Agradecemos a Chunxin Wang para a construção de pTSO2:: GUS e para fotos na Figura 1, Detlef Weigel para compartilhar o protocolo GUS-infalível, Paja Sijacic, e Courtney Hollender para comentários úteis. Pesquisa em laboratório Z. L s é apoiado pela National Science Foundation EUA (IOB0616096 e MCB0744752). ZL é parcialmente financiado pela Universidade de Maryland Estação Experimental Agrícola.

I. Floral-dip transformação de Arabidopsis thaliana <ol><li> Aproximadamente 1 mês antes da transformação, semear Arabidopsis para 4-8 vasos (5 polegadas potes quadrados), com cerca de 10-15 plantas por vaso. Para plantas com fertilidade normal, 4 potes de plantas por construir plasmídeo será suficiente. Se alguém tem de transformar Arabidopsis mutantes com a fertilidade reduzida, aumento do número de potes em conformidade. </li><li> As plantas são cultivadas em condições normais (16 horas luz e 8 horas escuro a 22 ° C). Se as plantas são cultivadas em dia mais curto ou mais baixa temperatura, eles vão demorar mais tempo a flor e mais tempo para estar pronto para a transformação. Cuidados com as plantas bom garantir plantas saudáveis, que são essenciais para a transformação de sucesso. </li><li> Quando as plantas têm apenas parafusados ​​e começou a florescer. Eles estão agora prontos para a transformação. Para aumentar a eficiência de transformação, 3-4 dias antes da transformação, corte os ramos principais da inflorescência, logo que eles têm aparafusado para incentivar mais a formação secundária atirar. Regar as plantas um dia antes da transformação. Isso garante que o solo não irá absorver media infiltração muito durante floral-dipping. </li><li> Dois dias antes da transformação, inocular LB 40ml contendo antibióticos apropriados (no caso de pTSO2:: GUS, 50 mg / ml kanamicina) com Agrobacterium tumefaciens GV3101 carregando o pTSO2:: GUS construir. O pTSO2:: GUS é clonado no vetor pBIN20 que possui NPTII gene que confere resistência à canamicina 3. Crescer durante a noite em uma incubadora de agitação a 28-30 ° C. </li><li> No dia seguinte, 8ml transferência da cultura durante a noite em 400ml de LB contendo canamicina 50 mcg / ml. Crescer durante a noite em uma incubadora de agitação a 28-30 ° C. </li><li> No dia da transformação, quando o OD 600 da cultura Agrobacterium atinge aproximadamente 0,8 (OD entre 0,5 para 1 600 são aceitáveis), girar a cultura Agrobacterium durante a noite a por 8 minutos a 5000rpm. </li><li> Ressuspender o sedimento em Agrobacterium 1 L (litro) de meios de infiltração (10mM MgCl 2, 5% de sacarose, 0,44 mm 6-benziladenina (BA), 0,3% Silwet L-77, 1x solução Gamborg s vitaminas e água esterilizada). O meio não precisa ser autoclavados, mas precisa ser feito fresco. Despeje a 1 L médio de infiltração Agrobacterium contendo em um prato (como 8 &quot;x15&quot; x2 &quot;prato de vidro Pyrex). </li><li> Inverter o pote (plantas não vai cair) e mergulhe todas as partes aéreas das plantas na solução de infiltração, segure por 5 minutos um pote de cada vez. Embora todas as partes aéreas da planta estão imersos no meio de infiltração, evitar que o solo absorva qualquer um dos meios de infiltração para reduzir as chances de crescimento de fungos no solo. </li><li> Após imersão, colocar todos os potes mergulhado em seu lado em várias camadas de papel toalha em uma bandeja. As toalhas de papel absorver quantidade de acesso dos meios de comunicação de infiltração. Cubra a bandeja com filme plástico para garantir alta umidade. Coloque a bandeja em câmara de crescimento vegetal. </li><li> No dia seguinte, retire a capa de plástico e toalhas de papel, e potes de lugar na vertical. Não regue as plantas para 4-5 mais dias. Depois, manter as plantas normalmente até que definir sementes e coletar sementes a granel. </li><li> Preparar placas (placa de petri 150x15mm) com meio MS contendo 50 mg / ml kanamicina (peso 2.2g Murashige e Skoog sal basal sem vitamina, dissolver em 500 ml de água pH, para 5,8 com NaOH 1M, acrescentar 4 gramas de ágar, autoclave, fresco e adicionar canamicina a 50 mg / ml concentração final). Placas são mantidos em 4 ° C até ser necessário. </li><li> Selecionar para resistentes aos antibióticos plantas transgênicas, é preciso 20 mil sementes de tela no mínimo. 