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1.
Nat Commun ; 11(1): 5043, 2020 10 07.
Artigo em Inglês | MEDLINE | ID: mdl-33028863

RESUMO

Human T-cell lymphotropic virus type 1 (HTLV-1) is a deltaretrovirus and the most oncogenic pathogen. Many of the ~20 million HTLV-1 infected people will develop severe leukaemia or an ALS-like motor disease, unless a therapy becomes available. A key step in the establishment of infection is the integration of viral genetic material into the host genome, catalysed by the retroviral integrase (IN) enzyme. Here, we use X-ray crystallography and single-particle cryo-electron microscopy to determine the structure of the functional deltaretroviral IN assembled on viral DNA ends and bound to the B56γ subunit of its human host factor, protein phosphatase 2 A. The structure reveals a tetrameric IN assembly bound to two molecules of the phosphatase via a conserved short linear motif. Insight into the deltaretroviral intasome and its interaction with the host will be crucial for understanding the pattern of integration events in infected individuals and therefore bears important clinical implications.


Assuntos
Vírus Linfotrópico T Tipo 1 Humano/patogenicidade , Integrases/ultraestrutura , Proteína Fosfatase 2/ultraestrutura , Vírus Linfotrópico T Tipo 1 de Símios/enzimologia , Proteínas Virais/ultraestrutura , Integração Viral , Motivos de Aminoácidos/genética , Clonagem Molecular , Microscopia Crioeletrônica , Cristalografia por Raios X , DNA Viral/metabolismo , DNA Viral/ultraestrutura , Vírus Linfotrópico T Tipo 1 Humano/enzimologia , Vírus Linfotrópico T Tipo 1 Humano/genética , Humanos , Integrases/genética , Integrases/metabolismo , Leucemia-Linfoma de Células T do Adulto/patologia , Leucemia-Linfoma de Células T do Adulto/virologia , Simulação de Acoplamento Molecular , Mutagênese Sítio-Dirigida , Paraparesia Espástica Tropical/patologia , Paraparesia Espástica Tropical/virologia , Multimerização Proteica , Proteína Fosfatase 2/genética , Proteína Fosfatase 2/metabolismo , Estrutura Quaternária de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura , Homologia de Sequência de Aminoácidos , Vírus Linfotrópico T Tipo 1 de Símios/genética , Imagem Individual de Molécula , Proteínas Virais/genética , Proteínas Virais/metabolismo
2.
PLoS One ; 15(10): e0236616, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33044964

RESUMO

Asexual blood stages of the malaria parasite are readily amenable to genetic modification via homologous recombination, allowing functional studies of parasite genes that are not essential in this part of the life cycle. However, conventional reverse genetics cannot be applied for the functional analysis of genes that are essential during asexual blood-stage replication. Various strategies have been developed for conditional mutagenesis of Plasmodium, including recombinase-based gene deletion, regulatable promoters, and mRNA or protein destabilization systems. Among these, the dimerisable Cre (DiCre) recombinase system has emerged as a powerful approach for conditional gene deletion in P. falciparum. In this system, the bacteriophage Cre is expressed in the form of two separate, enzymatically inactive polypeptides, each fused to a different rapamycin-binding protein. Rapamycin-induced heterodimerization of the two components restores recombinase activity. We have implemented the DiCre system in the rodent malaria parasite P. berghei, and show that rapamycin-induced excision of floxed DNA sequences can be achieved with very high efficiency in both mammalian and mosquito parasite stages. This tool can be used to investigate the function of essential genes not only in asexual blood stages, but also in other parts of the malaria parasite life cycle.


