sensitivity against ischemia/reperfusion injury.


Assuntos
Catepsina D/deficiência , Túbulos Renais Proximais/enzimologia , Túbulos Renais Proximais/patologia , Traumatismo por Reperfusão/enzimologia , Traumatismo por Reperfusão/patologia , Animais , Autofagia , Catepsina D/metabolismo , Células Epiteliais/metabolismo , Células Epiteliais/ultraestrutura , Integrases/metabolismo , Camundongos
7.
Nat Commun ; 10(1): 1937, 2019 04 26.
Artigo em Inglês | MEDLINE | ID: mdl-31028261

RESUMO

The development of site-specific recombinases (SSRs) as genome editing agents is limited by the difficulty of altering their native DNA specificities. Here we describe Rec-seq, a method for revealing the DNA specificity determinants and potential off-target substrates of SSRs in a comprehensive and unbiased manner. We applied Rec-seq to characterize the DNA specificity determinants of several natural and evolved SSRs including Cre, evolved variants of Cre, and other SSR family members. Rec-seq profiling of these enzymes and mutants thereof revealed previously uncharacterized SSR interactions, including specificity determinants not evident from SSR:DNA structures. Finally, we used Rec-seq specificity profiles to predict off-target substrates of Tre and Brec1 recombinases, including endogenous human genomic sequences, and confirmed their ability to recombine these off-target sequences in human cells. These findings establish Rec-seq as a high-resolution method for rapidly characterizing the DNA specificity of recombinases with single-nucleotide resolution, and for informing their further development.


Assuntos
DNA Nucleotidiltransferases/genética , DNA/genética , Edição de Genes/métodos , Genoma Humano , Integrases/genética , Sequência de Bases , Clonagem Molecular , DNA/metabolismo , DNA Nucleotidiltransferases/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Células HEK293 , Humanos , Integrases/metabolismo , Oligodesoxirribonucleotídeos/síntese química , Oligodesoxirribonucleotídeos/genética , Oligodesoxirribonucleotídeos/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Recombinação Genética
8.
Methods Mol Biol ; 1936: 249-274, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30820904

RESUMO

Cell-type-specific gene targeting with the Cre/loxP system has become an indispensable technique in experimental neuroscience, particularly for the study of late-born glial cells that make myelin. A plethora of conditional mutants and Cre-expressing mouse lines is now available to the research community that allows laboratories to readily engage in in vivo analyses of oligodendrocytes and their precursor cells. This chapter summarizes concepts and strategies in targeting myelinating glial cells in mice for mutagenesis or imaging, and provides an overview of the most important Cre driver lines successfully used in this rapidly growing field.


Assuntos
Marcação de Genes/métodos , Integrases/metabolismo , Oligodendroglia/citologia , Tamoxifeno/farmacologia , Animais , Linhagem da Célula , Regulação da Expressão Gênica/efeitos dos fármacos , Integrases/genética , Camundongos , Camundongos Transgênicos , Mutagênese , Especificidade de Órgãos , Transgenes
9.
Molecules ; 24(6)2019 Mar 26.
Artigo em Inglês | MEDLINE | ID: mdl-30917606

RESUMO

The genetic modification of the mouse genome using the cre-lox system has been an invaluable tool in deciphering gene and protein function in a temporal and/or spatial manner. However, it has its pitfalls, as researchers have shown that the unregulated expression of cre recombinase can cause DNA damage, the consequences of which can be very detrimental to mouse health. Previously published literature on the most utilized cardiac-specific cre, αMHC-cre, mouse model exhibited a nonlethal hypertrophic cardiomyopathy (HCM) with aging. However, using the same αMHC-cre mice, we observed a cardiac pathology, resulting in complete lethality by 11 months of age. Echocardiography and histology revealed that the αMHC-cre mice were displaying symptoms of dilated cardiomyopathy (DCM) by seven months of age, which ultimately led to their demise in the absence of any HCM at any age. Molecular analysis showed that this phenotype was associated with the DNA damage response through the downregulation of activated p38 and increased expression of JNK, p53, and Bax, known inducers of myocyte death resulting in fibrosis. Our data urges strong caution when interpreting the phenotypic impact of gene responses using αMHC-cre mice, since a lethal DCM was induced by the cre driver in an age-dependent manner in this commonly utilized model system.


