Name: co-localization of cell lineage markers and the tomato signal
Uploaded: 2016-12-28T14:00:00.000+00:00
Duration: 10 min 56 s
Description: Watch this Scientific Journal Video about Co-localization of Cell Lineage Markers and the Tomato Signal at JoVE.com

This study was supported by NIH grant DE025014 to JQF.

Todos os protocolos foram revisados ​​e aprovados pelo Comitê Institucional de Animal Care and Use (IACUC) da Texas A &amp; M University College of Dentistry. Reprodução 1. animal <ol><li> Use três modelos animais neste estudo. Para investigar o destino dos condrócitos embrionárias na formação côndilo, primeiro uso Col10a1-Cre 15 ratos e passe com Rosa26 tdTomato (B6; 129S6- Gt (ROSA) 26Sor tm9 (CAG-tdTomato) Hze / J) ratos para obter Col10a1- Cre e Rosa26 tdTomato ratinhos. Em seguida, atravessar esses camundongos com 2.3Col1a1-GFP ratos 16. Use Col10a1-Cre; Rosa26 tdTomato; Ratinhos 2.3Col1a1-GFP para executar as experiências de rastreio linhagem celular (Figura 1B). </li><li> Para estudar o destino dos condrócitos pós-natal, siga o mesmo procedimento como no passo 1.1, mas usar o ERT2 agrecan-cre (Agg-Cre ERT2) 17,18 linha e manter a Agg-Cre ERT2; Rosa26 tdTomato; Ratinhos 2.3Col1a1-GFP para executar as experiências de rastreio linhagem celular (Figura 1C). Injectar tamoxifeno no dia pós-natal 14. </li><li> Para combinar a linhagem celular de rastreamento técnica com imunofluorescência, atravessar ratos Agg-Cre ERT2 e camundongos Rosa26 tdTomato. Use ratinhos que transportam ambos os genes na experiência e injectar as tamoxifeno no dia pós-natal 3 (Figura 1D). </li></ol> 2. Preparação de Material <ol><li> Dissolve-se o pó de tamoxifeno em 10% de etanol e óleo de milho a 90% a uma concentração de 10 mg / ml. A dosagem para injecção é de 75 mg / kg. </li><li> Dissolve-se o pó de paraformaldeído (PFA) em PBS a uma concentração de 4%, com 2 M de hidrato de sódio para ajustar o valor do pH para 7,4. Use 4% de solução PFA para fixar o tecido (o côndilo mandibular e perna são usados ​​aqui) após o sacrifício. Devido à suatoxicidade, lidar com PFA na capa com luvas e uma máscara. </li><li> Dissolve-se o pó de EDTA em água destilada a uma concentração de 10%, com 2 M de hidrato de sódio para ajustar o valor do pH para 7,4. Use 10% EDTA a descalcificação do côndilo mandibular e perna. </li><li> Dissolve-se o pó de sacarose em PBS a concentrações de 15% e 30%. Use a solução de sacarose para desidratar o tecido após descalcificação. </li><li> Dissolve-se o pó de hialuronidase em PBS a uma concentração de 2 mg / ml, pH 5,0. Esta solução é para a recuperação de antigénio de imunofluorescência. </li><li> Utilizar um tubo de 1,5 ml para preparar uma solução de bloqueio que contém 3% de albumina de soro bovino (BSA) e soro de cabra a 20% em PBS para a coloração ou Runx2 DMP1 imunofluorescente. </li><li> Utilizar um tubo de 1,5 mL, para preparar uma solução de anticorpo primário que contém soro de cabra a 2% em PBS para a coloração ou Runx2 DMP1 imunofluorescente. A concentração do anticorpo primário é de 1: 400 para Runx2 e 1: 100 para DMP1. </li><li> Utilizar um tubo de 1,5 mL, para preparara solução de IgG de coelho como controlo para a coloração de imunofluorescência para evitar resultados positivos falsos. A solução contém soro de cabra a 2% em PBS. A concentração do IgG de coelho de controlo Runx2 é de 1: 400; para DMP1, 1: 100. Realizar o experimento e controle coloração simultaneamente. </li><li> Utilizar um tubo de 1,5 ml para preparar uma solução de anticorpo secundário que contém soro de cabra a 2% em PBS para a coloração ou Runx2 DMP1 imunofluorescente. Utilizar o anticorpo secundário com uma diluição de 1: 500. </li></ol> 3. Deslize Preparação para microscopia confocal (Lineage celular Rastreamento Apenas, nenhuma combinação com Imunofluorescência) <ol><li> Injectar tamoxifeno na-cre Agg ERT2 ratos em um ponto de tempo favorável. <ol><li> Em primeiro lugar, remover um mouse de sua gaiola. Em seguida, use o polegar eo dedo indicador esquerdo para agarrar a pele na parte de trás do rato e entregá-lo, expondo o abdômen. Use a mão direita para segurar a seringa. O ponto de entrada óptima para a injecção é a Left ou lado direito do hipogástrio, evitando o fígado e bexiga. </li><li> Manter a seringa paralelo com as pernas traseiras do mouse e injectar por via intraperitoneal. A dosagem para injecção é de 75 mg / kg. Os pesos dos ratos nas idades de 2 semanas, 3 semanas e 4 semanas de idade são de aproximadamente 7-9 g, 11-13 g, e 16-18 g, respectivamente. </li></ol></li><li> Anestesiar os ratos com uma combinação Xilazina / Ketaset. <ol><li> Para preparar a solução de trabalho, em primeiro lugar diluir a xilazina e Ketaset com água destilada a uma concentração de 1 mg / ml e 5 mg / ml, respectivamente. Em seguida, misturar a xilazina e Ketaset em uma proporção de 1: 2. A dosagem para injecção é de 30 ul / g. </li><li> Injectar como no passo 3.1. Confirmar a anestesia por beliscar o tornozelo do mouse. O mouse é inconsciente, se não tem reação. </li></ol></li><li> Perfundir os