0,1 grama é de cerca de 5000 sementes. Peso e estimar a quantidade de sementes. </li><li> Esterilizar sementes por lavá-los em 15 tubos Falcon ml com etanol 70% por 30 segundos. Lave as sementes com água estéril uma vez. Embeba as sementes por 30 segundos em uma solução contendo diluição de 1:10 de loja, comprei lixívia a 5% (Clorox). Enxaguar as sementes com água estéril 3-6 vezes. Na capa estéril, pipeta cerca de 5000 sementes em cada placa de MS (canamicina), espalhar as sementes uniformemente sobre a placa, selar as placas com fita Micropore 3M para evitar a contaminação. </li><li> Incubar as placas a 4 ° C no escuro por 3-4 dias, em seguida, transferir as placas para uma câmara (poderia ser a câmara de crescimento mesma planta). Após cerca de 14 dias, as mudas transgênicas ficar verde, mas os não-transgênicos são empalidecendo e morrer. Transferência das mudas transgênicas para o solo, diminuindo puxar as raízes para fora do meio e colocou-os no solo. </li></ol> II. Examinando pTSO2:: padrões de expressão GUS <ol><li> Colheita pTSO2:: GUS mudas transgênicas e colocá-los em acetona 90% a frio em um frasco de cintilação de vidro ou tubos de microcentrífugaque são mantidos no gelo. Placas de poliestireno de microtitulação (não polipropileno) também pode ser usado se analisa um grande número de amostras. </li><li> Depois de todas as amostras são colhidas, coloque os frascos em temperatura ambiente por 20 minutos. Durante este tempo compõem tampão fresco sem coloração x-Glic (0,2% Triton x-100, 50 mM tampão pH7.2 NaHPO 4, 2mM ferrocianeto de potássio, potássio ferricianeto 2mM) e colocar no gelo. </li><li> Remover acetona a partir das amostras e adicione o buffer de coloração. </li><li> Make up x-Glic solução corante (0,2% Triton x-100, 50 mM NaHPO 4 buffer pH7.2, potássio 2mM Ferrocianeto, potássio 2mM ferricianeto, 2mM x-glic). Remova o tampão de coloração de amostras e adicione o buffer de coloração contendo x-Glic às amostras. </li><li> Infiltrar as amostras sob vácuo por 15 a 20 minutos. Liberar o vácuo devagar e verifique se todas as amostras afundar sob a superfície da solução corante. Se necessário, repita a infiltração até que todas as amostras após afundar o vácuo é liberado. </li><li> Incubar as amostras a 37 ° C durante a noite no buffer coloração com x-Glic. </li><li> Remover amostras de incubadora e remover manchas de buffer. Lavar as amostras em série de etanol sucessivas (20%, 35% e etanol 50%) em temperatura ambiente por 30 minutos cada mudança. </li><li> Corrigir os tecidos em FAA fixador (etanol 50%, formaldeído 5%, 10% de ácido acético, água de descanso) por 30 minutos em temperatura ambiente. Remover FAA e adicionar etanol 70%. Neste ponto, os tecidos podem ser visualizados e fotografados em um microscópio de dissecação equipado com uma câmera digital. A cor azul escura indica onde TSO2 é expressa (Figura 1). Alternativamente, os tecidos podem ser armazenados a 4 ° C para posterior visualização. <img src="/files/ftp_upload/1952/1952fig1.jpg" alt="Figura 1" /> Figura 1 pTSO2:.: GUS expressão em mudas transgênicas (A), raízes lateral (B) e ponta da raiz (C). Note-se que TSO2 promotor é altamente ativo em tecidos jovens e dividindo. O padrão de expressão esporádica mostrado em (C) é típico de genes expressos em fases específicas do ciclo celular. </li></ol> Resultados representante Quando feito corretamente, a eficiência de transformação deve ser de aproximadamente 0,1-0,2%. Em outra palavra, deve-se obter mudas transgênicas 10/05 por triagem cada 5000 sementes. Em média, cerca de 100 linhagens transgênicas podem ser obtidas através da transformação 4 potes de plantas do tipo selvagem. Para mudas transgênicas contendo o pTSO2:: GUS construir 3, cor azul escuro refletindo a atividade GUS é encontrada em células que se dividem ativamente incluindo folhas jovens, rebentos, ponta da raiz, e lateral de raiz primórdios (Figura 1). O padrão não-uniforme esporádicos é característico do ciclo celular fase específica de expressão.