Assuntos
Envelhecimento/genética , Cardiomiopatia Dilatada/diagnóstico por imagem , Integrases/metabolismo , Cadeias Pesadas de Miosina/genética , Envelhecimento/metabolismo , Animais , Cardiomiopatia Dilatada/genética , Cardiomiopatia Dilatada/metabolismo , Dano ao DNA , Modelos Animais de Doenças , Ecocardiografia , Regulação da Expressão Gênica , Genes Letais , Integrases/genética , Camundongos , Cadeias Pesadas de Miosina/metabolismo , Fenótipo
10.
Cell Mol Gastroenterol Hepatol ; 7(3): 533-554, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30827941

RESUMO

BACKGROUND & AIMS: Loss of leucine-rich repeat-containing G-protein-coupled receptor 5-positive crypt base columnar cells provides permissive conditions for different facultative stem cell populations to dedifferentiate and repopulate the stem cell compartment. In this study, we used a defensin α4-Cre recombinase (Defa4Cre) line to define the potential of Paneth cells to dedifferentiate and contribute to intestinal stem cell (ISC) maintenance during normal homeostasis and after intestinal injury. METHODS: Small intestine and enteroids from Defa4Cre;Rosa26 tandem dimer Tomato (tdTomato), a red fluoresent protein, (or Rosa26 Enhanced Yellow Fluorescent Protein (EYFP)) reporter, Notch gain-of-function (Defa4Cre;Rosa26 Notch Intracellular Domain (NICD)-ires-nuclear Green Fluorescent Protein (nGFP) and Defa4Cre;Rosa26reverse tetracycline transactivator-ires Enhanced Green Fluorescent Protein (EGFP);TetONICD), A Disintegrin and Metalloproteinase domain-containing protein 10 (ADAM10) loss-of-function (Defa4Cre;ADAM10flox/flox), and Adenomatous polyposis coli (APC) inactivation (Defa4Cre;APCflox/flox) mice were analyzed. Doxorubicin treatment was used as an acute intestinal injury model. Lineage tracing, proliferation, and differentiation were assessed in vitro and in vivo. RESULTS: Defa4Cre-expressing cells are fated to become mature Paneth cells and do not contribute to ISC maintenance during normal homeostasis in vivo. However, spontaneous lineage tracing was observed in enteroids, and fluorescent-activated cell sorter-sorted Defa4Cre-marked cells showed clonogenic enteroid growth. Notch activation in Defa4Cre-expressing cells caused dedifferentiation to multipotent ISCs in vivo and was required for adenoma formation. ADAM10 deletion had no significant effect on crypt homeostasis. However, after acute doxorubicin-induced injury, Defa4Cre-expressing cells contributed to regeneration in an ADAM10-Notch-dependent manner. CONCLUSIONS: Our studies have shown that Defa4Cre-expressing Paneth cells possess cellular plasticity, can dedifferentiate into multipotent stem cells upon Notch activation, and can contribute to intestinal regeneration in an acute injury model.


Assuntos
Plasticidade Celular , Integrases/metabolismo , Intestinos/lesões , Intestinos/patologia , Celulas de Paneth/metabolismo , Receptores Notch/metabolismo , alfa-Defensinas/metabolismo , Proteína ADAM10/metabolismo , Adenoma/patologia , Proteína da Polipose Adenomatosa do Colo/metabolismo , Alelos , Animais , Desdiferenciação Celular , Linhagem da Célula , Células Clonais , Doxorrubicina , Deleção de Genes , Homeostase , Hiperplasia , Camundongos , Mitose , Células-Tronco Multipotentes/metabolismo , Organoides/crescimento & desenvolvimento , Organoides/patologia , Regeneração
11.
Nat Commun ; 10(1): 1444, 2019 03 29.
Artigo em Inglês | MEDLINE | ID: mdl-30926899

RESUMO

The phosphatase Shp-2 was implicated in NK cell development and functions due to its interaction with NK inhibitory receptors, but its exact role in NK cells is still unclear. Here we show, using mice conditionally deficient for Shp-2 in the NK lineage, that NK cell development and responsiveness are largely unaffected. Instead, we find that Shp-2 serves mainly to enforce NK cell responses to activation by IL-15 and IL-2. Shp-2-deficient NK cells have reduced proliferation and survival when treated with high dose IL-15 or IL-2. Mechanistically, Shp-2 deficiency hampers acute IL-15 stimulation-induced raise in glycolytic and respiration rates, and causes a dramatic defect in ERK activation. Moreover, inhibition of the ERK and mTOR cascades largely phenocopies the defect observed in the absence of Shp-2. Together, our data reveal a critical function of Shp-2 as a molecular nexus bridging acute IL-15 signaling with downstream metabolic burst and NK cell expansion.