ratos com PFA 4% após anestesia. <ol><li> Após o mouse perde a consciência, corrigir as quatro pernas do mouse em uma placa de entirely expor o abdômen. </li><li> Saturar o abdômen com etanol 70% e fazer uma excisão da parte inferior do abdómen para o pescoço ao longo da linha média. Comprima e, simultaneamente, puxa a pele para os lados laterais para revelar a membrana peritoneal. Use tesouras de dissecação para fazer uma excisão longitudinal. </li><li> Cortar e remover as costelas frente para expor o coração. Perfurar o coração do ventrículo esquerdo com uma seringa G 22, segure a seringa, e, simultaneamente, cortar uma abertura na aurícula direita. </li><li> Lentamente injetar a PFA 4%, o que é perfundido ao longo do sistema cardiovascular enquanto as ondas de sangue para fora do corte da aurícula direita. O volume do PFA para perfusão é de 1 ml / grama. Execute esta etapa em uma cabine de segurança biológica Classe I que é difícil-canalizado para o sistema de escape edifício. </li></ol></li><li> Retire a pele do rato e colocar todo o corpo para um tubo de centrífuga de polipropileno de 50 ml que contém 40 ml de 4% de PFA para fixar durante a noite a 4 ° C. </li><li>Use tesouras de dissecação e # 3 e # 5 fórceps para remover cuidadosamente a mandíbula e perna traseira do corpo e para remover os músculos e tendões na superfície. Execute esta etapa em uma cabine de segurança biológica Classe I que é difícil-canalizado para o sistema de escape edifício. </li><li> Cortar a mandíbula em duas peças na região distal do terceiro molar. Do mesmo modo, cortar o fémur e a tíbia, na parte média da diáfise os para expor a cavidade da medula óssea, a fim de acelerar a descalcificação. Colocar a parte que inclui o processo de côndilo e condilar e juntamente com a perna traseira em 40 ml de 10% de EDTA para descalcificar a 4 ° C durante 2 - 4 dias num tubo de centrífuga de polipropileno de 50 ml. </li><li> Utilizar 50 ml de 15% de sacarose para desidratar o côndilo e pata traseira durante a noite a 4 ° C num tubo de centrífuga de polipropileno de 50 ml. </li><li> Usar 30% de sacarose para desidratar o côndilo e pata traseira durante a noite a 4 ° C em um tubo de 50 ml de centrífuga de polipropileno. </li><li> Ao longo do plano sagital, incorporar a amostra comOutubro sobre a placa de corte na máquina de criocorte. <ol><li> Horizontalmente deitou a cabeça da mandíbula ou a pata traseira no molde de montagem. Mergulhe o tecido em outubro e deixá-lo na máquina criocorte até o outubro congela. </li><li> Monte o bloco de outubro sobre a placa de corte. Esperar por cerca de 15 minutos antes de cortar para garantir que o OCT é completamente congelado. </li></ol></li><li> Cortar a cabeça da mandíbula e perna em seções de 10 mícrons. Recolher as secções em lâminas e armazenar a -20 ° C. </li><li> Incubar as lâminas numa câmara de 37 ° C para remover a água antes da coloração. </li><li> Lavar as lâminas duas vezes com água destilada durante 5 minutos. </li><li> Limpe a água em torno de cada seção. Use uma caneta barreira hidrofóbica para circundar as seções e soltar DAPI ou solução de montagem anti-fade não-fluorescente dentro do círculo. Estabeleço com cuidado a tampa de deslizamento. </li></ol> 4. imuno coloração para Runx2 e DMP1 NOTA: O IgG controlo é necessário para coloração imuno-histoquímica para evitar sinais de falsos-positivos. A coloração para os grupos experimentais e de controlo necessita de ser realizados simultaneamente. <ol><li> Incubar as lâminas numa câmara de 37 ° C para remover a água antes da coloração. </li><li> Lavar as lâminas duas vezes com água destilada. </li><li> Use a caneta barreira hidrofóbica para circundar todas as seções no slide. A partir deste passo, adicionar toda a solução preparada para dentro do círculo para cobrir completamente as secções. </li><li> Trate as seções com hialuronidase em câmara húmida a 37 ° C durante 30 minutos. O volume da solução (em passos de 4,5 - 4,8) depende do tamanho da secção. Use 50 ml de solução para o côndilo e 100 ul para o osso longo. Lavar com PBST (PBS que contém 0,1% de Tween 20), três vezes. </li><li> Preparar e aplicar a solução de bloqueio a cada secção e incubar numa câmara húmida durante 1 h à temperatura ambiente. </li><li> Incubar as seções com primárioA solução de anticorpo (Runx2 de coelho anti-rato, coelho ou anti-ratinho DMP1) a 4 ° C durante a noite. Lavar com PBS três vezes. </li><li> Incubam-se as secções com solução de anticorpo secundário (anticorpo de cabra anti-coelho, Alexa Fluor 488) durante 2 horas à temperatura ambiente. Lavar com PBS três vezes. </li><li> Limpe a água ao redor da seção e soltar DAPI para dentro do círculo para cobrir a seção sobre o slide. Estabeleço com cuidado a tampa de deslizamento. </li></ol> 5. Microscopia Confocal <ol><li> Capturar imagens de células fluorescentes utilizando um microscópio confocal em comprimentos de onda que vão de 488 uM (verde) a 561 uM (vermelho). Tomar várias imagens empilhadas a 200 Hz (dimensões de 1.024 × 1.024) 19 utilizando 10X, 20X e 63X lentes. </li></ol>