<ol>
	<li>Clough, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Floral+dip:+agrobacterium-mediated+germ+line+transformation.">Floral dip: agrobacterium-mediated germ line transformation.</a> Methods Mol Biol. 286, 91-91 (2005).</li><li>Clough, S. J., Bent, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Floral+dip:+a+simplified+method+for+Agrobacterium-mediated+transformation+of+Arabidopsis+thaliana.">Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana.</a> Plant J. 16 (6), 735-735 (1998).</li><li>Wang, C., Liu, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arabidopsis+ribonucleotide+reductases+are+critical+for+cell+cycle+progression,+DNA+damage+repair,+and+plant+development.">Arabidopsis ribonucleotide reductases are critical for cell cycle progression, DNA damage repair, and plant development.</a> Plant Cell. 18 (2), 350-350 (2006).</li></ol>

Não há conflitos de interesse declarados.

A eficiência de transformação é determinada por muitos fatores diferentes, que são discutidos abaixo: <ol><li> A saúde das plantas e sua idade é de importância primordial. Cerca de um mês de idade plantas produzindo inúmeros botões florais imaturos são ideais. Aparando os galhos primária para encorajar a formação secundária dia 04/03 tiro antes da transformação irá aumentar a eficiência de transformação. Plantas mais velhas dará origem a transformantes, mas a uma taxa inferior. </li><li> Ferti...

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floral-dip transformation of arabidopsis thaliana to examine ptso2::&beta;-glucuronidase reporter gene expression

A capacidade de introduzir genes estranhos em um organismo é a base para a biologia moderna e biotecnologia. Na planta de floração modelo<em&gt; Arabidopsis thaliana</em&gt;, O floral-dip método de transformação<sup&gt; 02/01</sup&gt; Substituiu todos os métodos anteriores devido à sua simplicidade, eficiência e baixo custo. Especificamente, brotos de flores jovens<em&gt; Arabidopsis</emPlantas&gt; são mergulhados em uma solução de<em&gt; Agrobacterium tumefaciens</em&gt; Carregando construções específicas de plasmídeo. Após imersão, as plantas são devolvidos para o crescimento normal e sementes de rendimento, uma pequena porcentagem dos quais são transformados com o gene estranho e pode ser selecionado para em meio contendo antibióticos. Este método floral-dip significativamente facilitado<em&gt; Arabidopsis</em&gt; Pesquisa e contribuíram muito para nossa compreensão da função dos genes da planta. Neste estudo, usamos o floral-dip método para transformar um gene repórter,<em&gt; Β-glucuronidase (GUS)</em&gt;, Sob o controle de<em&gt; TSO2</em&gt; Promotor.<em&gt; TSO2</em&gt;, Codificação para a ribonucleotídeo redutase (RNR) subunidade pequena<sup&gt; 3</sup&gt;, É um gene do ciclo celular regulados essencial para a biossíntese dNDP na fase S do ciclo celular. Exame de<em&gt; GUS</em&gt; Expressão em transgênicos<em&gt; Arabidopsis</em&gt; Mudas mostra que<em&gt; TSO2</em&gt; É expressa em divisão ativa tecidos. O método relatado experimental e materiais podem ser facilmente adaptadas não só para a investigação, mas também para a educação na escola e na faculdade.

Watch this Scientific Journal Video about Floral-dip Transformation of Arabidopsis thaliana to Examine pTSO2::Î²-glucuronidase Reporter Gene Expression at JoVE.com

Floral-dip Transformation of Arabidopsis thaliana to Examine pTSO2::&amp;#946;-glucuronidase Reporter Gene Expression

Este artigo ilustra o floral-dip método de<em&gt; Agrobacterium tumefaciens</em&gt; Mediada por transformação de<em&gt; Arabidopsis thaliana</em&gt;. Pela introdução de um ciclo celular regulados promotor-repórter,<em&gt; PTSO2:: β-glucuronidase (GUS)</em&gt;, Em<em&gt; Arabidopsis</em&gt;, Que ilustra como um detecta GUS expressão repórter em mudas transgênicas.