Assuntos
Deleção de Genes , Edição de Genes , Genes de Protozoários/genética , Integrases/metabolismo , Malária/parasitologia , Mutagênese , Plasmodium berghei/genética , Animais , Feminino , Integrases/química , Integrases/genética , Estágios do Ciclo de Vida , Malária/genética , Malária/metabolismo , Camundongos , Camundongos Endogâmicos C57BL
3.
Virology ; 548: 160-167, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32838937

RESUMO

Filamentous Inoviridae phages integrate into the chromosome of plant pathogens Xanthomonas as prophages, but their diversity and integrative mechanism are not completely understood. A proviral Cf2 sequence of 6454 bases from Xanthomonas citri genome was revived as infectious virions able to lysogenize its host. Unlike other Xanthomonas phages (Cf1c, φLf, Xf109, XacF1), Cf2 phage has RstA/RstB replication protein, and its attP has XerD binding arm and dif central region but lacks XerC binding arm. XerC+/Xf109 and XerD+/Cf2 attPs are in the opposite direction in phage genomes. Moreover, XerCD binding and XerD catalysis for strand exchange are necessary for site-specific integration of XerD+/Cf2 and XerC+/Xf109 attPs. Taken together, these results provide a new insight into the mechanism of XerCD-mediated recombination at XerD + attP.


Assuntos
Proteínas de Bactérias/metabolismo , Bacteriófagos/fisiologia , Inovirus/fisiologia , Integrases/metabolismo , Xanthomonas/enzimologia , Xanthomonas/virologia , Sítios de Ligação Microbiológicos , Proteínas de Bactérias/genética , Bacteriófagos/genética , Genoma Bacteriano , Inovirus/genética , Integrases/genética , Lisogenia , Integração Viral , Xanthomonas/genética
4.
Mol Cell ; 79(5): 857-869.e3, 2020 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-32681820

RESUMO

Sister-chromatid cohesion describes the orderly association of newly replicated DNA molecules behind replication forks. It plays an essential role in the maintenance and faithful transmission of genetic information. Cohesion is created by DNA topological links and proteinaceous bridges, whose formation and deposition could be potentially affected by many processes. Current knowledge on cohesion has been mainly gained by fluorescence microscopy observation. However, the resolution limit of microscopy and the restricted number of genomic positions that can be simultaneously visualized considerably hampered progress. Here, we present a high-throughput methodology to monitor sister-chromatid contacts (Hi-SC2). Using the multi-chromosomal Vibrio cholerae bacterium as a model, we show that Hi-SC2 permits to monitor local variations in sister-chromatid cohesion at a high resolution over a whole genome.


Assuntos
Cromátides/fisiologia , Técnicas Genéticas , Vibrio cholerae/genética , Cromossomos Bacterianos/fisiologia , Replicação do DNA , DNA Bacteriano , Sequenciamento de Nucleotídeos em Larga Escala , Integrases/metabolismo , Conformação de Ácido Nucleico
5.
Nat Commun ; 11(1): 3708, 2020 07 24.
Artigo em Inglês | MEDLINE | ID: mdl-32709899

RESUMO

The Cre-loxP recombination system is a powerful tool for genetic manipulation. However, there are widely recognized limitations with chemically inducible Cre-loxP systems, and the UV and blue-light induced systems have phototoxicity and minimal capacity for deep tissue penetration. Here, we develop a far-red light-induced split Cre-loxP system (FISC system) based on a bacteriophytochrome optogenetic system and split-Cre recombinase, enabling optogenetical regulation of genome engineering in vivo solely by utilizing a far-red light (FRL). The FISC system exhibits low background and no detectable photocytotoxicity, while offering efficient FRL-induced DNA recombination. Our in vivo studies showcase the strong organ-penetration capacity of FISC system, markedly outperforming two blue-light-based Cre systems for recombination induction in the liver. Demonstrating its strong clinical relevance, we successfully deploy a FISC system using adeno-associated virus (AAV) delivery. Thus, the FISC system expands the optogenetic toolbox for DNA recombination to achieve spatiotemporally controlled, non-invasive genome engineering in living systems.


Assuntos
Engenharia Genética , Integrases/metabolismo , Integrases/efeitos da radiação , Luz , Recombinação Genética , Animais , Linhagem Celular , Sobrevivência Celular , Dependovirus/genética , Vetores Genéticos , Genoma , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Optogenética , Receptor EphB3
6.
PLoS One ; 15(7): e0236741, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32730300