Assuntos
MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Células Matadoras Naturais/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismo , Receptores de Interleucina-15/metabolismo , Animais , Antígenos Ly/metabolismo , Contagem de Células , Proliferação de Células/efeitos dos fármacos , Tamanho Celular/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Integrases/metabolismo , Interleucina-15/farmacologia , Células Matadoras Naturais/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Muromegalovirus/fisiologia , Receptor 1 Desencadeador da Citotoxicidade Natural/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 11/deficiência , Serina-Treonina Quinases TOR/metabolismo
12.
Mol Cell ; 73(4): 727-737.e3, 2019 02 21.
Artigo em Inglês | MEDLINE | ID: mdl-30709710

RESUMO

CRISPR-Cas immunity requires integration of short, foreign DNA fragments into the host genome at the CRISPR locus, a site consisting of alternating repeat sequences and foreign-derived spacers. In most CRISPR systems, the proteins Cas1 and Cas2 form the integration complex and are both essential for DNA acquisition. Most type V-C and V-D systems lack the cas2 gene and have unusually short CRISPR repeats and spacers. Here, we show that a mini-integrase comprising the type V-C Cas1 protein alone catalyzes DNA integration with a preference for short (17- to 19-base-pair) DNA fragments. The mini-integrase has weak specificity for the CRISPR array. We present evidence that the Cas1 proteins form a tetramer for integration. Our findings support a model of a minimal integrase with an internal ruler mechanism that favors shorter repeats and spacers. This minimal integrase may represent the function of the ancestral Cas1 prior to Cas2 adoption.


Assuntos
Proteínas Associadas a CRISPR/genética , Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , DNA Bacteriano/genética , Endodesoxirribonucleases/genética , Endonucleases/genética , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Edição de Genes/métodos , Integrases/genética , Pareamento de Bases , Proteínas Associadas a CRISPR/metabolismo , DNA Bacteriano/metabolismo , Endodesoxirribonucleases/metabolismo , Endonucleases/metabolismo , Escherichia coli/enzimologia , Proteínas de Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Integrases/metabolismo , Motivos de Nucleotídeos , Especificidade por Substrato
13.
Dis Model Mech ; 12(2)2019 02 12.
Artigo em Inglês | MEDLINE | ID: mdl-30760495

RESUMO

Lung cancer is the leading cause of cancer-related death. Two-thirds of cases are diagnosed at an advanced stage that is refractory to curative treatment. Therefore, strategies for the early detection of lung cancer are urgently sought. Total circulating free DNA (cfDNA) and tumour-derived circulating tumour DNA (ctDNA) are emerging as important biomarkers within a 'liquid biopsy' for monitoring human disease progression and response to therapy. Owing to the late clinical diagnosis of lung adenocarcinoma, the potential for cfDNA and ctDNA as early detection biomarkers remains unexplored. Here, using a Cre-regulated genetically engineered mouse model of lung adenocarcinoma development, driven by KrasG12D (the KrasLSL-G12D mouse), we serially tracked the release of cfDNA/ctDNA and compared this with tumour burden as determined by micro-computed tomography (CT). To monitor ctDNA, a droplet digital PCR assay was developed to permit discrimination of the KrasLox-G12D allele from the KrasLSL-G12D and KrasWT alleles. We show that micro-CT correlates with endpoint histology and is able to detect pre-malignant tumours with a combined volume larger than 7 mm3 Changes in cfDNA/ctDNA levels correlate with micro-CT measurements in longitudinal sampling and are able to monitor the emergence of lesions before the adenoma-adenocarcinoma transition. Potentially, this work has implications for the early detection of human lung adenocarcinoma using ctDNA/cfDNA profiling.A video abstract for this article is available at https://youtu.be/Ku8xJJyGs3UThis article has an associated First Person interview with the joint first authors of the paper.