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The authors declare that they have no competing financial interests or other conflicts of interest.

Devido às limitações tecnológicas, é sempre difícil de investigar o comportamento de células in vivo. No entanto, a técnica de linhagem celular de rastreamento está provando ser uma ferramenta poderosa para estudar biologia celular 7-9. Neste estudo, nós melhorar ainda mais este protocolo, combinando-a com a imunofluorescência. Deste modo, o destino da célula pode ser definido por vários marcadores relacionados, que alarga a aplicação do rastreio linhagem. Além disso, esta co-localiza...

Durante o desenvolvimento, a formação de osso endocondral responde por mais de 80% do volume do esqueleto. Acredita-se que começa com a apoptose de condrócitos hipertróf icos. Subsequentemente, as células da medula óssea subjacente invadir e iniciar angiogénese, seguido por nova deposição óssea por marrow- óssea e células derivadas de periósteo 1,2. O destino de células de condrócitos hipertróficas (HCs), no entanto, tem sido um tema de debate durante décadas 3. Inicialmente, foram consideradas HCs ser o final da via de diferenciação de condrócitos, e a apoptose foi geralmente considerado para ser o destino final de HCs. Agora, alguns investigadores sugerem que pelo menos alguns HCs poderia sobreviver e contribuir para a formação de osso endocondral. Embora eles proposto que o crescimento dos condrócitos placa tinha a capacidade de transdiferenciar em osteoblastos com base em ultra-estrutura, coloração imuno-histoquímica, e estudos in vitro, 46, nenhum destes métodos were definitiva em condrócitos demonstrando contribuição para a linhagem dos osteoblastos. A técnica de linhagem celular de rastreamento fornece uma maneira mais rigorosa para estudar o destino celular. Resumidamente falando, uma enzima recombinase, o qual é expresso apenas em um tipo específico de célula, estimula a expressão do gene repórter. Desta forma, este tipo de célula e seus descendentes são permanentemente marcado 7. O sistema Cre-loxP é comumente usado no rastreamento de linhagem. Cre (a enzima recombinase) vai excisar a sequência de paragem entre os dois locais loxP e activar o repórter em uma linha celular específica (Figura 1A). Em alguns casos, o investigador pode escolher um ponto de tempo favorável para activar Cre usando uma droga, tal como tamoxifeno, causando Cre para fundir a uma forma modificada do receptor de estrogénio (Cre ERT2) 8. repórteres fluorescentes tornaram-se o padrão na linhagem rastreamento experimentos porque reduzem drasticamente a complexidadee melhorar a precisão e eficiência da célula destino traçado 8,9. tdTomato está se tornando a melhor escolha entre os repórteres fluorescentes uma vez que tem o mais brilhante proteína fluorescente ea epifluorescência mais forte, tornando-se facilmente visualizado 7 (Figura 1A). Ao utilizar o Rosa26 tdTomato linhagem sistema de detecção, o nosso grupo e outros investigadores mostraram que HCs pode alterar o seu fenótipo em células ósseas durante o desenvolvimento 10-14. Para conseguir isso, nós desenvolvemos dois conjuntos de rastreamento combinações com Rosa26 tdtomato (expressão omnipresente em todas as células) / Cre (específico para condrócitos) ratos: 2.3Col1a1-GFP (específico para osteoblastos) e imunofluorescência (específico para células ósseas). Os dados demonstram que ambos os métodos são meios viáveis para estudar o destino celular in vivo.