RESUMO

Aryl hydrocarbon receptor (AHR) agonists such as dioxin have been associated with obesity and the development of diabetes. Whole-body Ahr knockout mice on high-fat diet (HFD) have been shown to resist obesity and hepatic steatosis. Tissue-specific knockout of Ahr in mature adipocytes via adiponectin-Cre exacerbates obesity while knockout in liver increases steatosis without having significant effects on obesity. Our previous studies demonstrated that treatment of subcutaneous preadipocytes with exogenous or endogenous AHR agonists disrupts maturation into functional adipocytes in vitro. Here, we used platelet-derived growth factor receptor alpha (Pdgfrα)-Cre mice, a Cre model previously established to knock out genes in preadipocyte lineages and other cell types, but not liver cells, to further define AHR's role in obesity. We demonstrate that Pdgfrα-Cre Ahr-floxed (Ahrfl/fl) knockout mice are protected from HFD-induced obesity compared to non-knockout Ahrfl/fl mice (control mice). The Pdgfrα-Cre Ahrfl/fl knockout mice were also protected from increased adiposity, enlargement of adipocyte size, and liver steatosis while on the HFD compared to control mice. On a regular control diet, knockout and non-knockout mice showed no differences in weight gain, indicating the protective phenotype arises only when animals are challenged by a HFD. At the cellular level, cultured cells from brown adipose tissue (BAT) of Pdgfrα-Cre Ahrfl/fl mice were more responsive than cells from controls to transcriptional activation of the thermogenic uncoupling protein 1 (Ucp1) gene by norepinephrine, suggesting an ability to burn more energy under certain conditions. Collectively, our results show that knockout of Ahr mediated by Pdgfrα-Cre is protective against diet-induced obesity and suggest a mechanism by which enhanced UCP1 activity within BAT might confer these effects.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/fisiologia , Dieta Hiperlipídica/efeitos adversos , Fígado Gorduroso/prevenção & controle , Integrases/metabolismo , Obesidade/prevenção & controle , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/fisiologia , Receptores de Hidrocarboneto Arílico/fisiologia , Adiposidade , Animais , Metabolismo Energético , Fígado Gorduroso/etiologia , Fígado Gorduroso/patologia , Feminino , Resistência à Insulina , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Obesidade/etiologia , Obesidade/patologia , Termogênese
7.
J Vis Exp ; (160)2020 06 11.
Artigo em Inglês | MEDLINE | ID: mdl-32597835

RESUMO

Myocardial infarction (MI) is a leading cause of morbidity and mortality in the Western world. In the past decade, gene therapy has become a promising treatment option for heart disease, owing to its efficiency and exceptional therapeutic effects. In an effort to repair the damaged tissue post-MI, various studies have employed DNA-based or viral gene therapy but have faced considerable hurdles due to the poor and uncontrolled expression of the delivered genes, edema, arrhythmia, and cardiac hypertrophy. Synthetic modified mRNA (modRNA) presents a novel gene therapy approach that offers high, transient, safe, nonimmunogenic, and controlled mRNA delivery to the heart tissue without any risk of genomic integration. Due to these remarkable characteristics combined with its bell-shaped pharmacokinetics in the heart, modRNA has become an attractive approach for the treatment of heart disease. However, to increase its effectiveness in vivo, a consistent and reliable delivery method needs to be followed. Hence, to maximize modRNA delivery efficiency and yield consistency in modRNA use for in vivo applications, an optimized method of preparation and delivery of modRNA intracardiac injection in a mouse MI model is presented. This protocol will make modRNA delivery more accessible for basic and translational research.


Assuntos
Técnicas de Transferência de Genes , Infarto do Miocárdio/genética , Infarto do Miocárdio/terapia , RNA Mensageiro/administração & dosagem , RNA Mensageiro/uso terapêutico , Animais , Modelos Animais de Doenças , Terapia Genética/métodos , Injeções , Integrases/metabolismo , Ligadura , Luciferases/metabolismo , Camundongos , Infarto do Miocárdio/cirurgia , Miocárdio/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reprodutibilidade dos Testes
8.
Nucleic Acids Res ; 48(12): 6413-6430, 2020 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-32479633