Floral-dip Transformação de Arabidopsis thaliana Examine a PTSO2:: β-glucuronidase Expressão Gênica Reporter

The ability to introduce foreign genes into an organism is the foundation for modern biology and biotechnology. In the model flowering plant Arabidopsis ...

floral-dip-transformation-arabidopsis-thaliana-to-examine-ptso2

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Biology

Floral-dip Transformation of Arabidopsis thaliana to Examine pTSO2::&beta;-glucuronidase Reporter Gene Expression

35.8K visualizações.  University of Maryland College Park. Este artigo ilustra o floral-dip método de Agrobacterium tumefaciens Mediada por transformação de Arabidopsis thaliana. Pela introdução de um ciclo celular regulados promotor-repórter, PTSO2:: β-glucuronidase (GUS), Em Arabidopsis, Que ilustra como um detecta GUS expressão repórter em mudas transgênicas.

Vídeo: Floral-dip Transformation of Arabidopsis thaliana to Examine pTSO2::&#946;-glucuronidase Reporter Gene Expression

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell  electroporation system and Gene Pulser  electroporation buffer were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

Elaboração de culturas de células primárias hematopoéticas de medula óssea de camundongos para Eletroporação

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

Biologia

Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell electroporation system and Gene Pulser electroporation buffer (Bio-Rad) were specifically developed to easily transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells. We will demonstrate how to perform a simple experiment to quickly identify the best electroporation conditions. We will demonstrate how to run several samples through a range of electroporation conditions so that an experiment can be conducted at the same time as optimization is performed. We will also show how optimal conditions identified using 96-well electroporation plates can be used with standard electroporation cuvettes, facilitating the switch from electroporation plates to electroporation cuvettes while maintaining the same electroporation efficiency. In the video, we will also discuss some of the key factors that can lead to the success or failure of electroporation experiments.

This procedure shows how to use the Gene Pulser MXcell electroporation system to rapidly and easily identify the best electroporation conditions for mouse embryonic fibroblasts (MEFs) or other primary cells. Considerations for troubleshooting are also discussed in the associated video.

Usando o Sistema de Eletroporação Gene Pulser MXcell para transfectar pilhas com Alta Eficiência

Using an Automated Cell Counter to Simplify Gene Expression Studies: siRNA Knockdown of IL-4 Dependent Gene Expression in Namalwa Cells

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only to study the effects of downregulating single genes, but also to interrogate signaling pathways and other complex interaction networks.  These pathway analyses require both the use of relevant cellular models and methods that cause less perturbation to the cellular physiology.  Electroporation is increasingly being used as an effective way to introduce siRNA and other nucleic acids into difficult to transfect cell lines and primary cells without altering the signaling pathway under investigation. There are multiple critical steps to a successful siRNA experiment, and there are ways to simplify the work while improving the data quality at several experimental stages.  To help you get started with your siRNA mediated gene knockdown project, we will demonstrate how to perform a pathway study complete from collecting and counting the cells prior to electroporation through post transfection real-time PCR gene expression analysis.  The following study investigates the role of the transcriptional activator STAT6 in IL-4 dependent gene expression of CCL17 in a Burkitt lymphoma cell line (Namalwa).  The techniques demonstrated are useful for a wide range of siRNA-based experiments on both adherent and suspension cells.  We will also show how to streamline cell counting with the TC10 automated cell counter, how to electroporate multiple samples simultaneously using the MXcell electroporation system, and how to simultaneously assess RNA quality and quantity with the Experion automated electrophoresis system.

This procedure describes a quick and easy workflow to introduce siRNA into difficult to transfect cell lines and follow gene expression by real-time PCR. Use of an automated cell counter, multi-well electroporation plate, and automated electrophoresis station provide quick and reliable results without the need for expensive robotic handling.

Usando um contador de células automatizadas para simplificar Estudos da Expressão Gênica: Knockdown siRNA de Expressão IL-4 Gene Dependente em células NAMALWA

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only ...