Assuntos
Ácidos Nucleicos Livres/sangue , Detecção Precoce de Câncer , Neoplasias Pulmonares/sangue , Neoplasias Pulmonares/diagnóstico , Mutação/genética , Lesões Pré-Cancerosas/sangue , Lesões Pré-Cancerosas/diagnóstico , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Alelos , Animais , Modelos Animais de Doenças , Feminino , Integrases/metabolismo , Neoplasias Pulmonares/diagnóstico por imagem , Neoplasias Pulmonares/patologia , Masculino , Camundongos Endogâmicos C57BL , Lesões Pré-Cancerosas/diagnóstico por imagem , Lesões Pré-Cancerosas/patologia , Proteínas Proto-Oncogênicas p21(ras)/sangue , Recombinação Genética/genética , Reprodutibilidade dos Testes , Carga Tumoral , Microtomografia por Raio-X
14.
Methods Mol Biol ; 1926: 3-21, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30742258

RESUMO

For studies of gene function during development, it can be very useful to generate mosaic embryos in which a small subset of cells in a given cell lineage lacks a gene of interest and carries a marker that allows the mutant cells to be specifically visualized and compared to wild-type cells. Several methods have been used to generate genetically mosaic mouse kidneys for such studies. These include (1) chimeric embryos generated using embryonic stem cells, (2) chimeric renal organoids generated by dissociation and reaggregation of the fetal kidneys, (3) generation of a knockout allele with a built-in reporter gene, (4) mosaic analysis with double markers (MADM), and (5) mosaic mutant analysis with spatial and temporal control of recombination (MASTR). In this chapter, these five methods are described, and their advantages and disadvantages are discussed.


Assuntos
Rim/citologia , Rim/embriologia , Organoides/citologia , Animais , DNA Nucleotidiltransferases/genética , DNA Nucleotidiltransferases/metabolismo , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Integrases/genética , Integrases/metabolismo , Rim/metabolismo , Camundongos , Camundongos Knockout , Organoides/metabolismo
15.
Genetics ; 211(4): 1155-1177, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30765420

RESUMO

To understand gene function, the cre/loxP conditional system is the most powerful available for temporal and spatial control of expression in mouse. However, the research community requires more cre recombinase expressing transgenic mouse strains (cre-drivers) that restrict expression to specific cell types. To address these problems, a high-throughput method for large-scale production that produces high-quality results is necessary. Further, endogenous promoters need to be chosen that drive cell type specific expression, or we need to further focus the expression by manipulating the promoter. Here we test the suitability of using knock-ins at the docking site 5' of Hprt for rapid development of numerous cre-driver strains focused on expression in adulthood, using an improved cre tamoxifen inducible allele (icre/ERT2), and testing a novel inducible-first, constitutive-ready allele (icre/f3/ERT2/f3). In addition, we test two types of promoters either to capture an endogenous expression pattern (MaxiPromoters), or to restrict expression further using minimal promoter element(s) designed for expression in restricted cell types (MiniPromoters). We provide new cre-driver mouse strains with applicability for brain and eye research. In addition, we demonstrate the feasibility and applicability of using the locus 5' of Hprt for the rapid generation of substantial numbers of cre-driver strains. We also provide a new inducible-first constitutive-ready allele to further speed cre-driver generation. Finally, all these strains are available to the research community through The Jackson Laboratory.


Assuntos
Encéfalo/metabolismo , Olho/metabolismo , Técnicas de Introdução de Genes/métodos , Camundongos Transgênicos/genética , Tamoxifeno/farmacologia , Ativação Transcricional/efeitos dos fármacos , Animais , Efeito Fundador , Hipoxantina Fosforribosiltransferase/genética , Hipoxantina Fosforribosiltransferase/metabolismo , Integrases/genética , Integrases/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Regiões Promotoras Genéticas
16.
Brain ; 142(2): 362-375, 2019 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-30601941