RESUMO

Streptomyces phage ϕC31 integrase (Int)-a large serine site-specific recombinase-is autonomous for phage integration (attP x attB recombination) but is dependent on the phage coded gp3, a recombination directionality factor (RDF), for prophage excision (attL x attR recombination). A previously described activating mutation, E449K, induces Int to perform attL x attR recombination in the absence of gp3, albeit with lower efficiency. E449K has no adverse effect on the competence of Int for attP x attB recombination. Int(E449K) resembles Int in gp3 mediated stimulation of attL x attR recombination and inhibition of attP x attB recombination. Using single-molecule analyses, we examined the mechanism by which E449K activates Int for gp3-independent attL x attR recombination. The contribution of E449K is both thermodynamic and kinetic. First, the mutation modulates the relative abundance of Int bound attL-attR site complexes, favoring pre-synaptic (PS) complexes over non-productively bound complexes. Roughly half of the synaptic complexes formed from Int(E449K) pre-synaptic complexes are recombination competent. By contrast, Int yields only inactive synapses. Second, E449K accelerates the dissociation of non-productively bound complexes and inactive synaptic complexes formed by Int. The extra opportunities afforded to Int(E499K) in reattempting synapse formation enhances the probability of success at fruitful synapsis.


Assuntos
Mutação com Ganho de Função , Integrases/metabolismo , Siphoviridae/enzimologia , Proteínas Virais/metabolismo , Proteínas de Ligação a DNA/metabolismo , Integrases/química , Integrases/genética , Cinética , Simulação de Dinâmica Molecular , Ligação Proteica , Recombinação Genética , Siphoviridae/genética , Proteínas Virais/química , Proteínas Virais/genética
9.
Am J Physiol Heart Circ Physiol ; 319(2): H349-H358, 2020 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-32589443

RESUMO

Here, we report the generation of a Cre-recombinase (iCre) transgenic rat, where iCre is driven using a vascular endothelial-cadherin (CDH5) promoter. The CDH5 promoter was cloned from rat pulmonary microvascular endothelial cells and demonstrated ~60% similarity to the murine counterpart. The cloned rat promoter was 2,508 bp, it extended 79 bp beyond the transcription start site, and it was 22,923 bp upstream of the translation start site. The novel promoter was cloned upstream of codon-optimized iCre and subcloned into a Sleeping Beauty transposon vector for transpositional transgenesis in Sprague-Dawley rats. Transgenic founders were generated and selected for iCre expression. Crossing the CDH5-iCre rat with a tdTomato reporter rat resulted in progeny displaying endothelium-restricted fluorescence. tdTomato fluorescence was prominent in major arteries and veins, and it was similar in males and females. Quantitative analysis of the carotid artery and the jugular vein revealed that, on average, more than 50% of the vascular surface area exhibited strong fluorescence. tdTomato fluorescence was observed in the circulations of every tissue tested. The microcirculation in all tissues tested displayed homogenous fluorescence. Fluorescence was examined across young (6-7.5 mo), middle (14-16.5 mo), and old age (17-19.5 mo) groups. Although tdTomato fluorescence was seen in middle- and old-age animals, the intensity of the fluorescence was significantly reduced compared with that seen in the young rats. Thus, this endothelium-restricted transgenic rat offers a novel platform to test endothelial microheterogeneity within all vascular segments, and it provides exceptional resolution of endothelium within-organ microcirculation for application to translational disease models.NEW & NOTEWORTHY The use of transgenic mice has been instrumental in advancing molecular insight of physiological processes, yet these models oftentimes do not faithfully recapitulate human physiology and pathophysiology. Rat models better replicate some human conditions, like Group 1 pulmonary arterial hypertension. Here, we report the development of an endothelial cell-restricted transgenic reporter rat that has broad application to vascular biology. This first-in-kind model offers exceptional endothelium-restricted tdTomato expression, in both conduit vessels and the microcirculations of organs.


Assuntos
Antígenos CD/genética , Caderinas/genética , Células Endoteliais/metabolismo , Genes Reporter , Integrases/genética , Proteínas Luminescentes/genética , Regiões Promotoras Genéticas , Fatores Etários , Animais , Feminino , Regulação da Expressão Gênica , Integrases/metabolismo , Proteínas Luminescentes/biossíntese , Masculino , Microcirculação , Ratos Sprague-Dawley , Ratos Transgênicos , Distribuição Tecidual , Transposases/genética , Transposases/metabolismo
10.
Nat Commun ; 11(1): 3121, 2020 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-32561747

