Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 1.656.104
Filtrar
Mais filtros








Intervalo de ano de publicação
1.
Braz. j. biol ; 84: e256691, 2024. tab, ilus
Artigo em Inglês | VETINDEX | ID: biblio-1374664

RESUMO

microRNAs (miRNAs) are recognized as diabetes mellitus type 2 (T2DM) biomarkers useful for disease metabolism comprehension and have great potential as therapeutics targets. BDNF and IGF1 increased expression are highly involved in the benefits of insulin and glucose paths, however, they are down-regulated in insulin resistance conditions, while their expression increase is correlated to the improvement of glucose and insulin metabolism. Studies suggest the microRNA regulation of these genes in several different contexts, providing a novel investigation approach for comprehending T2DM metabolism and revealing potential therapeutic targets. In the present study, we investigate in different animal models (human, rat, and mouse) miRNAs that target BDNF and IGF1 in skeletal muscle tissue with T2DM physiological conditions. Bioinformatics tools and databases were used to miRNA prediction, molecular homology, experimental validation of interactions, expression in the studied physiological condition, and network interaction. The findings showed three miRNAs candidates for IGF1(miR-29a, miR-29b, and miR-29c) and one for BDNF (miR-206). The experimental evaluations and the search for the expression in skeletal muscle from T2DM subjects confirmed the predicted interaction between miRNA-mRNA for miR-29b and miR-206 through human, rat, and mouse models. This interaction was reaffirmed in multiple network analyses. In conclusion, our results show the regulation relationship between miR-29b and miR-206 with the investigated genes, in several tissues, suggesting an inhibition pattern. Nevertheless, these data show a large number of possible interaction physiological processes, for future biotechnological prospects.


Os microRNAs (miRNAs) são reconhecidos como biomarcadores do diabetes mellitus tipo 2 (DM2), úteis para a compreensão do metabolismo da doença, e possuem grande potencial como alvos terapêuticos. O aumento da expressão de BDNF e IGF1 está altamente envolvido nos benefícios as vias de insulina e glicose, porém, são regulados negativamente em condições de resistência à insulina, enquanto seu aumento de expressão está correlacionado com a melhora do metabolismo da glicose e da insulina. Estudos sugerem a regulação desses genes por microRNA em vários contextos diferentes, proporcionando uma nova abordagem de investigação para compreender o metabolismo do DM2 e revelar potenciais alvos terapêuticos. No presente estudo, investigamos em diferentes modelos animais (humanos, ratos e camundongos) miRNAs que têm como alvo BDNF e IGF1 em tecido muscular esquelético com condições fisiológicas de DM2. As análises foram realizadas utilizando ferramentas de bioinformática e bancos de dados para predição de miRNA, homologia molecular, validação experimental de interações, expressão na condição fisiológica estudada e interação em rede. Os resultados mostraram três candidatos a miRNAs para IGF1 (miR-29a, miR-29b e miR-29c) e um para BDNF (miR-206). As avaliações experimentais e a busca pela expressão no músculo esquelético de indivíduos com DM2 confirmaram a interação prevista entre miRNA-mRNA para miR-29b e miR-206 através de modelos humanos, ratos e camundongos. Essa interação foi reafirmada em múltiplas análises de rede. Em conclusão, nossos resultados mostram a relação de regulação entre miR-29b e miR-206 com os genes investigados, em diversos tecidos, sugerindo um padrão de inibição. Contudo, esses dados mostram um grande número de possíveis processos fisiológicos de interação para perspectivas biotecnológicas.