<table><tbody><tr><td>Tamoxifen&amp;nbsp;</td><td>Sigma</td><td>T5648</td><td>activate the Cre event&amp;nbsp;</td></tr><tr><td>Paraformaldehyde</td><td>Sigma&amp;nbsp;</td><td>P6148</td><td>fix the sample</td></tr><tr><td>Ethylenediaminetetraacetic acid</td><td>Alfa Aesar</td><td>A10713</td><td>decalcify the hard tissue</td></tr><tr><td>Sucrose&amp;nbsp;</td><td>Sigma</td><td>S0389</td><td>dehydrate the tissue</td></tr><tr><td>Hyaluronidase from bovine testes&amp;nbsp;</td><td>Sigma</td><td>H4272</td><td>retrieve antigen for immunochemical staining</td></tr><tr><td>Bovine serum albumin</td><td>Sigma&amp;nbsp;</td><td>A3059</td><td>blocking solution&amp;nbsp;</td></tr><tr><td>primary antibody for Runx2&amp;nbsp;</td><td>Cell Signal</td><td>D1L7F</td><td>primary antibody for immunochemical staining</td></tr><tr><td>primary antibody for DMP1</td><td>provided by Dr. Chunlin Qin</td><td></td><td>primary antibody for immunochemical staining</td></tr><tr><td>anti-rabbit IgG</td><td>Sigma</td><td>18140</td><td>control for immunochemical staining</td></tr><tr><td>secondary antibody&amp;nbsp;</td><td>Invitrogen</td><td>A11008</td><td>second antibody for immunochemical staining</td></tr><tr><td>OCT</td><td>Tissue-Tek</td><td>4583</td><td>embed the sample for frozen section</td></tr><tr><td>Tween 20</td><td>Fisher Scientific</td><td>BP337</td><td>PBST</td></tr><tr><td>non-fluorescing antifade mountant</td><td>Life technologies</td><td>P36934</td><td>mounting slides</td></tr><tr><td>DAPI</td><td>Life technologies</td><td>P36931</td><td>nuclear staining</td></tr><tr><td>Hydrophobic Barrier Pen</td><td>Vector Laboratories</td><td></td><td>circle the section on the slide for for immunochemical staining&amp;nbsp;</td></tr><tr><td>Xylazine</td><td>AnaSed</td><td></td><td>anesthetization&amp;nbsp;</td></tr><tr><td>Ketaset&amp;nbsp;</td><td>Zoetis</td><td></td><td>anesthetization&amp;nbsp;</td></tr><tr><td>cryosection machine</td><td>Leica</td><td>CM1860 UV</td><td></td></tr><tr><td>confocal microscope</td><td>Leica</td><td>DM6000 CFS&amp;nbsp;</td><td></td></tr></tbody></table>

co-localization of cell lineage markers and the tomato signal

O sistema de rastreamento de linhagem celular foi usado predominantemente em estudos de biologia do desenvolvimento. A utilização da recombinase Cre permite a activação do repórter em uma linha celular específica e toda a progénie. Aqui, utilizou-se a linhagem de células rastreio técnica para demonstrar que os condrócitos transformar directamente em osteoblastos e os osteócitos durante ossos longos e desenvolvimento côndilo mandibular utilizando dois tipos de Cre, Col10a1-Cre e agrecan-Cre ERT2 (Agg-Cre ERT2), cruzados com Rosa26 tdTomato. Ambos Col10 e agrecano marcadores são bem reconhecido para condrócitos. Nesta base, nós desenvolvemos uma nova linhagem de células método de rastreio em conjunto com o destino celular imuno-fluorescente para definir por meio da análise da expressão de marcadores de células específicos. Runx2 (um marcador de células osteogênicas em início de carreira) e protein1 matriz de dentina (DMP1; um marcador para células em estágio final osteogênicos) foramutilizado para identificar as células de osso derivadas de condrócitos e o seu estado de diferenciação. Esta combinação não só amplia a aplicação de rastreio linhagem celular, mas também simplifica a geração de ratinhos compostos. Mais importante, os status de número, a localização e diferenciação de progênie de células-mãe são exibidos simultaneamente, fornecendo mais informações do que a linhagem celular de rastreamento sozinho. Em conclusão, a co-aplicação de técnicas de rastreio de linhagem celular e de imunofluorescência é uma ferramenta poderosa para investigar a biologia de células in vivo.

Condrócitos Diretamente transformar em células ósseas (osteoblastos e osteócitos) no Mandibular Condyle e Long Bone. Agrecano, um gene crítico para chondrogenesis, se expressa principalmente no início e maduros condrócitos 18. Como resultado, a injecção de tamoxifeno às 2 semanas de idade em Agg-Cre ERT2; Camundongos Rosa26 tdTomato activado...

Watch this Scientific Journal Video about Co-localization of Cell Lineage Markers and the Tomato Signal at JoVE.com

Co-localization of Cell Lineage Markers and the Tomato Signal

Nós desenvolvemos dois conjuntos de rastreamento combinações em Rosa26 tdtomato (ubiquamente expressa em todas as células) / Cre (especificamente expresso em condrócitos) camundongos: um com 2.3Col1a1-GFP (específico para osteoblastos) e um com imunofluorescência (específico para células ósseas). Os dados demonstram a transformação directa de condrócitos em células ósseas.