Investigating Tissue- and Organ-specific Phytochrome Responses using FACS-assisted Cell-type Specific Expression Profiling in Arabidopsis thaliana

Light mediates an array of developmental and adaptive processes throughout the life cycle of a plant. Plants utilize light-absorbing molecules called photoreceptors to sense and adapt to light. The red/far-red light-absorbing phytochrome photoreceptors have been studied extensively. Phytochromes exist as a family of proteins with distinct and overlapping functions in all higher plant systems in which they have been studied1. Phytochrome-mediated light responses, which range from seed germination through flowering and senescence, are often localized to specific plant tissues or organs2. Despite the discovery and elucidation of individual and redundant phytochrome functions through mutational analyses, conclusive reports on distinct sites of photoperception and the molecular mechanisms of localized pools of phytochromes that mediate spatial-specific phytochrome responses are limited. We designed experiments based on the hypotheses that specific sites of phytochrome photoperception regulate tissue- and organ-specific aspects of photomorphogenesis, and that localized phytochrome pools engage distinct subsets of downstream target genes in cell-to-cell signaling. We developed a biochemical approach to selectively reduce functional phytochromes in an organ- or tissue-specific manner within transgenic plants. Our studies are based on a bipartite enhancer-trap approach that results in transactivation of the expression of a gene under control of the Upstream Activation Sequence (UAS) element by the transcriptional activator GAL43. The biliverdin reductase (BVR) gene under the control of the UAS is silently maintained in the absence of GAL4 transactivation in the UAS-BVR parent4. Genetic crosses between a UAS-BVR transgenic line and a GAL4-GFP enhancer trap line result in specific expression of the BVR gene in cells marked by GFP expression4. BVR accumulation in Arabidopsis plants results in phytochrome chromophore deficiency in planta5-7. Thus, transgenic plants that we have produced exhibit GAL4-dependent activation of the BVR gene, resulting in the biochemical inactivation of phytochrome, as well as GAL4-dependent GFP expression. Photobiological and molecular genetic analyses of BVR transgenic lines are yielding insight into tissue- and organ-specific phytochrome-mediated responses that have been associated with corresponding sites of photoperception4, 7, 8. Fluorescence Activated Cell Sorting (FACS) of GFP-positive, enhancer-trap-induced BVR-expressing plant protoplasts coupled with cell-type-specific gene expression profiling through microarray analysis is being used to identify putative downstream target genes involved in mediating spatial-specific phytochrome responses. This research is expanding our understanding of sites of light perception, the mechanisms through which various tissues or organs cooperate in light-regulated plant growth and development, and advancing the molecular dissection of complex phytochrome-mediated cell-to-cell signaling cascades.

Investigating Tissue- and Organ-specific Phytochrome Responses using FACS-assisted Cell-type Specific Expression Profiling in Arabidopsis thaliana

The molecular basis of spatial-specific phytochrome responses is being investigated using transgenic plants that exhibit tissue- and organ-specific phytochrome deficiencies. The isolation of specific cells exhibiting induced phytochrome chromophore depletion by Fluorescence-Activated Cell Sorting followed by microarray analyses is being utilized to identify genes involved in spatial-specific phytochrome responses.

Investigando Tecido e órgãos específicos Responses Fitocromo usando FACS assistida Célula tipo Profiling expressão específica no Arabidopsis thaliana</em

Light mediates an array of developmental and adaptive processes throughout the life cycle of a plant. Plants utilize light-absorbing molecules called ...

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.
Telomerase is a specialized reverse transcriptase1 that adds short DNA repeats, called telomeres, to the 3' end of linear chromosomes2 that serve to protect them from end-to-end fusion and degradation. Following DNA replication, a short segment is lost at the end of the chromosome3 and without telomerase, cells continue dividing until eventually reaching their Hayflick Limit4. Additionally, telomerase is dormant in most somatic cells5 in adults, but is active in cancer cells6 where it promotes cell immortality7.
The minimal telomerase enzyme consists of two core components: the protein subunit (TERT), which comprises the catalytic subunit of the enzyme and an integral RNA component (TER), which contains the template TERT uses to synthesize telomeres8,9. Prior to 2008, only structures for individual telomerase domains had been solved10,11. A major breakthrough in this field came from the determination of the crystal structure of the active12, catalytic subunit of T. castaneum telomerase, TcTERT1.
Here, we present methods for producing large quantities of the active, soluble TcTERT for structural and biochemical studies, and the reconstitution of the telomerase RNP complex in vitro for telomerase activity assays. An overview of the experimental methods used is shown in Figure 1.