RESUMO

Integration of the reverse-transcribed viral DNA into host chromosomes is a critical step in the life-cycle of retroviruses, including an oncogenic delta(δ)-retrovirus human T-cell leukemia virus type-1 (HTLV-1). Retroviral integrase forms a higher order nucleoprotein assembly (intasome) to catalyze the integration reaction, in which the roles of host factors remain poorly understood. Here, we use cryo-electron microscopy to visualize the HTLV-1 intasome at 3.7-Šresolution. The structure together with functional analyses reveal that the B56γ (B'γ) subunit of an essential host enzyme, protein phosphatase 2 A (PP2A), is repurposed as an integral component of the intasome to mediate HTLV-1 integration. Our studies reveal a key host-virus interaction underlying the replication of an important human pathogen and highlight divergent integration strategies of retroviruses.


Assuntos
Interações Hospedeiro-Patógeno/genética , Vírus Linfotrópico T Tipo 1 Humano/genética , Integrases/metabolismo , Proteína Fosfatase 2/genética , Proteínas Virais/metabolismo , Integração Viral/genética , Microscopia Crioeletrônica , DNA Viral/metabolismo , Células HEK293 , Vírus Linfotrópico T Tipo 1 Humano/enzimologia , Humanos , Integrases/ultraestrutura , Modelos Moleculares , Mutação Puntual , Ligação Proteica/genética , Proteína Fosfatase 2/metabolismo , Proteína Fosfatase 2/ultraestrutura , Proteínas Virais/ultraestrutura
11.
Nat Commun ; 11(1): 2141, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-32358538

RESUMO

Optogenetic genome engineering tools enable spatiotemporal control of gene expression and provide new insight into biological function. Here, we report the new version of genetically encoded photoactivatable (PA) Cre recombinase, PA-Cre 3.0. To improve PA-Cre technology, we compare light-dimerization tools and optimize for mammalian expression using a CAG promoter, Magnets, and 2A self-cleaving peptide. To prevent background recombination caused by the high sequence similarity in the dimerization domains, we modify the codons for mouse gene targeting and viral production. Overall, these modifications significantly reduce dark leak activity and improve blue-light induction developing our new version, PA-Cre 3.0. As a resource, we have generated and validated AAV-PA-Cre 3.0 as well as two mouse lines that can conditionally express PA-Cre 3.0. Together these new tools will facilitate further biological and biomedical research.


Assuntos
Integrases/metabolismo , Recombinação Genética/genética , Animais , Códon/genética , Engenharia Genética/métodos , Integrases/genética , Luz , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Optogenética , Regiões Promotoras Genéticas/genética , Regiões Promotoras Genéticas/efeitos da radiação , Recombinação Genética/efeitos da radiação
12.
Nat Struct Mol Biol ; 27(5): 489-499, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32367067

RESUMO

Cas1 integrase associates with Cas2 to insert short DNA fragments into a CRISPR array, establishing nucleic acid memory in prokaryotes. Here we applied single-molecule FRET methods to the Enterococcus faecalis (Efa) Cas1-Cas2 system to establish a kinetic framework describing target-searching, integration, and post-synapsis events. EfaCas1-Cas2 on its own is not able to find the CRISPR repeat in the CRISPR array; it only does so after prespacer loading. The leader sequence adjacent to the repeat further stabilizes EfaCas1-Cas2 contacts, enabling leader-side integration and subsequent spacer-side integration. The resulting post-synaptic complex (PSC) has a surprisingly short mean lifetime. Remarkably, transcription effectively resolves the PSC, and we predict that this is a conserved mechanism that ensures efficient and directional spacer integration in many CRISPR systems. Overall, our study provides a complete model of spacer acquisition, which can be harnessed for DNA-based information storage and cell lineage tracing technologies.


Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Enterococcus faecalis/enzimologia , Integrases/metabolismo , Eletroporação , Enterococcus faecalis/genética , Escherichia coli/genética , Transferência Ressonante de Energia de Fluorescência , Integrases/genética , Cinética , Microrganismos Geneticamente Modificados , Mutação , Transcrição Genética
13.
Nat Commun ; 11(1): 1825, 2020 04 14.
Artigo em Inglês | MEDLINE | ID: mdl-32286280

RESUMO

Pineoblastoma is a rare pediatric cancer induced by germline mutations in the tumor suppressors RB1 or DICER1. Presence of leptomeningeal metastases is indicative of poor prognosis. Here we report that inactivation of Rb plus p53 via a WAP-Cre transgene, commonly used to target the mammary gland during pregnancy, induces metastatic pineoblastoma resembling the human disease with 100% penetrance. A stabilizing mutation rather than deletion of p53 accelerates metastatic dissemination. Deletion of Dicer1 plus p53 via WAP-Cre also predisposes to pineoblastoma, albeit with lower penetrance. In silico analysis predicts tricyclic antidepressants such as nortriptyline as potential therapeutics for both pineoblastoma models. Nortriptyline disrupts the lysosome, leading to accumulation of non-functional autophagosome, cathepsin B release and pineoblastoma cell death. Nortriptyline further synergizes with the antineoplastic drug gemcitabine to effectively suppress pineoblastoma in our preclinical models, offering new modality for this lethal childhood malignancy.


Assuntos
Mutação em Linhagem Germinativa/genética , Lisossomos/metabolismo , Pinealoma/tratamento farmacológico , Pinealoma/genética , Animais , Autofagossomos/efeitos dos fármacos , Autofagossomos/metabolismo , Autofagossomos/ultraestrutura , Autofagia/efeitos dos fármacos , Análise por Conglomerados , Modelos Animais de Doenças , Deleção de Genes , Humanos , Integrases/metabolismo , Estimativa de Kaplan-Meier , Lisossomos/efeitos dos fármacos , Camundongos , Metástase Neoplásica , Nortriptilina/farmacologia , Nortriptilina/uso terapêutico , Pinealoma/patologia , Pinealoma/ultraestrutura , Proteína do Retinoblastoma/metabolismo , Proteína Supressora de Tumor p53/metabolismo
14.
Biochim Biophys Acta Gene Regul Mech ; 1863(8): 194568, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32344203

RESUMO

One of the widely used applications of the popular Cre-loxP method for targeted recombination is the permanent activation of marker genes, such as reporter genes or antibiotic resistance genes, by excision of a preceding transcriptional stop signal. The STOP cassette consists of three identical SV40-derived poly(A) signal repeats and is flanked by two loxP sites. We found that in addition to complete loxP-mediated recombination, limiting levels of the Cre recombinase also cause incomplete recombination of the STOP cassette. Partial recombination leads to the loss of only one or two of the three identical poly(A) repeats with recombination breakpoints always precisely matching the end/start of each poly(A) signal repeat without any relevant similarity to the canonical or known cryptic loxP sequences, suggesting that this type of Cre-mediated recombination is loxP-independent. Incomplete deletion of the STOP cassette results in partial read-through transcription, explaining at least some of the variability often observed in marker gene expression from an otherwise identical locus.


Assuntos
Integrases/genética , Integrases/metabolismo , Recombinação Genética , Animais , Células CHO , Cricetulus , Feminino , Expressão Gênica , Genes Reporter/genética , Marcadores Genéticos , Rim , Masculino , Camundongos , Transcriptoma
15.
Nat Methods ; 17(5): 541-550, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32313222

RESUMO

Recombinant adeno-associated viruses (rAAVs) are efficient gene delivery vectors via intravenous delivery; however, natural serotypes display a finite set of tropisms. To expand their utility, we evolved AAV capsids to efficiently transduce specific cell types in adult mouse brains. Building upon our Cre-recombination-based AAV targeted evolution (CREATE) platform, we developed Multiplexed-CREATE (M-CREATE) to identify variants of interest in a given selection landscape through multiple positive and negative selection criteria. M-CREATE incorporates next-generation sequencing, synthetic library generation and a dedicated analysis pipeline. We have identified capsid variants that can transduce the central nervous system broadly, exhibit bias toward vascular cells and astrocytes, target neurons with greater specificity or cross the blood-brain barrier across diverse murine strains. Collectively, the M-CREATE methodology accelerates the discovery of capsids for use in neuroscience and gene-therapy applications.