Co-localização de marcadores de células de linhagem e o sinal de tomate

The cell lineage tracing system has been used predominantly in developmental biology studies. The use of Cre recombinase allows for the activation of the ...

co-localization-of-cell-lineage-markers-and-the-tomato-signal

Universidad San Sebastian

Research

JoVE Journal

Developmental Biology

12.1K visualizações.  Texas A&M University College of Dentistry.  Nós desenvolvemos dois conjuntos de rastreamento combinações em Rosa26 tdtomato (ubiquamente expressa em todas as células) / Cre (especificamente expresso em condrócitos) camundongos: um com 2.3Col1a1-GFP (específico para osteoblastos) e um com imunofluorescência (específico para células ósseas). Os dados demonstram a transformação directa de condrócitos em células ósseas. 

Vídeo: Co-localization of Cell Lineage Markers and the Tomato Signal

A Method for Lineage Tracing of Corneal Cells Using Multi-color Fluorescent Reporter Mice

Lineage tracing experiments define the origin, fate and behavior of cells in a specific tissue or organism. This technique has been successfully applied for many decades, revealing seminal findings in developmental biology. More recently, it was adopted by stem cell biologists to identify and track different stem cell populations with minimal experimental intervention. The recent developments in mouse genetics, the availability of a large number of mouse strains, and the advancements in fluorescent microscopy allow the straightforward design of powerful lineage tracing systems for various tissues with basic expertise, using commercially available tools. We have recently taken advantage of this powerful methodology to explore the origin and fate of stem cells at the ocular surface using R26R-Confetti mouse. This model offers a multi-color genetic system, for the expression of 4 fluorescent genes in a random manner. Here we describe the principles of this methodology and provide an adaptable protocol for designing lineage tracing experiments; specifically for the corneal epithelium as well as for other tissues.

In this article we describe the principles for designing and performing a multi-color lineage tracing experiment using R26R-Confetti mice. We provide a specific protocol for tracking corneal epithelial cells which can be modified for other tissues of interest.

Um método para Lineage Tracing de células da córnea Usando Multi-cor repórter fluorescente Mice

Lineage tracing experiments define the origin, fate and behavior of cells in a specific tissue or organism. This technique has been successfully applied ...

Biologia do Desenvolvimento

Cell Lineage Analyses and Gene Function Studies Using Twin-spot MARCM

Mosaic analysis with a repressible cell marker (MARCM) is a positive mosaic labeling system that has been widely applied in Drosophila neurobiological studies to depict intricate morphologies and to manipulate the function of genes in subsets of neurons within otherwise unmarked and unperturbed organisms. Genetic mosaics generated in the MARCM system are mediated through site-specific recombination between homologous chromosomes within dividing precursor cells to produce both marked (MARCM clones) and unmarked daughter cells during mitosis. An extension of the MARCM method, called twin-spot MARCM (tsMARCM), labels both of the twin cells derived from a common progenitor with two distinct colors. This technique was developed to enable the retrieval of useful information from both hemi-lineages. By comprehensively analyzing different pairs of tsMARCM clones, the tsMARCM system permits high-resolution neural lineage mapping to reveal the exact birth-order of the labeled neurons produced from common progenitor cells. Furthermore, the tsMARCM system also extends gene function studies by permitting the phenotypic analysis of identical neurons of different animals. Here, we describe how to apply the tsMARCM system to facilitate studies of neural development in Drosophila.

Here, we present a protocol for a mosaic labeling technique that permits the visualization of neurons derived from a common progenitor cell in two distinct colors. This facilitates neural lineage analysis with the capability of birth-dating individual neurons and studying gene function in the same neurons of different individuals.

Análises celular linhagem e estudos da função de genes utilizando marcm Twin-ponto

Mosaic analysis with a repressible cell marker (MARCM) is a positive mosaic labeling system that has been widely applied in Drosophila neurobiological ...

Confocal Live Imaging of Shoot Apical Meristems from Different Plant Species

The shoot apical meristem (SAM) functions as a conserved stem cell reservoir and it generates almost all aboveground tissues during the postembryonic development. The activity and morphology of SAMs determine important agronomic traits, such as shoot architecture, size and number of reproductive organs, and most importantly, grain yield. Here, we provide a detailed protocol for analyzing both the surface morphology and the internal cellular structure of the living SAMs from different species through laser scanning confocal microscope. The whole procedure from the sample preparation to the acquisition of high resolution three-dimensional (3D) images can be accomplished within as short as 20 minutes. We demonstrate that this protocol is highly efficient for studying not only the inflorescence SAMs of the model species but also the vegetative meristems from different crops, providing a simple but powerful tool to study the organization and development of meristems across different plant species.

This protocol presents how to live image and analyze the shoot apical meristems from different plant species using laser scanning confocal microscopy.

Confocal ao vivo de imagens de Shoot sub-apical de diferentes espécies de plantas

The shoot apical meristem (SAM) functions as a conserved stem cell reservoir and it generates almost all aboveground tissues during the postembryonic ...