Assuntos
Humanos , Camundongos , Ratos , Resistência à Insulina , Biomarcadores , Terapia Genética , Diabetes Mellitus Tipo 2/metabolismo
2.
Braz. j. biol ; 84: e260091, 2024. tab, ilus
Artigo em Inglês | VETINDEX | ID: biblio-1374650

RESUMO

Epilepsy is one of the most common neurological disorders affecting most social, economic and biological aspects of human life. Most patients with epilepsy have uncontrolled seizures and drug side effects despite the medications. Patients with epilepsy often have problems with attention, memory, and information processing speed, which may be due to seizures, underlying causes, or anticonvulsants. Therefore, improving seizure control and reducing or changing the anti-epileptic drugs can solve these problems, but these problems will not be solved in most cases. In this work, we looked at the effects of pioglitazone, a Peroxisome Proliferator-Activated Receptor agonist used to treat type 2 diabetes, on pilocarpine-induced seizures in mice. The Racine scale was used to classify pilocarpine-induced convulsions. After that, all of the animals were beheaded, and the brain and hippocampus were dissected. Finally, biochemical techniques were used to determine the levels of Malondialdehyde and Catalase activity, as well as Superoxide Dismutase and Glutathione Reductase in the hippocampus. The results of this investigation suggest that pioglitazone's antioxidant action may play a key role in its neuroprotective properties against pilocarpine-induced seizure neuronal damage.


A epilepsia é um dos distúrbios neurológicos mais comuns que afetam a maioria dos aspectos sociais, econômicos e biológicos da vida humana. A maioria dos pacientes com epilepsia tem convulsões não controladas e apresenta efeitos colaterais de medicamentos. Pacientes com epilepsia, geralmente, têm problemas de atenção, memória e velocidade de processamento de informações, ocasionados por convulsões, causas subjacentes ou anticonvulsivantes. Portanto, melhorar o controle das crises e reduzir ou alterar as drogas antiepilépticas pode resolver esses problemas, mas, na maioria dos casos, eles não serão resolvidos. Neste trabalho, analisamos os efeitos da pioglitazona, um agonista do receptor ativado por proliferador de peroxissoma usado para tratar diabetes tipo 2, em convulsões induzidas por pilocarpina em camundongos. A escala de Racine foi usada para classificar as convulsões induzidas pela pilocarpina. Em seguida, todos os animais foram decapitados, e o cérebro e o hipocampo foram dissecados. Finalmente, técnicas bioquímicas foram utilizadas para determinar os níveis de atividade do malondialdeído e da catalase, bem como da superóxido dismutase e glutationa redutase no hipocampo. Os resultados desta investigação sugerem que a ação antioxidante da pioglitazona pode desempenhar um papel fundamental em suas propriedades neuroprotetoras contra o dano neuronal convulsivo induzido pela pilocarpina.


Assuntos
Camundongos , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , Epilepsia , Pioglitazona/uso terapêutico , Anticonvulsivantes
3.
Braz. j. biol ; 84: e250556, 2024. ilus
Artigo em Inglês | LILACS, VETINDEX | ID: biblio-1360208

RESUMO

Exosomes are 30-120nm bio particles transferred from donor to recipient cells leading to modification in their regulatory mechanisms depending upon the coded message in the form of loaded biomolecule. Cancer cells derived exosomes the true representatives of the parent cells have been found to modify the tumor surrounding/distinct regions and participate in metastasis, angiogenesis and immune suppression. Tis study was aimed to study the effects of tumor mice derived exosomes on the normal mice spleen isolated T cells by using co-culture experiments and flow cytometer analysis. We mainly focused on some of the T cells population and cytokines including IFN-γ, FOXP3+ regulatory T (Treg) cells and KI67 (proliferation marker). Overall results indicated random changes in different set of experiments, where the cancer derived exosomes reduced the IFN-γ expression in both CD4 and CD8 T cells, similarly the Treg cells were also found decreased in the presence of cancer exosomes. No significant changes were observed on the Ki67 marker expression. Such studies are helpful in understanding the role of cancer exosomes in immune cells suppression in tumor microenvironment. Cancer exosomes will need to be validated in vivo and in vitro on a molecular scale in detail for clinical applications.