RESUMO

De novo mutations of the sodium channel gene SCN8A result in an epileptic encephalopathy with refractory seizures, developmental delay, and elevated risk of sudden death. p.Arg1872Trp is a recurrent de novo SCN8A mutation reported in 14 unrelated individuals with epileptic encephalopathy that included seizure onset in the prenatal or infantile period and severe verbal and ambulatory comorbidities. The major biophysical effect of the mutation was previously shown to be impaired channel inactivation accompanied by increased current density. We have generated a conditional mouse mutation in which expression of this severe gain-of-function mutation is dependent upon Cre recombinase. Global activation of p.Arg1872Trp by EIIa-Cre resulted in convulsive seizures and lethality at 2 weeks of age. Neural activation of the p.Arg1872Trp mutation by Nestin-Cre also resulted in early onset seizures and death. Restriction of p.Arg1872Trp expression to excitatory neurons using Emx1-Cre recapitulated seizures and juvenile lethality between 1 and 2 months of age. In contrast, activation of p.Arg1872Trp in inhibitory neurons by Gad2-Cre or Dlx5/6-Cre did not induce seizures or overt neurological dysfunction. The sodium channel modulator GS967/Prax330 prolonged survival of mice with global expression of R1872W and also modulated the activity of the mutant channel in transfected cells. Activation of the p.Arg1872Trp mutation in adult mice was sufficient to generate seizures and death, indicating that successful therapy will require lifelong treatment. These findings provide insight into the pathogenic mechanism of this gain-of-function mutation of SCN8A and identify excitatory neurons as critical targets for therapeutic intervention.


Assuntos
Encefalopatias/genética , Potenciais Pós-Sinápticos Excitadores/fisiologia , Integrases/genética , Canal de Sódio Disparado por Voltagem NAV1.6/genética , Neurônios/fisiologia , Prosencéfalo/fisiologia , Animais , Encefalopatias/patologia , Células Cultivadas , Feminino , Mutação com Ganho de Função/genética , Integrases/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neurônios/patologia , Técnicas de Cultura de Órgãos , Prosencéfalo/patologia
17.
Nat Neurosci ; 22(3): 460-469, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30692687

RESUMO

Memories of fearful events can last a lifetime. The prelimbic (PL) cortex, a subregion of prefrontal cortex, plays a critical role in fear memory retrieval over time. Most studies have focused on acquisition, consolidation, and retrieval of recent memories, but much less is known about the neural mechanisms of remote memory. Using a new knock-in mouse for activity-dependent genetic labeling (TRAP2), we demonstrate that neuronal ensembles in the PL cortex are dynamic. PL neurons TRAPed during later memory retrievals are more likely to be reactivated and make larger behavioral contributions to remote memory retrieval compared to those TRAPed during learning or early memory retrieval. PL activity during learning is required to initiate this time-dependent reorganization in PL ensembles underlying memory retrieval. Finally, while neurons TRAPed during earlier and later retrievals have similar broad projections throughout the brain, PL neurons TRAPed later have a stronger functional recruitment of cortical targets.


Assuntos
Córtex Cerebral/fisiologia , Memória de Longo Prazo/fisiologia , Rememoração Mental/fisiologia , Neurônios/fisiologia , Córtex Pré-Frontal/fisiologia , Animais , Condicionamento Clássico , Medo , Integrases/metabolismo , Aprendizagem/fisiologia , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Tamoxifeno/administração & dosagem
18.
Nature ; 566(7742): 105-109, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30675057

RESUMO

A gene drive biases the transmission of one of the two copies of a gene such that it is inherited more frequently than by random segregation. Highly efficient gene drive systems have recently been developed in insects, which leverage the sequence-targeted DNA cleavage activity of CRISPR-Cas9 and endogenous homology-directed repair mechanisms to convert heterozygous genotypes to homozygosity1-4. If implemented in laboratory rodents, similar systems would enable the rapid assembly of currently impractical genotypes that involve multiple homozygous genes (for example, to model multigenic human diseases). To our knowledge, however, such a system has not yet been demonstrated in mammals. Here we use an active genetic element that encodes a guide RNA, which is embedded in the mouse tyrosinase (Tyr) gene, to evaluate whether targeted gene conversion can occur when CRISPR-Cas9 is active in the early embryo or in the developing germline. Although Cas9 efficiently induces double-stranded DNA breaks in the early embryo and male germline, these breaks are not corrected by homology-directed repair. By contrast, Cas9 expression limited to the female germline induces double-stranded breaks that are corrected by homology-directed repair, which copies the active genetic element from the donor to the receiver chromosome and increases its rate of inheritance in the next generation. These results demonstrate the feasibility of CRISPR-Cas9-mediated systems that bias inheritance of desired alleles in mice and that have the potential to transform the use of rodent models in basic and biomedical research.


