Damos las gracias a Chunxin Wang para la construcción de pTSO2:: GUS y las fotos en la Figura 1, Detlef Weigel para compartir el protocolo de GUS-a prueba de tontos, Sijacic Paja y Hollender Courtney por sus valiosos comentarios. Investigación en el laboratorio Z. L s es apoyado por los EE.UU. National Science Foundation (IOB0616096 y MCB0744752). ZL es parcialmente financiado por la Universidad de Maryland Estación Experimental Agrícola.

I. floral inmersión en la transformación de Arabidopsis thaliana <ol><li> Aproximadamente un mes antes de la transformación, sembrar las semillas de Arabidopsis en 4-8 botes (5 ollas pulgada cuadrada), con aproximadamente 10-15 plantas por maceta. Para las plantas con fertilidad normal, 4 macetas de plantas por construir el plásmido será suficiente. Si uno tiene que transformar Arabidopsis mutante con reducción de la fertilidad, aumentar el número de macetas en consecuencia. </li><li> Las plantas se cultivan en condiciones estándar (16 horas luz y 8 h oscuridad a 22 ° C). Si las plantas se cultivan en día más corto o más baja temperatura, se tardará más tiempo en flor y más tiempo para estar listo para la transformación. Buen cuidado de las plantas se asegurará de plantas sanas, que son esenciales para la transformación exitosa. </li><li> Cuando las plantas tienen sólo atornilladas y comenzado a florecer. Ahora están listos para la transformación. Para aumentar la eficiencia de transformación, 3-4 días antes de la transformación, corte los tallos inflorescencia principal tan pronto como se han atornillado a fomentar la formación de brotes más secundario. Regar las plantas el día antes de la transformación. Esto asegura que el suelo no tomar los medios de infiltración en exceso durante floral inmersión. </li><li> Dos días antes de la transformación, inocular 40 ml de LB con antibióticos apropiados (en el caso de pTSO2:: GUS, 50 mg / ml de kanamicina) con Agrobacterium tumefaciens cepa GV3101 que lleva el pTSO2:: GUS construir. El pTSO2:: GUS se clona en el vector pBIN20 que posee genes NPTII que confiere resistencia a Kanamicina 3. Crecen durante la noche en una incubadora de agitación a 28-30 ° C. </li><li> Al día siguiente, 8 ml de transferencia de la cultura durante la noche en 400 ml de LB con 50 mg / ml kanamicina. Crecen durante la noche en una incubadora de agitación a 28-30 ° C. </li><li> En el día de la transformación, cuando el OD 600 del cultivo de Agrobacterium llega a aproximadamente 0,8 (OD 600 entre 0,5 a 1 son aceptables), la desaceleración de la cultura durante la noche en el Agrobacterium durante 8 minutos a 5000 rpm. </li><li> Resuspender el precipitado en un Agrobacterium L (litro) de los medios de infiltración (10 mM MgCl 2, 5% de sacarosa, 0.44mM 6-benciladenina (BA), un 0,3% Silwet L-77, la solución de 1x Gamborg s vitaminas y agua esterilizada). El medio no tiene por qué ser esterilizados en autoclave, pero tiene que ser fresca. Vierta el 1 L Agrobacterium que contienen medio de infiltración en un plato (por ejemplo, 8 &quot;x15&quot; x2 &quot;Pyrex plato de cristal). </li><li> Invertir la olla (las plantas no se caiga) y sumergir todas las partes aéreas de las plantas en la solución de infiltración, mantener durante 5 minutos una olla a la vez. Mientras que todas las partes aéreas de la planta se encuentran inmersos en el medio de la infiltración, evitar que el suelo absorba la infiltración de los medios de comunicación para reducir las posibilidades de crecimiento de hongos en el suelo. </li><li> Después de la inmersión, el lugar todos los utensilios de inmersión de su lado en varias capas de toallas de papel en una bandeja. Las toallas de papel absorben la cantidad de acceso de los medios de infiltración. Cubra la bandeja con papel de plástico para asegurar una alta humedad. Coloque la bandeja en la cámara de crecimiento de las plantas. </li><li> Al día siguiente, retire la envoltura de plástico y toallas de papel, y las ollas a cabo en posición vertical. No riegue las plantas durante cuatro o cinco días más. A continuación, mantener las plantas con normalidad hasta que se pusieron las semillas y recolectar las semillas a granel. </li><li> En placas (150x15mm placa de Petri) con un medio MS con 50 mg / ml de kanamicina (2,2 g de peso medio de Murashige y Skoog basal sin sal vitamina, se disuelven en 500 ml de agua, el pH a 5.8 con NaOH 1M, añadir 4 gramos de agar, autoclave, fresco y añadir a la kanamicina a 50 mg / ml concentración final). Las placas se mantienen en 4 ° C hasta que se necesite. </li><li> Para seleccionar resistentes a los antibióticos de plantas transgénicas, se necesita a la pantalla de 20.000 semillas en el mínimo. 