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.

Na reconstituição in vitro da atividade T. Telomerase castaneum

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more ...

Histochemical Staining of Arabidopsis thaliana Secondary Cell Wall Elements

Arabidopsis thaliana is a model organism commonly used to understand and manipulate various cellular processes in plants, and it has been used extensively in the study of secondary cell wall formation. Secondary cell wall deposition occurs after the primary cell wall is laid down, a process carried out exclusively by specialized cells such as those forming vessel and fiber tissues. Most secondary cell walls are composed of cellulose (40&#8211;50%), hemicellulose (25&#8211;30%), and lignin (20&#8211;30%). Several mutations affecting secondary cell wall biosynthesis have been isolated, and the corresponding mutants may or may not exhibit obvious biochemical composition changes or visual phenotypes since these mutations could be masked by compensatory responses. Staining procedures have historically been used to show differences on a cellular basis. These methods are exclusively visual means of analysis; nevertheless their role in rapid and critical analysis is of great importance. Congo red and calcofluor white are stains used to detect polysaccharides, whereas M&#228;ule and phloroglucinol are commonly used to determine differences in lignin, and toluidine blue O is used to differentially stain polysaccharides and lignin. The seemingly simple techniques of sectioning, staining, and imaging can be a challenge for beginners. Starting with sample preparation using the A. thaliana model, this study details the protocols of a variety of staining methodologies that can be easily implemented for observation of cell and tissue organization in secondary cell walls of plants.

Histochemical Staining of Arabidopsis thaliana Secondary Cell Wall Elements

Plant cell wall composition varies between tissue types and can include lignin, cellulose, hemicelluloses, and pectin. Various staining techniques have been developed to visualize differences at the cell-type level. This paper is a compilation of commonly used cell wall staining techniques.

Coloração histoquímica de<em&gt; Arabidopsis thaliana</em&gt; Celular Secundária parede Elements

Arabidopsis thaliana is a model organism commonly used to understand and manipulate various cellular processes in plants, and it has been used extensively ...

Analysis of Global RNA Synthesis at the Single Cell Level following Hypoxia

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a particular transcriptional program in order to mount an appropriate and coordinated cellular response. The cell possesses several oxygen sensor enzymes that require molecular oxygen as cofactor for their activity. These range from prolyl-hydroxylases to histone demethylases. The majority of studies analyzing cellular responses to hypoxia are based on cellular populations and average studies, and as such single cell analysis of hypoxic cells are seldom performed. Here we describe a method of analysis of global RNA synthesis at the single cell level in hypoxia by using Click-iT RNA imaging kits in an oxygen controlled workstation, followed by microscopy analysis and quantification.&nbsp; Using cancer cells exposed to hypoxia for different lengths of time, RNA is labeled and measured in each cell. This analysis allows the visualization of temporal and cell-to-cell changes in global RNA synthesis following hypoxic stress.

We describe a technique for analysis of global RNA synthesis in hypoxia using imaging. Click-chemistry labeling of RNA has not previously been performed under hypoxia and allows visualization of global RNA changes at the single cell level. This approach complements the existing averaged RNA techniques, allowing direct visualization of cell-to-cell changes in global RNA synthesis.

Análise da Global RNA Síntese no nível da célula única seguinte hipóxia

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a ...

Floral-Dip Transformation of Flax (Linum usitatissimum) to Generate Transgenic Progenies with a High Transformation Rate

Agrobacterium-mediated plant transformation via floral-dip is a widely used technique in the field of plant transformation and has been reported to be successful for many plant species. However, flax (Linum usitatissimum) transformation by floral-dip has not been reported. The goal of this protocol is to establish that Agrobacterium and the floral-dip method can be used to generate transgenic flax. We show that this technique is simple, inexpensive, efficient, and more importantly, gives a higher transformation rate than the current available methods of flax transformation.
In summary, inflorescences of flax were dipped in a solution of Agrobacterium carrying a binary vector plasmid (T-DNA fragment plus the Linum Insertion Sequence, LIS-1) for 1 - 2 min. The plants were laid flat on their side for 24 hr. Then, plants were maintained under normal growth conditions until the next treatment. The process of dipping was repeated 2 - 3 times, with approximately 10 - 14 day intervals between dipping. The T1 seeds were collected and germinated on soil. After approximately two weeks, treated progenies were tested by direct PCR; 2 - 3 leaves were used per plant plus the appropriate T-DNA primers. Positive transformants were selected and grown to maturity. The transformation rate was unexpectedly high, with 50 - 60% of the seeds from treated plants being positive transformants. This is a higher transformation rate than those reported for Arabidopsis thaliana and other plant species, using floral-dip transformation. It is also the highest, which has been reported so far, for flax transformation using other methods for transformation.