Os exossomos são biopartículas de 30-120 nm transferidas de células doadoras para células receptoras, levando à modificação em seus mecanismos reguladores, dependendo da mensagem codificada na forma de biomolécula carregada. Verificou-se que exossomos derivados de células cancerosas ­ os verdadeiros representantes das células-mãe ­ modificam as regiões circundantes / distintas do tumor e participam da metástase, angiogênese e imunossupressão. Este estudo teve como objetivo estudar os efeitos de exossomos derivados de camundongos com tumor nas células T isoladas de baço de camundongos normais, usando experimentos de cocultura e análise de citômetro de fluxo. Concentrou-se, principalmente, em algumas populações de células T e citocinas, incluindo IFN-γ, células T reguladoras FOXP3 + (Treg) e KI67 (marcador de proliferação). Os resultados gerais indicaram mudanças aleatórias em diferentes conjuntos de experimentos, em que os exossomos derivados de câncer reduziram a expressão de IFN-γ em células T CD4 e CD8, da mesma forma que as células Treg também foram encontradas diminuídas na presença de exossomos de câncer. Nenhuma mudança significativa foi observada na expressão do marcador Ki67. Esses dados são úteis para a compreensão do papel dos exossomos do câncer na supressão de células do sistema imunológico no microambiente tumoral. Exossomos de câncer precisarão ser validados in vivo e in vitro em escala molecular com detalhes para aplicações clínicas.


Assuntos
Animais , Camundongos , Exossomos , Microambiente Tumoral , Sistema Imunitário , Metástase Neoplásica , Neoplasias
4.
Med Gas Res ; 13(1): 23-28, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-35946219

RESUMO

Demyelination of the cerebral white matter is the most common pathological change after carbon monoxide (CO) poisoning. Notch signaling, the mechanism underlying the differentiation of astrocytes and oligodendrocytes, is critical to remyelination of the white matter after brain lesion. The purpose of this work was to determine the effects of hyperbaric oxygen (HBO) on Notch signaling pathway after CO poisoning for the explanation of the protective effects of HBO on CO-poisoning-related cerebral white matter demyelination. The male C57 BL/6 mice with severe CO poisoning were treated by HBO. And HBO therapy shortened the escape latency and improved the body mass after CO poisoning. HBO therapy also significantly suppressed protein and mRNA levels of Notch1 and Hes5 after CO poisoning. Our findings suggested that HBO could suppress the activation of Notch signaling pathway after CO poisoning, which is the mechanism underlying the neuroprotection of HBO on demyelination after severe CO poisoning.


Assuntos
Intoxicação por Monóxido de Carbono , Doenças Desmielinizantes , Oxigenoterapia Hiperbárica , Animais , Intoxicação por Monóxido de Carbono/terapia , Doenças Desmielinizantes/induzido quimicamente , Doenças Desmielinizantes/terapia , Masculino , Camundongos , Oxigênio , Transdução de Sinais
5.
Methods Mol Biol ; 2578: 161-175, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36152286

RESUMO

Peptide microarray provides the ability to miniaturize, parallelize, and automate high-throughput screening substrate specificities of enzymes, profiling of multiple enzyme activities, discovery of disease biomarkers, and development of drugs. Matrix metalloproteinases (MMPs) are demonstrated as important biomarkers of tumor invasion and metastasis. Herein, a peptide microarray-based fluorescence assay is proposed to profile multiple MMPs (MMP-1, MMP-2, MMP-3, MMP-7, MMP-9, and MMP-13) activities in the culture medium of four human osteosarcoma (OS) cells and in the progression of OS by using the mouse-bearing xenograft OSs including U-2OS and Saos-2 human. This method has excellent selectivity and sensitivity, which enables to detect the activities of cellular secreted MMP-1, MMP-2, MMP-3, MMP-7, MMP-9, and MMP-13 with limit of detection downs to 10 pM, 30 pM, 113 pM, 13 pM, 93 pM, and 12 pM, respectively. Furthermore, it is demonstrated that the activity pattern of MMPs is serum closely relevant to the disease progression and type of tumor.