Visualization of IL-22-expressing Lymphocytes Using Reporter Mice

Reporter mice have been widely used to observe the localization of expression of targeted genes. This protocol focuses on a strategy to establish a new transgenic reporter mouse model. We chose to visualize interleukin (IL) 22 gene expression because this cytokine has important activities in the intestine, where it contributes to repair tissues damaged by inflammation. Reporter systems offer considerable advantages over other methods of identifying products in vivo. In the case of IL-22, other studies had first isolated cells from tissues and then re-stimulated the cells in vitro. IL-22, which is normally secreted, was trapped inside cells using a drug, and intracellular staining was used to visualize it. This method identifies cells capable of producing IL-22, but it does not determine whether they were doing so in vivo. The reporter design includes inserting a gene for a fluorescent protein (tdTomato) into the IL-22 gene in such a way that the fluorescent protein cannot be secreted and therefore remains trapped inside the producing cells in vivo. Fluorescent producers can then be visualized in tissue sections or by ex vivo analysis through flow cytometry. The actual construction process for the reporter included recombineering a bacterial artificial chromosome that contained the IL-22 gene. This engineered chromosome was then introduced into the mouse genome. Homeostatic IL-22 reporter expression was observed in different mouse tissues, including the spleen, thymus, lymph nodes, Peyer&#39;s patch, and intestine, by flow cytometry analysis. Colitis was induced by T-cell (CD4+CD45RBhigh) transfer, and reporter expression was visualized. Positive T cells were first present in the mesenteric lymph nodes, and then they accumulated inside the lamina propria of the distal small intestine and colon tissues. The strategy using BACs gave good-fidelity reporter expression compared to IL-22 expression, and it is simpler than knock-in procedures.

We describe here a transgenic reporter mouse model to visualize the IL-22-producing cells inside different mouse tissues. This method can be used to track the location of other cytokines or secretary proteins in the mouse.

Visualização de IL-expressando-22 Linfócitos Usando ratos repórter

Reporter mice have been widely used to observe the localization of expression of targeted genes. This protocol focuses on a strategy to establish a new ...

Imunologia e Infecção

Identification of Plasmodesmal Localization Sequences in Proteins In Planta

Plasmodesmata (Pd) are cell-to-cell connections that function as gateways through which small and large molecules are transported between plant cells. Whereas Pd transport of small molecules, such as ions and water, is presumed to occur passively, cell-to-cell transport of biological macromolecules, such proteins, most likely occurs via an active mechanism that involves specific targeting signals on the transported molecule. The scarcity of identified plasmodesmata (Pd) localization signals (PLSs) has severely restricted the understanding of protein-sorting pathways involved in plant cell-to-cell macromolecular transport and communication. From a wealth of plant endogenous and viral proteins known to traffic through Pd, only three PLSs have been reported to date, all of them from endogenous plant proteins. Thus, it is important to develop a reliable and systematic experimental strategy to identify a functional PLS sequence, that is both necessary and sufficient for Pd targeting, directly in the living plant cells. Here, we describe one such strategy using as a paradigm the cell-to-cell movement protein (MP) of the Tobacco mosaic virus (TMV). These experiments, that identified and characterized the first plant viral PLS, can be adapted for discovery of PLS sequences in most Pd-targeted proteins.

Identification of Plasmodesmal Localization Sequences in Proteins In Planta

Plant intercellular connections, the plasmodesmata (Pd), play central roles in plant physiology and plant-virus interactions. Critical to Pd transport are sorting signals that direct proteins to Pd. However, our knowledge about these sequences is still in its infancy. We describe a strategy to identify Pd localization signals in Pd-targeted proteins.

Identificação de sequências de localização Plasmodesmal em proteínas em Planta

Plasmodesmata (Pd) are cell-to-cell connections that function as gateways through which small and large molecules are transported between plant cells. ...

In Situ Hybridization for the Precise Localization of Transcripts in Plants

With the advances in genomics research of the past decade, plant biology has seen numerous studies presenting large-scale quantitative analyses of gene expression. Microarray and next generation sequencing approaches are being used to investigate developmental, physiological and stress response processes, dissect epigenetic and small RNA pathways, and build large gene regulatory networks1-3. While these techniques facilitate the simultaneous analysis of large gene sets, they typically provide a very limited spatiotemporal resolution of gene expression changes. This limitation can be partially overcome by using either profiling method in conjunction with lasermicrodissection or fluorescence-activated cell sorting4-7. However, to fully understand the biological role of a gene, knowledge of its spatiotemporal pattern of expression at a cellular resolution is essential. Particularly, when studying development or the effects of environmental stimuli and mutants can the detailed analysis of a gene's expression pattern become essential. For instance, subtle quantitative differences in the expression levels of key regulatory genes can lead to dramatic phenotypes when associated with the loss or gain of expression in specific cell types. 
Several methods are routinely used for the detailed examination of gene expression patterns. One is through analysis of transgenic reporter lines. Such analysis can, however, become time-consuming when analyzing multiple genes or working in plants recalcitrant to transformation. Moreover, an independent validation to ensure that the transgene expression pattern mimics that of the endogenous gene is typically required. Immunohistochemical protein localization or mRNA in situ hybridization present relatively fast alternatives for the direct visualization of gene expression within cells and tissues. The latter has the distinct advantage that it can be readily used on any gene of interest. In situ hybridization allows detection of target mRNAs in cells by hybridization with a labeled anti-sense RNA probe obtained by in vitro transcription of the gene of interest. 
Here we outline a protocol for the in situ localization of gene expression in plants that is highly sensitivity and specific. It is optimized for use with paraformaldehyde fixed, paraffin-embedded sections, which give excellent preservation of histology, and DIG-labeled probes that are visualized by immuno-detection and alkaline-phosphatase colorimetric reaction. This protocol has been successfully applied to a number of tissues from a wide range of plant species, and can be used to analyze expression of mRNAs as well as small RNAs8-14.