Floral-Dip Transformation of Flax Linum usitatissimum to Generate Transgenic Progenies with a High Transformation Rate

Here, we present a protocol to transform flax using Agrobacterium-mediated plant transformation via floral-dip. This protocol is simple to perform and inexpensive, yet yields a higher transformation rate than the current available methods for flax transformation.

Floral-Dip Transformação de linho (<em&gt; Linum usitatissimum</em&gt;) Para gerar Transgênicos Progênies com uma alta taxa de Transformação

Agrobacterium-mediated plant transformation via floral-dip is a widely used technique in the field of plant transformation and has been reported to be ...

Visualizing Stromule Frequency with Fluorescence Microscopy

Stromules, or "stroma-filled tubules", are narrow, tubular extensions from the surface of the chloroplast that are universally observed in plant cells but whose functions remain mysterious. Alongside growing attention on the role of chloroplasts in coordinating plant responses to stress, interest in stromules and their relationship to chloroplast signaling dynamics has increased in recent years, aided by advances in fluorescence microscopy and protein fluorophores that allow for rapid, accurate visualization of stromule dynamics. Here, we provide detailed protocols to assay stromule frequency in the epidermal chloroplasts of Nicotiana benthamiana, an excellent model system for investigating chloroplast stromule biology. We also provide methods for visualizing chloroplast stromules in vitro by extracting chloroplasts from leaves. Finally, we outline sampling strategies and statistical approaches to analyze differences in stromule frequencies in response to stimuli, such as environmental stress, chemical treatments, or gene silencing. Researchers can use these protocols as a starting point to develop new methods for innovative experiments to explore how and why chloroplasts make stromules.

Protocols to investigate the dynamics of chloroplast stromules, the stroma-filled tubules that extend from the surface of chloroplasts, are described.

Visualizando Frequency Stromule com microscopia de fluorescência

Stromules, or "stroma-filled tubules", are narrow, tubular extensions from the surface of the chloroplast that are universally observed in plant cells but ...

Investigação espaço-temporal da dinâmica ERL1 via imagem confocal

Image-Based Methods to Study Membrane Trafficking Events in Stomatal Lineage Cells

In eukaryotic cells, membrane components, including proteins and lipids, are spatiotemporally transported to their destination within the endomembrane system. This includes the secretory transport of newly synthesized proteins to the cell surface or the outside of the cell, the endocytic transport of extracellular cargoes or plasma membrane components into the cell, and the recycling or shuttling transport of cargoes between the subcellular organelles, etc. Membrane trafficking events are crucial to the development, growth, and environmental adaptation of all eukaryotic cells and, thus, are under stringent regulation. Cell-surface receptor kinases, which perceive ligand signals from the extracellular space, undergo both secretory and endocytic transport. Commonly used approaches to study the membrane trafficking events using a plasma membrane-localized leucine-rich-repeat receptor kinase, ERL1, are described here. The approaches include plant material preparation, pharmacological treatment, and confocal imaging setup. To monitor the spatiotemporal regulation of ERL1, this study describes the co-localization analysis between ERL1 and a multi-vesicular body marker protein, RFP-Ara7, the time series analysis of these two proteins, and the z-stack analysis of ERL1-YFP treated with the membrane trafficking inhibitors brefeldin A and wortmannin.

Autor em destaque: Métodos baseados em imagens para estudar eventos de tráfego de membrana em células de linhagem estomática  

Several commonly used methods are introduced here to study the membrane trafficking events of a plasma membrane receptor kinase. This manuscript describes detailed protocols including the plant material preparation, pharmacological treatment, and confocal imaging setup.

Métodos Baseados em Imagem para Estudo de Eventos de Tráfego de Membranas em Células de Linhagem Estomática

In eukaryotic cells, membrane components, including proteins and lipids, are spatiotemporally transported to their destination within the endomembrane ...