Assuntos
Neoplasias Ósseas , Nanotubos , Osteossarcoma , Óxido de Zinco , Animais , Neoplasias Ósseas/patologia , Fluorescência , Humanos , Metaloproteinase 1 da Matriz , Metaloproteinase 13 da Matriz , Metaloproteinase 2 da Matriz , Metaloproteinase 3 da Matriz , Metaloproteinase 7 da Matriz , Metaloproteinase 9 da Matriz , Camundongos , Osteossarcoma/patologia , Peptídeos , Polímeros
6.
Methods Mol Biol ; 2577: 65-81, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36173566

RESUMO

Accessible chromatin often represents gene regulatory elements, including promoters and enhancers, essential for gene expression. Assay for Transposase Accessible Chromatin sequencing (ATAC-seq) is one of the most popular techniques to investigate chromatin accessibility across the genome. Here we describe, step by step, a series of optimized experimental methods and bioinformatics pipelines for ATAC-seq analysis. As an example, we present an analysis of murine spermatogenic cells: a method to isolate germ cells, a reaction step using Tn5 transposase to insert sequencing adapters into accessible DNA, a library preparation method for high-throughput sequencing, and bioinformatics analysis of sequencing data. Overall, we introduce a framework of ATAC-seq analysis that can be applied to any cell population to identify cell-type-specific gene regulatory elements and their cis-regulatory networks.


Assuntos
Sequenciamento de Cromatina por Imunoprecipitação , Cromatina , Animais , Cromatina/genética , DNA/genética , Análise de Dados , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Camundongos , Análise de Sequência de DNA/métodos , Transposases/genética , Transposases/metabolismo
7.
Methods Mol Biol ; 2577: 83-92, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36173567

RESUMO

Cleavage Under Target & Release Using Nuclease (CUT&RUN) enables the detection of DNA regions that are bound by a protein of interest. This method is suitable for low-input materials because of the absence of an immunoprecipitation step. However, it sometimes fails when applying it to fragile cells, such as mouse oocytes. Here we describe our low-input CUT&RUN protocol optimized for mouse oocyte and preimplantation embryo samples in which the primary antibody and protein A-MNase binding steps are completed before the cells are bound to Concanavalin A-coated magnetic beads. This modification prevents crush of oocytes and early embryos and unwanted loss of chromatin during CUT&RUN procedures.


Assuntos
Blastocisto , Oócitos , Animais , Blastocisto/metabolismo , Cromatina/metabolismo , Cromossomos , Concanavalina A , Camundongos , Oócitos/metabolismo
8.
Methods Mol Biol ; 2577: 103-122, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36173569

RESUMO

The Spliced TetO REpeAt, MS2 repeat, and INtein sandwiched reporter Gene tag (STREAMING-tag) system enables imaging of nuclear localization as well as the transcription activity of a specific endogenous gene at sub-100-nm resolution in living cells. The use of this system combined with imaging of epigenome states enables a detailed analysis of the impact of epigenome status on transcriptional dynamics. In this chapter, we describe a method for quantifying distances between Nanog gene and clusters of cofactor BRD4 using the STREAMING-tag system in mouse embryonic stem cells.


Assuntos
Fatores de Transcrição , Transcrição Genética , Animais , Camundongos , Células-Tronco Embrionárias Murinas , Proteínas Nucleares , Tecnologia , Fatores de Transcrição/genética
9.
Methods Mol Biol ; 2577: 123-146, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36173570

RESUMO

Precise regulation of gene expression is integral in development. Emerging studies have highlighted that super-enhancers (SEs), which are clusters of multiple enhancers, play critical roles in regulating cell type-specific gene expression via 3D chromatin, thereby defining the cellular identities of given cells. Here we provide optimized bioinformatics pipelines to identify SEs and 3D chromatin contacts. Our pipelines encompass the processing of chromatin immunoprecipitation sequencing (ChIP-seq) data to identify SEs and the processing of genome-wide chromosome conformation capture (Hi-C) data. We can then infer long-range chromatin contacts between SEs and other genomic regions. This integrative computational approach, which can be applied to CUT&RUN and CUT&Tag, alternative technologies to ChIP-seq, allows us to identify genomic locations of SEs and their 3D genome configuration, whereby multiple SEs act in concert. We show an analysis of mouse spermatogenesis as an example of this application.