In Situ Hybridization for the Precise Localization of Transcripts in Plants

The in situ hybridization protocol described here allows a direct localization of mRNA and small RNA expression at the cellular level with high sensitivity and specificity. The procedure is optimized for paraffin-embedded plant tissue sections, is applicable to a wide range of plants and tissues, and can be completed within ten days.

Hibridização In Situ para a localização precisa de transcrições em Plantas

With the advances in genomics research of the past decade, plant biology has seen numerous studies presenting large-scale quantitative analyses of gene ...

Biologia

Cell Specific Analysis of Arabidopsis Leaves Using Fluorescence Activated Cell Sorting

After initiation of the leaf primordium, biomass accumulation is controlled mainly by cell proliferation and expansion in the leaves1. However, the Arabidopsis leaf is a complex organ made up of many different cell types and several structures. At the same time, the growing leaf contains cells at different stages of development, with the cells furthest from the petiole being the first to stop expanding and undergo senescence1. Different cells within the leaf are therefore dividing, elongating or differentiating; active, stressed or dead; and/or responding to stimuli in sub-sets of their cellular type at any one time. This makes genomic study of the leaf challenging: for example when analyzing expression data from whole leaves, signals from genetic networks operating in distinct cellular response zones or cell types will be confounded, resulting in an inaccurate profile being generated.
To address this, several methods have been described which enable studies of cell specific gene expression. These include laser-capture microdissection (LCM)2 or GFP expressing plants used for protoplast generation and subsequent fluorescence activated cell sorting (FACS)3,4, the recently described INTACT system for nuclear precipitation5 and immunoprecipitation of polysomes6.
FACS has been successfully used for a number of studies, including showing that the cell identity and distance from the root tip had a significant effect on the expression profiles of a large number of genes3,7. FACS of GFP lines have also been used to demonstrate cell-specific transcriptional regulation during root nitrogen responses and lateral root development8, salt stress9 auxin distribution in the root10 and to create a gene expression map of the Arabidopsis shoot apical meristem11. Although FACS has previously been used to sort Arabidopsis leaf derived protoplasts based on autofluorescence12,13, so far the use of FACS on Arabidopsis lines expressing GFP in the leaves has been very limited4. In the following protocol we describe a method for obtaining Arabidopsis leaf protoplasts that are compatible with FACS while minimizing the impact of the protoplast generation regime. We demonstrate the method using the KC464 Arabidopsis line, which express GFP in the adaxial epidermis14, the KC274 line, which express GFP in the vascular tissue14 and the TP382 Arabidopsis line, which express a double GFP construct linked to a nuclear localization signal in the guard cells (data not shown; Figure 2). We are currently using this method to study both cell-type specific expression during development and stress, as well as heterogeneous cell populations at various stages of senescence.

Cell Specific Analysis of Arabidopsis Leaves Using Fluorescence Activated Cell Sorting

A method for producing Arabidopsis leaf protoplasts that are compatible with fluorescence activated cell sorting (FACS), allowing for studies of specific cell populations. This method is compatible with any Arabidopsis line that expresses GFP in a subset of cells.

Análise de célula específico<em&gt; Arabidopsis</em&gt; Folhas utilizando ordenação células activadas por fluorescência

After initiation of the leaf primordium, biomass accumulation is controlled mainly by cell proliferation and expansion in the leaves1. However, the ...

Lineage Tracing of Inducible Fluorescently-Labeled Stem Cells in the Adult Mouse Brain

A telomerase reverse transcriptase (Tert) lineage-tracing mouse line was developed to investigate the behavior and fate of adult tissue stem cells, by crossing the &#39;Tet-On&#39; system oTet-Cre mouse with a novel reverse tetracycline transactivator (rtTA) transgene linked to the Tert promoter, which we have demonstrated marks a novel population of adult brain stem cells. Here, administration of the tetracycline derivative doxycycline to mTert-rtTA::oTet-Cre mice will indelibly mark a population of cells that express a 4.4 kb fragment of the promoter region of the gene Tert. When combined the Rosa-mTmG reporter, mTert-rtTA::oTet-Cre::Rosa-mTmG mice will express membrane tdTomato (mTomato) until doxycycline treatment induces the replacement of mTomato expression with membrane EGFP (mGFP) in cells that also express Tert. Therefore, when these triple-transgenic lineage tracing mice receive doxycycline (the &#34;pulse&#34; period during which TERT expressing cells are marked), these cells will become indelibly marked mGFP+ cells, which can be tracked for any desirable amount of time after doxycycline removal (the &#34;chase&#34; period), even if Tert expression is subsequently lost. Brains are then perfusion-fixed and processed for immunofluorescence and other downstream applications in order to interpret changes to stem cell activation, proliferation, lineage commitment, migration to various brain niches, and differentiation to mature cell types. Using this system, any rtTA mouse can be mated to oTet-Cre and a Rosa reporter to conduct doxycycline-inducible &#34;pulse-chase&#34; lineage&#160;tracing experiments using markers of stem cells.