0,1 gramos es de unos 5.000 semillas. Peso y estimar la cantidad de semillas. </li><li> Esterilizar las semillas mediante lavado en 15 ml tubo Falcon con etanol al 70% durante 30 segundos. Lavar las semillas con agua estéril una vez. Remoje las semillas durante 30 segundos en una solución que contiene una dilución de 1:10 de la tienda compró lejía al 5% (Clorox). Enjuague las semillas con agua estéril tres a seis veces. En campana estéril, una pipeta de 5000 semillas en cada Estado miembro (kanamicina) plato, esparcir las semillas de manera uniforme sobre la placa, sellar las placas con cinta 3M microporos para evitar la contaminación. </li><li> Las placas se incuban a 4 ° C en la oscuridad durante 3-4 días, a continuación, transferir las placas a una cámara (podría ser la cámara de crecimiento de la planta misma). Después de unos 14 días, las plantas transgénicas se mantienen verdes, pero los no-transgénicos son palidecer y morir. Transferencia de las plántulas transgénicas en el suelo al disminuir tirando de las raíces a partir del medio y los colocó en el suelo. </li></ol> II. Examinar pTSO2:: patrones de expresión de GUS <ol><li> Cosecha pTSO2:: GUS plantas transgénicas y ponerlas en acetona fría al 90% en un vial de centelleo de vidrio o tubos de microcentrífugaque se mantienen en hielo. Placas de poliestireno de microtitulación (no de polipropileno) también se puede utilizar si se analiza un gran número de muestras. </li><li> Después de todas las muestras se recogen, coloque los tubos a temperatura ambiente durante 20 minutos. Durante este tiempo, constituyen tinción de amortiguación fresco sin X-gluc (0,2% Triton X-100, 50 mm NaHPO 4 Buffer pH 7.2, 2 mM de potasio ferrocianuro potásico, 2 mM ferricianuro) y el lugar en el hielo. </li><li> Eliminar la acetona de las muestras y agregar el buffer de tinción. </li><li> Conforman X-gluc solución de tinción (0,2% Triton X-100, 50 mm NaHPO 4 Buffer pH 7.2, 2 mM de potasio ferrocianuro potásico, 2 mM ferricianuro, 2mm x-gluc). Eliminar el buffer de tinción de las muestras y agregar el buffer de tinción con X-gluc a las muestras. </li><li> Infiltrarse en las muestras bajo vacío durante 15 a 20 minutos. Cortar el vacío lentamente y verificar que todas las muestras se hunden bajo la superficie de la solución de tinción. Si es necesario, repetir la infiltración hasta que todas las muestras después de hundirse el vacío se libera. </li><li> Las muestras se incuban a 37 ° C durante la noche en el buffer de tinción con X-gluc. </li><li> Extraer muestras de la incubadora y eliminar tinción de amortiguación. Lavado de las muestras en series de etanol sucesivos (20%, 35% y 50% de etanol) a temperatura ambiente durante 30 minutos cada cambio. </li><li> Fijar los tejidos de la FAA fijador (50% etanol, 5% de formaldehído, ácido acético 10%, resto agua) durante 30 minutos a temperatura ambiente. Retire la FAA y añadir etanol al 70%. En este punto, los tejidos pueden ser visualizados y fotografiados en un microscopio equipado con una cámara digital. Un color azul oscuro indica que TSO2 se expresa (Figura 1). Por otra parte, los tejidos pueden ser almacenadas a 4 ° C para su posterior visualización. <img src="/files/ftp_upload/1952/1952fig1.jpg" alt="Figura 1" /> Figura 1 pTSO2:.: Expresión de GUS en plantas transgénicas (A), las raíces laterales (B), y la punta de la raíz (C). Tenga en cuenta que TSO2 promotor es muy activa en los tejidos de los jóvenes y se dividan. El patrón de expresión esporádica se muestra en (C) es típico de los genes que se expresan en determinadas fases del ciclo celular. </li></ol> Resultados representante Cuando se hace correctamente, la eficiencia de transformación debe ser aproximadamente de 0.1-0.2%. En una palabra, se debe obtener plantas transgénicas 5.10 por cada 5000 semillas de selección. En promedio, alrededor de 100 líneas transgénicas se pueden obtener mediante la transformación de 4 macetas de plantas silvestres. Para las plántulas transgénicas que contienen el pTSO2:: GUS construir 3, color azul oscuro que refleja la actividad de GUS se encuentra en las células en división activa como las hojas jóvenes, ápice del brote, punta de la raíz y la raíz lateral primordios (Figura 1). El patrón esporádico no uniforme es característico de la fase del ciclo celular específicos de la expresión.