Assuntos
Cromatina , Elementos Facilitadores Genéticos , Animais , Cromatina/genética , Imunoprecipitação da Cromatina , Sequenciamento de Cromatina por Imunoprecipitação , Biologia Computacional , Camundongos
10.
Methods Mol Biol ; 2577: 161-173, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36173572

RESUMO

Sperm chromatin compaction is physiologically essential for sperm to acquire the fertility. However, this unique structure composed of protamines makes us unable to solubilize the chromatin due to its resistance to sonication and enzymes usually used for chromatin fragmentation in somatic cells. Even when intense enzymatic treatment is applied, it appears to solubilize only certain portions of sperm chromatin presumably because of the heterogeneous properties. To overcome this issue, we previously developed a method to treat the sperm with recombinant nucleoplasmin, a protamine remover in fertilized embryos, followed by sonication. The nucleoplasmin treatment dramatically increased the efficiency of sperm chromatin solubilization, while a relatively large amount of recombinant nucleoplasmin was required. Here, we describe an improvement of nucleoplasmin method with a less amount of recombinant protein and a shorter reaction time.


Assuntos
Cromatina , Proteínas Nucleares , Animais , Cromatina/genética , Cromatina/metabolismo , Masculino , Camundongos , Proteínas Nucleares/metabolismo , Nucleoplasminas/metabolismo , Fosfoproteínas/metabolismo , Protaminas , Proteínas Recombinantes/metabolismo , Sêmen/metabolismo , Espermatozoides/metabolismo
11.
Methods Mol Biol ; 2577: 229-240, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36173577

RESUMO

The CRISPR-Cpf1 also known as Cas12a is an RNA-guided endonuclease similar to CRISPR-Cas9. Combining the CRISPR-Cpf1 with optogenetics technology, we have engineered photoactivatable Cpf1 (paCpf1) to precisely control the genome sequence in a spatiotemporal manner. We also identified spontaneously activated split Cpf1 and thereby developed a potent dCpf1 split activator, which has the potential to activate endogenous target genes. Here we describe a method for optogenetic endogenous genome editing using paCpf1 in mammalian cells. Furthermore, we show a method for endogenous gene activation using dCpf1 split activator in mammalian cells and mice.


Assuntos
Endonucleases , Edição de Genes , Animais , Sistemas CRISPR-Cas/genética , Endonucleases/genética , Endonucleases/metabolismo , Edição de Genes/métodos , Genoma , Mamíferos/metabolismo , Camundongos , RNA , Ativação Transcricional
12.
Methods Mol Biol ; 2577: 243-254, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36173578

RESUMO

In germ cell lines, including early preimplantation embryos, centromeres and pericentromeres are known to show a marked hypomethylation pattern compared to somatic cells. Elucidation of the biological function of this region-specific DNA hypomethylation state, region-specific epigenomic manipulation is essential as an analytical method. We have applied genome editing to show that region-specific DNA methylation can be effectively introduced by a fusion protein, TALE, which recognizes pericentromeres, and SssI, a bacterial CpG methyltransferase. This makes it possible to increase the DNA methylation state of the pericentromeres, which is normally about 20%, to about 60-75%, enabling comparative analysis of the developmental processes of normal embryos with hypomethylated pericentromeres and embryos that have been epigenetically edited to be hypermethylated. In this chapter, we describe a method for introducing DNA methylation into pericentromeres of early mouse embryos by expressing TALE-SssI fusion protein and a method for detecting DNA methylation.