The ability to permanently mark stem cells and their progeny with a fluorophore using an inducible transgenic lineage tracing mouse line allows for spatial and temporal analysis of activation, proliferation, migration, and/or differentiation in vivo. Lineage tracing can reveal novel information about lineage commitment, response to intervention(s), and multipotency.

Rastreamento de linhagem de células-tronco fluorescentes indutíveis no cérebro do rato adulto

A telomerase reverse transcriptase (Tert) lineage-tracing mouse line was developed to investigate the behavior and fate of adult tissue stem cells, by ...

Neurociência

Imaging Spatial Reorganization of a MAPK Signaling Pathway Using the Tobacco Transient Expression System

Visualization of dynamic signaling events in live cells has been a challenge. We expanded an established transient expression system, the biomolecular fluorescent complementation (BiFC) assay in tobacco epidermal cells, from testing protein-protein interaction to monitoring spatial distribution of signal transduction in plant cells. In this protocol, we used the BiFC assay to show that the interaction and the signaling between the Arabidopsis MAPKKK YODA and MAPK6 occur at the plasma membrane. When the scaffold protein BASL was co-expressed, the YODA-MAPK interaction redistributed and spatially co-polarized with CFP-BASL. This modified tobacco expression system allows for quick examination of signaling localization and dynamic changes (less than 4 days) and can accommodate multiple of fluorescent protein colors (at least three). We also presented detailed methods to quantify protein distribution (asymmetric spatial localization, or &#34;polarization&#34;) in tobacco cells. This advanced tobacco expression system has a potential to be used widely for quick testing of dynamic signaling events in live plant cells.

At the subcellular level, signaling events are dynamically modulated by developmental and environmental cues. Here we describe a protocol that employs the tobacco transient expression system to monitor dynamic protein-protein interaction and to disclose spatial organization of signal transduction in plant cells.

Imagiologia reorganização espacial de uma via de sinalização MAPK Utilizar o sistema de expressão transitória do Tabaco

Visualization of dynamic signaling events in live cells has been a challenge. We expanded an established transient expression system, the biomolecular ...

Lineage Tracing and Clonal Analysis in Developing Cerebral Cortex Using Mosaic Analysis with Double Markers (MADM)

Beginning from a limited pool of progenitors, the mammalian cerebral cortex forms highly organized functional neural circuits. However, the underlying cellular and molecular mechanisms regulating lineage transitions of neural stem cells (NSCs) and eventual production of neurons and glia in the developing neuroepithelium remains unclear. Methods to trace NSC division patterns and map the lineage of clonally related cells have advanced dramatically. However, many contemporary lineage tracing techniques suffer from the lack of cellular resolution of progeny cell fate, which is essential for deciphering progenitor cell division patterns. Presented is a protocol using mosaic analysis with double markers (MADM) to perform in vivo clonal analysis. MADM concomitantly manipulates individual progenitor cells and visualizes precise division patterns and lineage progression at unprecedented single cell resolution. MADM-based interchromosomal recombination events during the G2-X phase of mitosis, together with temporally inducible CreERT2, provide exact information on the birth dates of clones and their division patterns. Thus, MADM lineage tracing provides unprecedented qualitative and quantitative optical readouts of the proliferation mode of stem cell progenitors at the single cell level. MADM also allows for examination of the mechanisms and functional requirements of candidate genes in NSC lineage progression. This method is unique in that comparative analysis of control and mutant subclones can be performed in the same tissue environment in vivo. Here, the protocol is described in detail, and experimental paradigms to employ MADM for clonal analysis and lineage tracing in the developing cerebral cortex are demonstrated. Importantly, this protocol can be adapted to perform MADM clonal analysis in any murine stem cell niche, as long as the CreERT2 driver is present.

Lineage Tracing and Clonal Analysis in Developing Cerebral Cortex Using Mosaic Analysis with Double Markers MADM

A protocol to perform lineage tracing and functional genetic analysis of candidate genes at a single cell level using mosaic analysis with double markers (MADM) is presented. MADM clonal analysis provides a quantitative framework to measure the proliferative behavior, cellular output, and lineage relationship of individual progenitors and their daughter cells.

Rastreamento de linhagem e análise clonal no desenvolvimento de córtex cerebral usando análise de mosaico com marcadores duplos (MADM)

Beginning from a limited pool of progenitors, the mammalian cerebral cortex forms highly organized functional neural circuits. However, the underlying ...