Assuntos
Encéfalo/virologia , Proteínas do Capsídeo/metabolismo , Dependovirus/genética , Técnicas de Transferência de Genes , Engenharia Genética/métodos , Vetores Genéticos/genética , Integrases/metabolismo , Animais , Barreira Hematoencefálica/metabolismo , Proteínas do Capsídeo/genética , Feminino , Terapia Genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Especificidade de Órgãos , Tropismo Viral
16.
Sheng Li Xue Bao ; 72(2): 148-156, 2020 Apr 25.
Artigo em Chinês | MEDLINE | ID: mdl-32328608

RESUMO

The adrenal gland is an important endocrine organ of human body. CYP11B1 gene was specifically expressed in the zona fasciculata in adrenal cortex. In order to better study the function of genes specifically expressed in the zona fasciculata in adrenal cortex, the mice with Cre recombinase specifically expressed in the zona fasciculata in adrenal cortex were constructed. It was then confirmed that CYP11B1 was specifically expressed in adrenal glands. Then, using CRISPR/Cas9 technique, CYP11B1-2A-GfpCre recombinant vector was constructed and subsequently injected into the fertilized eggs of mice. It was confirmed that the Cre gene was mainly expressed in the zona fasciculata in adrenal cortex of CYP11B1Cre mice by using mTmG and LacZ staining. The CYP11B1Cre mice were then mated with cystathionine γ-lyase (CTH)f/f mice, thereby generating CTHf/f/CYP11B1Cre mice. It was also confirmed that CTH gene in the zona fasciculata in adrenal cortex was specifically knocked out in these mice. These results suggest that transgenic mice with specific Cre recombinase expression in the zona fasciculata in adrenal cortex were constructed successfully. This animal model can be a powerful tool for the study of the function of genes expressed in the zona fasciculata in adrenal cortex.


Assuntos
Córtex Suprarrenal/enzimologia , Integrases/metabolismo , Camundongos Transgênicos , Zona Fasciculada/enzimologia , Animais , Sistemas CRISPR-Cas , Cistationina gama-Liase/genética , Integrases/genética , Camundongos
17.
Invest Ophthalmol Vis Sci ; 61(3): 51, 2020 03 09.
Artigo em Inglês | MEDLINE | ID: mdl-32232350

RESUMO

Purpose: The lysozyme 2 (Lyz2 or LysM) cre mouse is extensively used to achieve genetic manipulation in myeloid cells and it has been widely employed in retinal research. However, LysM has been recently described to be expressed in brain neurons and there is a debate on whether it is also expressed by resident microglia in addition to infiltrating macrophages. Methods: We examined LysM-cre recombination in retinal tissue using a LysM-cre/tdTomato reporter mouse together with immunolabeling for several retinal cell markers. We further compared LysM-cre tdTomato recombination with that of Cdh5-cre driver, which is expressed in both endothelial and hematopoietic cells. Results: LysM-cre was strongly expressed in most microglia/resident macrophages in neonatal retinas (P8) and to a lesser extent in microglia of adult retinas. In addition, there was some neuronal recombination (8 %) of LysM-cre specifically in adult retinal ganglion cells and amacrine cells. After retinal ischemia-reperfusion injury, LysM-cre was strongly expressed in microglia/infiltrating macrophages. Cdh5-cre was expressed in endothelial and myeloid cells of P8 pups retinas. Unexpectedly, Cdh5 showed additional expression in adult mouse retinal ganglion cells and brain neurons. Conclusions: LysM-cre is expressed in macrophages and a subset of microglia together with a small but significant recombination of LysM-cre in the retinal neurons of adult mice. Cdh5 also showed some neuronal expression in both retina and brain of adult mice. These findings should be taken into consideration when interpreting results from central nervous system research using LysM-cre and Cdh5-cre mice.