Assuntos
Proteína 9 Associada à CRISPR/metabolismo , Sistemas CRISPR-Cas/genética , Conversão Gênica , Tecnologia de Impulso Genético/métodos , Mutação em Linhagem Germinativa/genética , Heterozigoto , Homozigoto , Alelos , Animais , Cruzamento , Proteína 9 Associada à CRISPR/genética , Cromossomos de Mamíferos/genética , Quebras de DNA de Cadeia Dupla , Modelos Animais de Doenças , Embrião de Mamíferos/enzimologia , Embrião de Mamíferos/metabolismo , Feminino , Integrases/genética , Integrases/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Monofenol Mono-Oxigenase/genética , RNA Guia/genética , Transgenes/genética
19.
Nat Protoc ; 14(2): 556-575, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30610240

RESUMO

A big challenge in proteomics is the identification of cell-type-specific proteomes in vivo. This protocol describes how to label, purify and identify cell-type-specific proteomes in living mice. To make this possible, we created a Cre-recombinase-inducible mouse line expressing a mutant methionyl-tRNA synthetase (L274G), which enables the labeling of nascent proteins with the non-canonical amino acid azidonorleucine (ANL). This amino acid can be conjugated to different affinity tags by click chemistry. After affinity purification (AP), the labeled proteins can be identified by tandem mass spectrometry (MS/MS). With this method, it is possible to identify cell-type-specific proteomes derived from living animals, which was not possible with any previously published method. The reduction in sample complexity achieved by this protocol allows for the detection of subtle changes in cell-type-specific protein content in response to environmental changes. This protocol can be completed in ~10 d (plus the time needed to generate the mouse lines, the desired labeling period and MS analysis).


Assuntos
Azidas/metabolismo , Química Click/métodos , Metionina tRNA Ligase/genética , Norleucina/análogos & derivados , Proteoma/isolamento & purificação , Proteômica/métodos , Coloração e Rotulagem/métodos , Animais , Expressão Gênica , Integrases/genética , Integrases/metabolismo , Metionina tRNA Ligase/metabolismo , Camundongos , Camundongos Transgênicos , Mutação , Norleucina/metabolismo , Especificidade de Órgãos , Proteoma/biossíntese , Proteoma/genética , Purificação por Afinidade em Tandem/métodos , Espectrometria de Massas em Tandem
20.
Metab Eng ; 52: 293-302, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30633974

RESUMO

The real value of gas-fermenting clostridia, capable of using CO and CO2, resides in their potential of being developed into cell factories to produce various bulk chemicals and fuels. This process requires rapid chromosomal integration of heterologous chemical biosynthetic pathways, which is impeded by the absence of genetic tools competent for efficient genome engineering in these anaerobes. Here, we developed a phage serine integrase-mediated site-specific genome engineering technique in Clostridium ljungdahlii, one of the major acetogenic gas-fermenting microbes. Two heterologous phage attachment/integration (Att/Int) systems (from Clostridium difficile and Streptomyces) were introduced into C. ljungdahlii and proven to be highly active, achieving efficient chromosomal integration of a whole donor vector via single-crossover recombination. Based on this, we further realized markerless chromosomal integration of target DNA fragments through a "dual integrase cassette exchange" (DICE) strategy with the assistance of the CRISPR-Cas9 editing system. As a proof of concept, a butyric acid production pathway from Clostridium acetobutylicum was integrated into the C. ljungdahlii genome without the introduction of extra markers, enabling stable expression of the pathway genes. The resulting engineered strain produced 1.01 g/L of butyric acid within 3 days by fermenting synthesis gas (CO2/CO). More importantly, the engineered strain showed good genetic stability and maintained butyric acid production ability after continuous subculturing. The system developed in this study overcomes the deficiencies of currently available genetic tools in the chromosomal integration of large DNA fragments (rapid, markerless and stable) in C. ljungdahlii, and may be extended to other Clostridium species.


Assuntos
Bacteriófagos/enzimologia , Bacteriófagos/genética , Clostridium/genética , Clostridium/metabolismo , Integrases/genética , Integrases/metabolismo , Engenharia Metabólica/métodos , Sítios de Ligação Microbiológicos/genética , Ácido Butírico/metabolismo , Sistemas CRISPR-Cas , DNA Bacteriano/genética , Fermentação , Genoma Bacteriano/genética , Redes e Vias Metabólicas/genética , Plasmídeos/genética , Serina/metabolismo
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