Assuntos
Blastocisto , Metilação de DNA , Animais , Blastocisto/metabolismo , DNA/metabolismo , Edição de Genes/métodos , Metiltransferases/metabolismo , Camundongos
13.
Methods Mol Biol ; 2577: 255-268, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36173579

RESUMO

Epigenetic regulatory mechanisms play an important role in gene silencing and genome stability; therefore, epigenetic mutations cause a variety of diseases. Analysis of the epigenome by next-generation sequencers has revealed many epigenetic mutations in various diseases such as cancer, obesity, diabetes, autism, allergies, immune diseases, and imprinting diseases. Unfortunately, it has been difficult to identify the causative epigenetic mutations because there has been no method to generate animals with target-specific epigenetic mutations. However, it has become possible to generate such animals due to the recent development of epigenome editing technology. Here, we introduce the generation of epigenome-edited mice by target-specific DNA demethylation.


Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Animais , Desmetilação do DNA , Metilação de DNA , Epigênese Genética , Epigenoma , Edição de Genes/métodos , Camundongos
14.
Methods Mol Biol ; 2577: 269-277, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36173580

RESUMO

A new technique called the dCas9-SunTag and scFv-TET1CD epigenome editing system has recently been developed to edit the DNA methylation status of specific genes. The transfection of an all-in-one vector containing this system into cells is feasible and induces the DNA demethylation of specific genes; however, due to the large size of the vector, difficulties are associated with its introduction into mice. We herein used a hydrodynamic tail vein injection (HTVi) to introduce the all-in-one vector into mice for in vivo epigenome editing. HTVi needs to be considered for inducing the targeted DNA demethylation of particular genes in the mouse liver.


Assuntos
Desmetilação do DNA , Cauda , Animais , Metilação de DNA , Hidrodinâmica , Fígado , Camundongos
15.
Methods Mol Biol ; 2559: 31-39, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36180624

RESUMO

Specific cell ablation by the diphtheria toxin (DT) system is widely used to analyze the in vivo function of target cells in mice. In this chapter, we describe the methods of depleting regulatory T cells (Tregs) systemically or selectively in the skin. Since it has been difficult to conclude the importance of tissue-residing Tregs with systemic Treg ablation, we sought to selectively deplete cutaneous Tregs to investigate their function in the skin without the depletion of Tregs in non-target organs. Here, we describe protocols for the depletion of Tregs by the DT system, and subsequent analysis of Tregs in the skin and skin-draining lymph node (dLN) by flow cytometry. This procedure of selective depletion of cutaneous Tregs can be applicable to other tissues and cells, to allow investigation of the role of tissue-resident cells in mice.


Assuntos
Toxina Diftérica , Linfócitos T Reguladores , Animais , Toxina Diftérica/farmacologia , Fatores de Transcrição Forkhead , Imunoterapia , Depleção Linfocítica/métodos , Camundongos , Camundongos Endogâmicos C57BL
16.
Methods Mol Biol ; 2559: 41-49, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36180625

RESUMO

Elucidation of the symbiotic relationship between the host and its gut microbiota is critically important for understanding host pathophysiology. Peripherally derived regulatory T cells (pTregs) are recognized as central to immune homeostasis in the intestine. Moreover, the gut microbiota nourishes the intestinal and systemic immune systems, including pTreg, via their metabolites and other components. Therefore, methods to detect pTreg as well as to analyze the interactions between the gut microbiota and pTreg are important for better understanding of the symbiotic relationship with these microorganisms. Here, we describe a protocol to isolate colonic lamina propria cells and analyze pTregs in mice.


Assuntos
Microbioma Gastrointestinal , Linfócitos T Reguladores , Animais , Colo , Mucosa Intestinal , Intestinos , Camundongos
17.
Methods Mol Biol ; 2559: 51-63, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36180626

RESUMO

Besides their important function in immune homeostasis, autoimmunity, and peripheral tolerance, regulatory T cells (Tregs) also play a crucial role in cancer immune evasion, by assisting tumors to shield from antitumor responses by effector cells. Tregs are recruited to infiltrate tumors and upon finding favourable conditions in the tumor microenvironment, proliferate and suppress effector T cell function thereby promoting tumor escape and growth. In human cancer patients and mouse models, a low ratio of effector T cells to Tregs is a key feature of this immune-suppressive tumor microenvironment and correlates with poor prognosis. This chapter describes protocols for the isolation of tumor-infiltrating lymphocytes (TILs) from solid tumors, their quantification, and phenotyping via flow cytometry to assess the effector T cell:Treg ratio and the expression of relevant markers.