Assuntos
Antígenos CD/metabolismo , Encéfalo/metabolismo , Caderinas/metabolismo , Integrases/metabolismo , Substâncias Luminescentes/metabolismo , Proteínas Luminescentes/metabolismo , Muramidase/metabolismo , Vasos Retinianos/metabolismo , Animais , Animais Recém-Nascidos , Pesquisa Biomédica , Diagnóstico por Imagem , Endotélio Vascular/metabolismo , Feminino , Genes Reporter , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Microglia/metabolismo , Neurônios/metabolismo , Recombinação Genética , Traumatismo por Reperfusão/metabolismo , Células Ganglionares da Retina/metabolismo
18.
Nucleic Acids Res ; 48(6): 2841-2852, 2020 04 06.
Artigo em Inglês | MEDLINE | ID: mdl-32112097

RESUMO

Base editing (BE) is a powerful tool for engineering single nucleotide variants (SNVs) and has been used to create targeted mutations in cell lines, organoids and animal models. Recent development of new BE enzymes has provided an extensive toolkit for genome modification; however, identifying and isolating edited cells for analysis has proven challenging. Here we report a 'Gene On' (GO) reporter system that indicates precise cytosine or adenine base editing in situ with high sensitivity and specificity. We test GO using an activatable GFP and use it to measure the kinetics, efficiency and PAM specificity of a range of new BE variants. Further, GO is flexible and can be easily adapted to induce expression of numerous genetically encoded markers, antibiotic resistance genes or enzymes, such as Cre recombinase. With these tools, GO can be exploited to functionally link BE events at endogenous genomic loci to cellular enzymatic activities in human and mouse cell lines and organoids. Thus, GO provides a powerful approach to increase the practicality and feasibility of implementing CRISPR BE in biomedical research.


Assuntos
Edição de Genes , Genes Reporter , Animais , Sequência de Bases , Linhagem Celular Tumoral , Resistência Microbiana a Medicamentos , Células HEK293 , Humanos , Integrases/metabolismo , Camundongos , Células NIH 3T3 , Recombinação Genética/genética
19.
J Vis Exp ; (155)2020 01 30.
Artigo em Inglês | MEDLINE | ID: mdl-32065159

RESUMO

Chlamydia trachomatis is an obligate intracellular pathogen that has been historically difficult to genetically manipulate. Definitive progress in elucidating the mechanisms that C. trachomatis use to create and maintain a privileged intracellular niche has been limited due to a lack of genetic tools. Fortunately, there have recently been several new advances in genetic manipulation techniques. Among these is the development of fluorescence-reported allelic exchange mutagenesis (FRAEM). This method allows targeted gene deletion coupled with insertion of a selection cassette encoding antibiotic resistance and green fluorescent protein (GFP). Reliance on this strategy can be complicated when targeting genes within polycistronic operons due to the potential of polar effects on downstream genes. Floxed cassette allelic exchange mutagenesis (FLAEM), the protocol for which is described here, was developed to alleviate cassette-induced polar effects. FLAEM utilizes Cre-loxP genome editing to remove the selection cassette after targeted deletion by allelic exchange. The resulting strains contain markerless gene deletions of one or more coding sequences. This technique facilitates direct assessment of gene function and expands the repertoire of tools for genetic manipulation in C. trachomatis.


Assuntos
Alelos , Chlamydia trachomatis/genética , Deleção de Genes , Mutagênese Insercional/genética , Mutagênese/genética , Sequência de Bases , DNA Bacteriano/genética , Genoma Bacteriano , Proteínas de Fluorescência Verde/genética , Integrases/metabolismo , Transformação Genética
20.
Eur J Immunol ; 50(4): 603-605, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32087088

RESUMO

A growing body of evidence suggests that Cre recombinase can be toxic to immune cells in various experimental settings. Cre recombinase toxicity is dependent on the level of Cre activity and may also interfere with cell proliferation. Here, we compared two different published tamoxifen-inducible CD4-CreERT2 mouse lines for their suitability to study the dynamics of T-follicular helper cell responses in vivo. Our data underscore that under certain circumstances inducible Cre toxicity (tamoxifen application results in translocation of preformed CreERT2 to the nucleus) interferes with cell survival and, therefore, necessitates careful interpretation of experimental data and the inclusion of appropriate controls. Interestingly, our data indicate that low expression of CreERT2 can still allow for efficient recombination in proliferating lymphocytes without causing excessive cell loss due to Cre toxicity.


Assuntos
Centro Germinativo/imunologia , Integrases/metabolismo , Linfócitos T Auxiliares-Indutores/imunologia , Animais , Linhagem Celular , Proliferação de Células , Sobrevivência Celular , Integrases/genética , Camundongos , Camundongos Transgênicos , Tamoxifeno/metabolismo
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