<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>

La eficiencia de transformación está determinada por muchos factores diferentes, que se analizan a continuación: <ol><li> La salud de las plantas y su edad son de vital importancia. Aproximadamente un mes las plantas produciendo numerosos botones florales inmaduros son ideales. Recorte de los brotes principales para fomentar la formación secundaria días 3-4 disparar antes de la transformación se incrementará la eficiencia de transformación. Las plantas más viejas dará lugar a transformantes, pero a un ritm...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>6-benzyladenine</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>852430</td>
<td>&nbsp;</td>
<tr>
<td>Silwet-77</td>
<td>&nbsp;</td>
<td>Crompton Corp.</td>
<td>&nbsp;</td>
<td>Toxic, wear gloves</td>
</tr>
<tr>
<td>Murashige and Skoog Basal medium (MS)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>M5519</td>
<td>&nbsp;</td>
<tr>
<td>Gamborg's vitamin solution 1000X</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>G1019</td>
<td>&nbsp;</td>
<tr>
<td>X-Gluc (5-bromo-4-chloro-3-indolyl &beta;-D-glucuronide cyclohexamine salt</td>
<td>&nbsp;</td>
<td>Biosynth</td>
<td>B-7300</td>
<td>Toxic, light sensitive keep in the dark</td>
</tr>
<tr>
<td>Micropore 3M Tape</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>19027761</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

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

La capacidad de introducir genes extraños en un organismo es la base de la biología moderna y la biotecnología. En el modelo de planta de flores<em&gt; Arabidopsis thaliana</em&gt;, El floral dip transformación del método<sup&gt; 2.1</sup&gt; Ha sustituido a todos los métodos anteriores, debido a su simplicidad, eficiencia y bajo costo. En concreto, los brotes jóvenes de la floración<em&gt; Arabidopsis</emPlantas&gt; se sumergen en una solución de<em&gt; Agrobacterium tumefaciens</em&gt; La realización específica construcciones de plásmidos. Después de la inmersión, las plantas se volvió a su normal crecimiento y rendimiento de semillas, un pequeño porcentaje de los cuales son transformados con el gen extraño y pueden ser seleccionados para el medio con antibióticos. Este método de inmersión floral facilitado considerablemente<em&gt; Arabidopsis</em&gt; Investigación y contribuyó enormemente a nuestra comprensión de la función de genes de plantas. En este estudio, utilizamos el método de inmersión floral para transformar un gen reportero,<em&gt; Β-glucuronidasa (GUS)</em&gt;, Bajo el control de<em&gt; TSO2</em&gt; Promotor.<em&gt; TSO2</em&gt;, Que codifica para la ribonucleótido reductasa (RNR) subunidad pequeña<sup&gt; 3</sup&gt;, Es un gen ciclo celular está regulado esencial para la biosíntesis de dNDP en la fase S del ciclo celular. El examen de<em&gt; GUS</em&gt; Expresión en plantas transgénicas<em&gt; Arabidopsis</em&gt; Plántulas muestra que<em&gt; TSO2</em&gt; Se expresa en los tejidos se dividen activamente. El método experimental reportado y los materiales pueden ser fácilmente adaptados no sólo a la investigación sino también para la educación en la escuela secundaria y superior.

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

En este artículo se ilustra el método de inmersión floral de<em&gt; Agrobacterium tumefaciens</em&gt; La transformación mediada de<em&gt; Arabidopsis thaliana</em&gt;. Mediante la introducción de un ciclo celular regulados promotor-reportero,<em&gt; PTSO2:: β-glucuronidasa (GUS)</em&gt;, En<em&gt; Arabidopsis</em&gt;, Que ilustra cómo se detecta la expresión de GUS en plantas transgénicas reportero.

Floral dip transformación de la Arabidopsis thaliana Para el Examen de PTSO2:: β-glucuronidasa Reporter Expresión Génica

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

Research

JoVE Journal

Biology

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

35.8K vistas.  University of Maryland College Park. En este artículo se ilustra el método de inmersión floral de Agrobacterium tumefaciens La transformación mediada de Arabidopsis thaliana. Mediante la introducción de un ciclo celular regulados promotor-reportero, PTSO2:: β-glucuronidasa (GUS), En Arabidopsis, Que ilustra cómo se detecta la expresión de GUS en plantas transgénicas reportero.

Video: 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.

La preparación de cultivos primarios de células hematopoyéticas de la médula ósea murina por electroporación

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

Investigación

Biología

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.

Utilizando el sistema de electroporación Gene Emisores MXcell para transfectar células primarias con alta eficiencia

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.

El uso de un contador de células automatizado para simplificar estudios de expresión génica: siRNA Derribo de IL-4 la expresión génica en células dependientes 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.

La investigación de los tejidos y órganos específicos de las respuestas fitocromo usando FACS asistida por células de tipo Perfiles de expresión específica en 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.

En la reconstitución in vitro de los activos T. castaneum telomerasa

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.

La tinción histoquímica de<em&gt; Arabidopsis thaliana</em&gt; Celda Secundaria Elements Pared

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.

Análisis de RNA Global Síntesis en el nivel de células individuales siguiente Hipoxia

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 transformación de lino (<em&gt; Linum usitatissimum</em&gt;) Para generar transgénicos progenies con una alta tasa de Transformación

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.

La visualización de frecuencia Stromule con microscopía de fluorescencia

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

Investigación espacio-temporal de la dinámica de ERL1 mediante imágenes confocales

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 destacado: Métodos basados en imágenes para estudiar los eventos de tráfico de membrana en células de linaje 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 basados en imágenes para estudiar los eventos de tráfico de membrana en células de linaje estomática

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