Assuntos
Neoplasias , Linfócitos T Reguladores , Animais , Humanos , Linfócitos do Interstício Tumoral , Camundongos , Neoplasias/patologia , Evasão Tumoral , Microambiente Tumoral
18.
Methods Mol Biol ; 2559: 67-77, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36180627

RESUMO

This chapter shows protocols for the differentiation of peripheral Treg (pTreg) from polyclonal and monoclonal CD4+ T cells. Polyclonal naïve CD4+ T cells can differentiate into pTreg upon adoptive transfer into Foxp3-diphtheria toxin receptor transgenic recipient mice in which endogenous Tregs are transiently depleted by administration of diphtheria toxin before adoptive transfer. Differentiation of monoclonal pTreg is induced through oral delivery of ovalbumin into RAG-deficient DO11.10 mice, in which T cells are ovalbumin specific. We show the isolation of naïve CD4+ T cells by flow cytometry, the administration of ovalbumin in drinking water, and the analysis tools, including an optional protocol for the enrichment of analysis samples in CD4+ T cells using a magnetic purification.


Assuntos
Água Potável , Linfócitos T Reguladores , Animais , Toxina Diftérica , Fatores de Transcrição Forkhead , Fator de Crescimento Semelhante a EGF de Ligação à Heparina , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Ovalbumina
19.
Methods Mol Biol ; 2559: 79-94, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36180628

RESUMO

The transcription factor Foxp3/FOXP3 orchestrates regulatory T (Treg) cell development and function by interacting with numerous target genes and partner proteins. Functional analysis of naturally occurring or engineered Foxp3/FOXP3 mutations has provided important insights into how the complex Foxp3/FOXP3-centered molecular network operates. Here, we describe detailed protocols for retroviral transduction of murine primary conventional CD4+ T cells to determine the impacts of Foxp3 mutations on the Treg-cell-like phenotype and function conferred by Foxp3.


Assuntos
Linfócitos T CD4-Positivos , Fatores de Transcrição Forkhead , Animais , Linfócitos T CD4-Positivos/metabolismo , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Regulação da Expressão Gênica , Camundongos , Retroviridae/genética , Retroviridae/metabolismo , Linfócitos T Reguladores/metabolismo
20.
Methods Mol Biol ; 2559: 95-114, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36180629

RESUMO

The lack of unambiguous Foxp3+ Treg cell-specific surface markers has prompted the development of various transgenic mouse lines with Foxp3-dependent reporter activity, which involved different fluorochromes and transgenic strategies, including coexpression of multiple transgenes, such as Cre recombinase. Since then, Foxp3 transcriptional reporter has proven to be an indispensable tool to identify and isolate viable Foxp3+ Treg cell populations. However, the physiologic Treg cell pool is functionally heterogeneous and consists of intrathymically (tTreg) and peripherally (pTreg) induced Treg cells, which may confound interpretation of data relying on indiscriminatory Foxp3-fluorochrome reporter expressed in all Treg cells. In this chapter, we describe how the dual Foxp3RFP/GFP reporter can be exploited to discriminate both developmental sublineages based on tTreg cell lineage-specific GFP/Cre recombinase activity, in conjunction with Foxp3-driven RFP expression in all Foxp3+ Treg cells, and provide guidelines for experimental design and implementation. We also elaborate on the possibility to exploit GFP/Cre expression of Foxp3RFP/GFP reporter mice for the manipulation of gene expression (activation and inactivation), such as lineage tracing and in vivo ablation of tTreg cells, while sparing pTreg cells.


Assuntos
Corantes Fluorescentes , Linfócitos T Reguladores , Animais , Linhagem da Célula/genética , Corantes Fluorescentes/metabolismo , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Camundongos , Camundongos Transgênicos , Linfócitos T Reguladores/metabolismo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA