Los autores desean agradecer al Dr. Chris Taylor (Universidad Estatal de Ohio) para la introducción de la tecnología de raíces peludas a nuestro laboratorio y los Dres. Senthil Subramanian (South Dakota State University), Juan Zhang (Ludong la Universidad de Shandong de China) para mejorar el protocolo. También agradecemos al Dr. Cheng Hao (Nanjing Universidad de Agricultura) para la ayuda en la toma de este video. Este trabajo es apoyado en parte por subvenciones del Departamento de Energía (DE-SC0001295), NSF (MCB-0923779) y el USDA (2010-65116-20514) a OY

El siguiente protocolo se ha utilizado para generar plantas peludas raíces compuesto en un modelo de leguminosas M. especies truncatula. Protocolos similares han sido adaptadas por lo menos ocho especies de plantas 1-4. Se utilizó M. plantas truncatula peludas raíces compuesto para estudiar las funciones de genes en las raíces y el desarrollo de nódulos. El protocolo se divide en cuatro secciones: 1) la preparación de materiales vegetales, 2) la generación de las plantas peludas raíz compuesta, 3) la infección simbiótica Rhizobium, y 4) la identificación transgénicos raíz. Se utilizó el vector binario que contiene la proteína verde fluorescente (GFP) de genes como reportero para la detección de la raíz transgénica en las plantas de compuestos 3. En comparación con los antibióticos basados ​​en la selección, las buenas prácticas agrarias basadas en detección es rápido, fácil y barata. En nuestra construcción, la ER-expresión optimizado gen gfp está impulsado por el promotor de la ubiquitina súper, que tiene fuertes señales de las buenas prácticas agrarias constitutiva en las raíces de transgénicos, que permite distinguir fácilmente entre las raíces de transgénicos y no transgénicos. 1. Preparación de materiales para plantas <ol><li> M. truncautla semillas se obtienen de las plantas cultivadas en un invernadero (50% de humedad relativa, 16 / 8 h luz / oscuridad, 22/18 ° C día / noche la temperatura). Las semillas maduras se pueden almacenar a 4 ° C. </li><li> Aproximadamente 100 semillas son escarificadas en 10 ml de ácido sulfúrico concentrado (95-98%, Sigma-Aldrich, MO) durante 10 minutos con agitación periódica. </li><li> Las semillas se lavan 5-7 veces con agua estéril con una suave agitación. </li><li> Las semillas se remojan en agua a temperatura ambiente durante 6 horas y se mantiene a 4 ° C durante 36-48 horas para sincronizar la germinación. </li><li> Aproximadamente el 20-30 semillas se extienden sobre papel de filtro húmedo, colocado en una placa de Petri. Se mantienen a 22 ° C, con 16 / 8 h luz / oscuridad ciclo, y 40 μmol.m -2. S -1 intensidad de la luz, durante 5-6 días. </li><li> Plántulas germinadas se transfieren en el suelo y se coloca en el invernadero durante un mes. </li></ol> 2. La generación de plantas peludas raíz Composite <ol><li> Construcciones binarias portadores de genes de interés se convirtieron en A. rhizogenes utilizando protocolos estándar. Hemos diseñado un conjunto de vectores binarios que contienen el gen de la GFP como un marcador que facilitar la detección de los tejidos transgénicos. Estos vectores contienen varios promotores y todos tienen sitios de puerta de enlace de la clonación (Invitrogen, Carlsbad, CA) por la sobre-expresión o el silenciamiento de determinados genes 3,5. </li><li> A. rhizogenes se cultivaron en medio LB 50 ml con la selección adecuada de antibióticos a 28 ° C durante 16-24 horas. </li><li> Las bacterias se recogen y se volvió a suspender a una concentración final de 600 OD = 0,3 en una solución de la planta libre de nitrógeno nutriente (Tabla 1 y 6). </li><li> Para apoyar la regeneración de las raíces peludas, una matriz de esterilización de apoyo (&quot;lana de roca&quot;, Hummert Internacional, Earth City, MO) se corta en tacos de unos 3 cm 3. Ponemos 4 se conecta a una placa de Petri. </li><li> Un agujero se metió en la parte superior de cada tapón usando una pipeta de punta para facilitar la inserción de los explantes. A. rhizogenes (5 ml) se añade a cada enchufe. </li><li> Una sección de disparar con 2-3 yemas axilares se extirpa con un corte oblicuo. El explante se inserta en el enchufe, y ha crecido a 22 ° C durante unas tres semanas. Nos reservamos el explantes Medicago sin riego durante los primeros 10 días, entonces, el agua con 5 ml de nitrógeno libre de solución cuando sea necesario. Descubrieron que al eliminar el meristemo apical podría disminuir la formación de raíces adventicias. </li></ol> 3. Simbiótica Rhizobium (Sinorhizobium meliloti) Infección <ol><li> Después de tres semanas, algunas raíces adventicias saldrá de la lana de roca. Las plantas de raíz compuesta peluda se transfieren a la arena o vermiculita (esterilizada), con o sin la lana de roca. Que se cultivan en el invernadero a 22 ° C, 16 / 8 h luz / oscuridad ciclo, y 300 μmol.m -2. S -1 intensidad de la luz durante una semana más. </li><li> Antes de rizobios infección, S. meliloti 1201 se cultivaron en 50 extracto de levadura-manitol ml de medio por 7 días a 30 ° C (Tabla 2 y 6,7). </li><li> Resuspendidas las células rizobios se diluyen a una concentración final de 600 OD = 0,08 con solución de nitrógeno libre de nutrición. Una suspensión fresca 10 ml de S. meliloti se aplicó a cada planta de compuestos para inducir la formación de nódulos. </li></ol> 4. Identificación de la raíz transgénica <ol><li> Dos semanas después de la inoculación de rizobios, las plantas están compuestas arrancadas por el lavado en agua. Las raíces se pueden separar fácilmente de arena o perlita en el agua. </li><li> Ponemos las raíces peludos bajo un microscopio UV (Nikon SMZ1500, excitación 460-500nm, 505nm dicroicas, la barrera más 510 Nm). Las raíces transgénicas GFP positivos y las raíces adventicias son las buenas prácticas agrarias negativo. A continuación, recoger las raíces according la señal de las buenas prácticas agrarias, y contar el número de nódulos, medir la longitud de la raíz, y calcular la densidad de las raíces laterales. Las raíces de las buenas prácticas agrarias negativos son utilizados como control. </li></ol> 5. Representante de Resultados En nuestro experimento, las raíces del pelo nuevo regeneradas a partir de los explantes en 2-3 semanas después de A. rhizogenes inoculación. Bajo el microscopio de rayos UV, las raíces transgénicas portadoras del gen GFP muestran una fuerte fluorescencia verde (Fig. 1). La cantidad de raíces regeneradas y la porción de las raíces de las buenas prácticas agrarias positivo dependerá de las condiciones de los explantes y el medio ambiente de crecimiento compuesta promedio plants.On, el 25% de las raíces se producen transgénicos raíces peludas 3. Para aumentar la eficiencia de transformación, 1) la condena cubierta de plástico, como se muestra en el video, es suficiente para mantener la humedad en la bandeja de crecimiento para los primeros días, y las plantas sólo requieren poca agua en los primeros días. El riego excesivo es perjudicial para la formación de raíces peludas, 2) la concentración de inoculante Agrobacterium es importante para la transformación. Cantidad excesiva de las células no son útiles para la formación de raíces peludas o formación de nódulos. Para la formación de nódulos, la solución de las necesidades de riego que se libre de nitrógeno, de lo contrario, los nódulos de pocos formulario. Las plantas de raíz compuesta peludos se pueden generar con los cotiledones o plántulas Entact como material de partida. Sólo modificaciones menores del protocolo anterior se ncessary para generar raíces peludas de otros tejidos 8. Es importante destacar que cada raíz peluda es un evento de transformación independientes. Por lo tanto, los fenotipos observados en una planta de compuestos son la suma de eventos transforamtion varios que necesitan ser confirmados por las repeticiones en varias plantas compuestas <img src="/files/ftp_upload/2633/2633fig1.jpg" alt="Figura 1" /> Figura 1. A. raíces transgénicas pueden ser resueltos a partir de raíces peludas. B. Los nódulos se formaron en las plantas de raíz compuesta peludo. Colocamos cuatro semanas de edad M. plantas truncatula peludas raíces en la UV-microscopio y las raíces GFP positivos pueden ser fácilmente identificados. (Flecha roja: La raíz de las buenas prácticas agrarias; Flecha amarilla: no GFP raíz) <table border="1"><tbody><tr><td> Valores </td><td> g/200ml </td><td> ml de caldo / litro </td></tr><tr><td> MgSO 4 .7 H 2 O </td><td> 12.3 </td><td> 2 </td></tr><tr><td> CaCl2 .2 H 2 O </td><td> 14.7 </td><td> 4 </td></tr><tr><td> K 2 HPO 4 .3 H 2 O </td><td> 6.8 </td><td> 1 </td></tr><tr><td> K 2 SO 4 </td><td> 11 </td><td> 4 </td></tr><tr><td> Fe Cl 3 .6 H 2 O </td><td> 0.49 </td><td> 2.5 </td></tr><tr><td> Micronutrientes </td><td> Ver más abajo </td><td> 1 </td></tr></tbody></table><table border="1"><tbody><tr><td> Micronutrientes </td><td> g por litro </td></tr><tr><td> H 3 BO 3 </td><td> 0.142 </td></tr><tr><td> MnSO 4. H 2 O </td><td> 0.077 </td></tr><tr><td> ZnSO 4 .7 H 2 O </td><td> 0,1725 </td></tr><tr><td> CuSO 4 .5 H 2 O </td><td> 0.037 </td></tr><tr><td> NaMoO 4. H 2 O </td><td> 0.024 </td></tr><tr><td> CoCl 2. H 2 O </td><td> 0,0025 </td></tr><tr><td> NiSO4 </td><td> 0.001 </td></tr></tbody></table> Tabla 1. Nitogen sin solución de la nutrición <table border="1"><tbody><tr><td> K 2 HPO4 </td><td> 0,5 g </td></tr><tr><td> NaCl </td><td> 0,1 g </td></tr><tr><td> MgSO 4 · 7H 2 O </td><td> 0,2 g </td></tr><tr><td> Extracto de levadura </td><td> 0,4 g </td></tr><tr><td> Manitol </td><td> 10g </td></tr><tr><td> pH = 6,8 </td><td></td></tr></tbody></table> Tabla 2. Medio de extracto de levadura manitol (por litro)

<ol>
	<li>Limpens, E., Ramos, J., Franken, C., Raz, V., Compaan, B., Franssen, H., Bisseling, T., Geurts, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+interference+in+Agrobacterium+rhizogenes-transformed+roots+of+Arabidopsis+and+Medicago+truncatula.">RNA interference in Agrobacterium rhizogenes-transformed roots of Arabidopsis and Medicago truncatula.</a> J. Exp. Bot. 55, 983-992 (2004).</li><li>Collier, R., Fuchs, B., Walter, N., Kevin, L. W., Taylor, C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ex+vitro+composite+plants:+an+inexpensive,+rapid+method+for+root+biology.">Ex vitro composite plants: an inexpensive, rapid method for root biology.</a> Plant J. 43, 449-457 (2005).</li><li>Zhang, J., Subramanian, S., Stacey, G., Yu, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flavones+and+flavonols+play+distinct+critical+roles+during+nodulation+of+Medicago+truncatula+by+Sinorhizobium+meliloti.">Flavones and flavonols play distinct critical roles during nodulation of Medicago truncatula by Sinorhizobium meliloti.</a> Plant J. 57, 171-183 (2009).</li><li>Boisson-Dernier, A., Chabaud, M., Garcia, F., Becard, G., Rosenberg, C., Barker, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Agrobacterium-rhizogenes-transformed+roots+of+Medicago+truncatula+for+the+study+of+nitrogen-fixing+and+endomycorrhizal+symbiotic+associations.">Agrobacterium-rhizogenes-transformed roots of Medicago truncatula for the study of nitrogen-fixing and endomycorrhizal symbiotic associations.</a> Mol. Plant-Microbe Interact. 14, 695-700 (2001).</li><li>Zhang, J., Subramanian, S., Zhang, Y., Yu, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flavone+synthases+from+Medicago+truncatula+are+flavanone-2-hydroxylases+and+are+important+for+nodulation.">Flavone synthases from Medicago truncatula are flavanone-2-hydroxylases and are important for nodulation.</a> Plant Physiol. 144, 741-751 .</li><li>Subramanian, S., Hu, X., Lu, G., Odelland, J., Yu, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+promoters+of+two+isoflavone+synthase+genes+respond+differentially+to+nodulation+and+defense+signals+in+transgenic+soybean+roots.">The promoters of two isoflavone synthase genes respond differentially to nodulation and defense signals in transgenic soybean roots.</a> Plant Mol Biol. 54, 623-639 (2004).</li><li>Vincent, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> A manual for the practical study of root nodule bacteria International Biological Program Handbook. , 1-13 (1970).</li><li>Subramanian, S., Graham, M. Y., Yu, O., Graham, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+Interference+of+Soybean+Isoflavone+Synthase+Genes+Leads+to+Silencing+in+Tissues+Distal+to+the+Transformation+Site+and+to+Enhanced+Susceptibility+to+Phytophthora+sojae.">RNA Interference of Soybean Isoflavone Synthase Genes Leads to Silencing in Tissues Distal to the Transformation Site and to Enhanced Susceptibility to Phytophthora sojae.</a> Plant Physiol. 137, 1345-1353 (2005).</li></ol>

La generación de raíces de las plantas compuestas peluda es un método rápido y fácil de obtener grandes cantidades de material transgénico para muchas especies de dicotiledóneas. Aunque este método no puede producir semillas transgénicas, se puede producir materiales transgénicos en unas pocas semanas. El método es especialmente adecuado para las plantas que tienen dificultades para establecer cultivos de tejidos o la generación de transformantes estables. Con los años, hemos utilizado esta tecnología para estudiar las funciones de genes, las funciones de promotor, los microARNs, las raíces y el desarrollo de las raíces laterales, la defensa y las respuestas de estrés abiótico, la nodulación y otros procesos simbióticos, las respuestas hormonales, perfil metabólico, el perfil de genes, el análisis proteómico, y otros procesos biológicos. El protocolo es robusto y replicable.

generation of composite plants in medicago truncatula used for nodulation assays

Al igual que en tumerfaciens Agrobacterium, rhizogenes puede transferir ADN extraño en células de las plantas sobre la base de las comunidades autónomas de raíz la inducción (Ri) del plásmido. A. rhizogenes puede causar la formación de raíces peludas en los tejidos vegetales y forma de las plantas compuestas después de la transformación. En estas plantas compuestas, algunas de las raíces regeneradas son transgénicos, llevando el tipo salvaje del T-ADN y el vector binario de ingeniería, mientras que los brotes siguen siendo los no transgénicos, que sirve para proporcionar energía y apoyar el crecimiento. Estas plantas de raíz compuesta peludo no productoras de semillas transgénicas, pero hay una serie de características importantes que hacen que estas plantas compuestos muy útiles en la investigación de las plantas. En primer lugar, con una amplia gama de huéspedes, A. rhizogenes puede transformar muchas especies de plantas, especialmente en las dicotiledóneas, lo que permite la ingeniería genética en una variedad de especies. En segundo lugar, A. rhizogenes infectar los tejidos y los explantes directamente, sin cultivos de tejidos antes de la transformación es necesaria para la obtención de plantas compuestas, lo que es ideal para la transformación de las especies recalcitrantes de la planta. Por otra parte, los tejidos transgénicos raíz se pueden generar en cuestión de semanas. De Medicago truncatula, podemos obtener las raíces de transgénicos en tan corto como tres semanas, más rápido de lo normal floral dip transformación de Arabidopsis. En general, la tecnología de raíces peludas planta compuesta es una herramienta versátil y útil para estudiar las funciones de genes y fenotipos relacionados con la raíz. Aquí se demuestra cómo las plantas peludas raíces compuesto puede ser utilizado para estudiar las interacciones planta-rizobio y la nodulación en el M. difíciles de transformar las especies truncatula.

Watch this Scientific Journal Video about Generation of Composite Plants in Medicago truncatula used for Nodulation Assays at JoVE.com

Generation of Composite Plants in Medicago truncatula used for Nodulation Assays

Se demuestra cómo las plantas peludas raíces compuesto puede ser utilizado para estudiar las interacciones planta-rizobio y la nodulación en las difíciles de transformar las especies<em&gt; Medicago truncatula</em&gt;.

Generación de plantas compuestas en Medicago truncatula Utilizado para los ensayos nodulación

Similar to Agrobacterium tumerfaciens, Agrobacterium rhizogenes can transfer foreign DNAs into plant cells based on the autonomous root-inducing (Ri) ...

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Research

JoVE Journal

Biology

Generation of Composite Plants in Medicago truncatula used for Nodulation Assays

15.9K vistas.  Donald Danforth Plant Science Center. Se demuestra cómo las plantas peludas raíces compuesto puede ser utilizado para estudiar las interacciones planta-rizobio y la nodulación en las difíciles de transformar las especies Medicago truncatula.

Video: Generation of Composite Plants in Medicago truncatula used for Nodulation Assays

Neurocircuit Assays for Seizures in Epilepsy Mutants of Drosophila

Drosophila melanogaster is a useful tool for studying seizure like activity. A variety of mutants in which seizures can be induced through either physical shock or electrical stimulation is available for study of various aspects of seizure activity and behavior. All flies, including wild-type, will undergo seizure-like activity if stimulated at a high enough voltage. Seizure like activity is an all-or-nothing response and each genotype has a specific seizure threshold. The seizure threshold of a specific genotype of fly can be altered either by treatment with a drug or by genetic suppression or enhancement. The threshold is easily measured by electrophysiology. Seizure-like activity can be induced via high frequency electrical stimulation delivered directly to the brain and recorded through the dorsal longitudinal muscles (DLMs) in the thorax. The DLMs are innervated by part of the giant fiber system. Starting with low voltage, high frequency stimulation, and subsequently raising the voltage in small increments, the seizure threshold for a single fly can be measured.

Neurocircuit Assays for Seizures in Epilepsy Mutants of Drosophila

Using high frequency electrical stimulation, seizure-like activity can be induced in Drosophila. This activity is easily recorded from the giant fiber system.

Neurocircuit ensayos para las convulsiones en la epilepsia de los mutantes Drosophila</em

Drosophila melanogaster is a useful tool for studying seizure like activity. A variety of mutants in which seizures can be induced through either physical ...

Investigación

Biología

Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

Schwann cells are one of the commonly used cells in repair strategies following spinal cord injuries. Schwann cells are capable of supporting axonal regeneration and sprouting by secreting growth factors 1,2 and providing growth promoting adhesion molecules 3 and extracellular matrix molecules 4. In addition they myelinate the demyelinated axons at the site of injury 5.
However following transplantation, Schwann cells do not migrate from the site of implant and do not intermingle with the host astrocytes 6,7. This results in formation of a sharp boundary between the Schwann cells and astrocytes, creating an obstacle for growing axons trying to exit the graft back into the host tissue proximally and distally. Astrocytes in contact with Schwann cells also undergo hypertrophy and up-regulate the inhibitory molecules 8-13.
In vitro assays have been used to model Schwann cell-astrocyte interactions and have been important in understanding the mechanism underlying the cellular behaviour.
These in vitro assays include boundary assay, where a co-culture is made using two different cells with each cell type occupying different territories with only a small gap separating the two cell fronts. As the cells divide and migrate, the two cellular fronts get closer to each other and finally collide. This allows the behaviour of the two cellular populations to be analyzed at the boundary. Another variation of the same technique is to mix the two cellular populations in culture and over time the two cell types segregate with Schwann cells clumped together as islands in between astrocytes together creating multiple Schwann-astrocyte boundaries.
The second assay used in studying the interaction of two cell types is the migration assay where cellular movement can be tracked on the surface of the other cell type monolayer 14,15. This assay is commonly known as inverted coverslip assay. Schwann cells are cultured on small glass fragments and they are inverted face down onto the surface of astrocyte monolayers and migration is assessed from the edge of coverslip.
Both assays have been instrumental in studying the underlying mechanisms involved in the cellular exclusion and boundary formation. Some of the molecules identified using these techniques include N-Cadherins 15, Chondroitin Sulphate proteoglycans(CSPGs) 16,17, FGF/Heparin 18, Eph/Ephrins19.
This article intends to describe boundary assay and migration assay in stepwise fashion and elucidate the possible technical problems that might occur.

Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

This article intends to describe in stepwise fashion the commonly used in vitro assays used in studying Schwann cell-asrtocyte interactions.

El análisis de Schwann-astrocito interacciones utilizando In Vitro Ensayos

Schwann cells are one of the commonly used cells in repair strategies following spinal cord injuries. Schwann cells are capable of supporting axonal ...

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.

Hibridación in situ para la localización precisa de las transcripciones de las plantas

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

Viability Assays for Cells in Culture

Manual cell counts on a microscope are a sensitive means of assessing cellular viability but are time-consuming and therefore expensive. Computerized viability assays are expensive in terms of equipment but can be faster and more objective than manual cell counts. The present report describes the use of three such viability assays. Two of these assays are infrared and one is luminescent. Both infrared assays rely on a 16&#160;bit Odyssey Imager. One infrared assay uses the DRAQ5 stain for nuclei combined with the Sapphire stain for cytosol and is visualized in the 700 nm channel. The other infrared assay, an In-Cell Western, uses antibodies against cytoskeletal proteins (&#945;-tubulin or microtubule associated protein 2) and labels them in the 800 nm channel. The third viability assay is a commonly used luminescent assay for ATP, but we use a quarter of the recommended volume to save on cost. These measurements are all linear and correlate with the number of cells plated, but vary in sensitivity. All three assays circumvent time-consuming microscopy and sample the entire well, thereby reducing sampling error. Finally, all of the assays can easily be completed within one day of the end of the experiment, allowing greater numbers of experiments to be performed within short timeframes. However, they all rely on the assumption that cell numbers remain in proportion to signal strength after treatments, an assumption that is sometimes not met, especially for cellular ATP. Furthermore, if cells increase or decrease in size after treatment, this might affect signal strength without affecting cell number. We conclude that all viability assays, including manual counts, suffer from a number of caveats, but that computerized viability assays are well worth the initial investment. Using all three assays together yields a comprehensive view of cellular structure and function.

Therapeutic compounds are often first examined in vitro with viability assays. Blind cell counts by a human observer can be highly sensitive to small changes in cell number but do not assess function. Computerized viability assays, as described here, can assess both structure and function in an objective manner.

Los ensayos de viabilidad de las células en cultivo

Manual cell counts on a microscope are a sensitive means of assessing cellular viability but are time-consuming and therefore expensive. Computerized ...

Efficient Generation Human Induced Pluripotent Stem Cells from Human Somatic Cells with Sendai-virus

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs include modeling pathogenesis of human genetic diseases, autologous cell therapy after gene correction, and personalized drug screening by providing a source of patient-specific and symptom relevant cells. However, there are several hurdles to overcome, such as eliminating the remaining reprogramming factor transgene expression after human iPSCs production. More importantly, residual transgene expression in undifferentiated human iPSCs could hamper proper differentiations and misguide the interpretation of disease-relevant in vitro phenotypes. With this reason, integration-free and/or transgene-free human iPSCs have been developed using several methods, such as adenovirus, the piggyBac system, minicircle vector, episomal vectors, direct protein delivery and synthesized mRNA. However, efficiency of reprogramming using integration-free methods is quite low in most cases.
Here, we present a method to isolate human iPSCs by using Sendai-virus (RNA virus) based reprogramming system. This reprogramming method shows consistent results and high efficiency in cost-effective manner.

Here, we present our established method to reprogram human somatic cells into transgene-free human iPSCs with Sendai virus, which shows consistent outcome and enhanced efficiency.

Eficiente generación, por actividad humana células madre pluripotentes de células somáticas humanas con virus Sendai-

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs ...

MALDI-Mass Spectrometric Imaging for the Investigation of Metabolites in Medicago truncatula Root Nodules

Most techniques used to study small molecules, such as pharmaceutical drugs or endogenous metabolites, employ tissue extracts which require the homogenization of the tissue of interest that could potentially cause changes in the metabolic pathways being studied1. Mass spectrometric imaging (MSI) is a powerful analytical tool that can provide spatial information of analytes within intact slices of biological tissue samples1-5. This technique has been used extensively to study various types of compounds including proteins, peptides, lipids, and small molecules such as endogenous metabolites. With matrix-assisted laser desorption/ionization (MALDI)-MSI, spatial distributions of multiple metabolites can be simultaneously detected. Herein, a method developed specifically for conducting untargeted metabolomics MSI experiments on legume roots and root nodules is presented which could reveal insights into the biological processes taking place. The method presented here shows a typical MSI workflow, from sample preparation to image acquisition, and focuses on the matrix application step, demonstrating several matrix application techniques that are useful for detecting small molecules. Once the MS images are generated, the analysis and identification of metabolites of interest is discussed and demonstrated. The standard workflow presented here can be easily modified for different tissue types, molecular species, and instrumentation.

MALDI-Mass Spectrometric Imaging for the Investigation of Metabolites in Medicago truncatula Root Nodules

Mass spectrometric imaging (MSI) is a powerful tool that can be used to discover and identify various chemical species in intact tissues, preserving the compounds in their native environments, which can provide new insights into biological processes. Herein a MSI method developed for the analysis of small molecules is described.&#160;

Imaging MALDI espectrometría de masas para la Investigación de metabolitos en<em&gt; Medicago truncatula</em&gt; nódulos de las raíces

Most techniques used to study small molecules, such as pharmaceutical drugs or endogenous metabolites, employ tissue extracts which require the ...

Measuring Spatial and Temporal Ca2+ Signals in Arabidopsis Plants

Developmental and environmental cues induce Ca2+ fluctuations in plant cells. Stimulus-specific spatial-temporal Ca2+ patterns are sensed by cellular Ca2+ binding proteins that initiate Ca2+ signaling cascades. However, we still know little about how stimulus specific Ca2+ signals are generated. The specificity of a Ca2+ signal may be attributed to the sophisticated regulation of the activities of Ca2+ channels and/or transporters in response to a given stimulus. To identify these cellular components and understand their functions, it is crucial to use systems that allow a sensitive and robust recording of Ca2+ signals at both the tissue and cellular levels. Genetically encoded Ca2+ indicators that are targeted to different cellular compartments have provided a platform for live cell confocal imaging of cellular Ca2+ signals. Here we describe instructions for the use of two Ca2+ detection systems: aequorin based FAS (film adhesive seedlings) luminescence Ca2+ imaging and case12 based live cell confocal fluorescence Ca2+ imaging. Luminescence imaging using the FAS system provides a simple, robust and sensitive detection of spatial and temporal Ca2+ signals at the tissue level, while live cell confocal imaging using Case12 provides simultaneous detection of cytosolic and nuclear Ca2+ signals at a high resolution.

Measuring Spatial and Temporal Ca2+ Signals in Arabidopsis Plants

Ca2+ signaling regulates diverse biological processes in plants. Here we present approaches for monitoring abiotic stress induced spatial and temporal Ca2+ signals in Arabidopsis cells and tissues using the genetically encoded Ca2+ indicators Aequorin or Case12.

Medición Espacial y Temporal Ca<sup&gt; 2 +</sup&gt; Las señales en plantas de Arabidopsis

Developmental and environmental cues induce Ca2+ fluctuations in plant cells. Stimulus-specific spatial-temporal Ca2+ patterns are sensed by cellular Ca2+ ...

MRM Microcoil Performance Calibration and Usage Demonstrated on Medicago truncatula Roots at 22 T

This protocol describes a signal-to-noise ratio (SNR) calibration and sample preparation method for solenoidal microcoils combined with biological samples, designed for high-resolution magnetic resonance imaging (MRI), also referred to as MR microscopy (MRM). It may be used at pre-clinical MRI spectrometers, demonstrated on Medicago truncatula root samples. Microcoils increase sensitivity by matching the size of the RF resonator to the size of the sample of interest, thereby enabling higher image resolutions in a given data acquisition time. Due to the relatively simple design, solenoidal microcoils are straightforward and cheap to construct and can be easily adapted to the sample requirements. Systematically, we explain how to calibrate new or home-built microcoils, using a reference solution. The calibration steps include: pulse power determination using a nutation curve; estimation of RF-field homogeneity; and calculating a volume-normalized signal-to-noise ratio (SNR) using standard pulse sequences. Important steps in sample preparation for small biological samples are discussed, as well as possible mitigating factors such as magnetic susceptibility differences. The applications of an optimized solenoid coil are demonstrated by high-resolution (13 x 13 x 13 &#956;m3, 2.2 pL) 3D imaging of a root sample.

MRM Microcoil Performance Calibration and Usage Demonstrated on Medicago truncatula Roots at 22 T

A protocol to study biological tissue at high spatial resolution using ultra-high field magnetic resonance microscopy (MRM) using microcoils is presented. Step-by-step instructions are provided for characterizing the microcoils. Finally, optimization of imaging is demonstrated on plant roots.

This protocol describes a signal-to-noise ratio (SNR) calibration and sample preparation method for solenoidal microcoils combined with biological ...

Laser-Capture Microdissection RNA-Sequencing for Spatial and Temporal Tissue-Specific Gene Expression Analysis in Plants

The development of a complex multicellular organism is governed by distinct cell types that have different transcriptional profiles. To identify transcriptional regulatory networks that govern developmental processes it is necessary to measure the spatial and temporal gene expression profiles of these individual cell types. Therefore, insight into the spatio-temporal control of gene expression is essential to gain understanding of how biological and developmental processes are regulated. Here, we describe a laser-capture microdissection (LCM) method to isolate small number of cells from three barley embryo organs over a time-course during germination followed by transcript profiling. The method consists of tissue fixation, tissue processing, paraffin embedding, sectioning, LCM and RNA extraction followed by real-time PCR or RNA-seq. This method has enabled us to obtain spatial and temporal profiles of seed organ transcriptomes from varying numbers of cells (tens to hundreds), providing much greater tissue-specificity than typical bulk-tissue analyses. From these data we were able to define and compare transcriptional regulatory networks as well as predict candidate regulatory transcription factors for individual tissues. The method should be applicable to other plant tissues with minimal optimization.

Presented here is a protocol for laser-capture microdissection (LCM) of plant tissues. LCM is a microscopic technique for isolating areas of tissue in a contamination-free manner. The procedure includes tissue fixation, paraffin embedding, sectioning, LCM and RNA extraction. RNA is used in the downstream tissue-specific, temporally resolved analysis of transcriptomes.

Secuenciación de ARN-Secuenciación de ARN-Captura láser para Análisis de Expresión Genética Espacial y Temporal Específica de Tejidos en Plantas

The development of a complex multicellular organism is governed by distinct cell types that have different transcriptional profiles. To identify ...

Microscopy Techniques for Interpreting Fungal Colonization in Mycoheterotrophic Plants Tissues and Symbiotic Germination of Seeds

Structural botany is an indispensable perspective to fully understand the ecology, physiology, development, and evolution of plants. When researching mycoheterotrophic plants (i.e., plants that obtain carbon from fungi), remarkable aspects of their structural adaptations, the patterns of tissue colonization by fungi, and the morphoanatomy of subterranean organs can enlighten their developmental strategies and their relationships with hyphae, the source of nutrients. Another important role of symbiotic fungi is related to the germination of orchid seeds; all Orchidaceae species are mycoheterotrophic during germination and seedling stage (initial mycoheterotrophy), even the ones that photosynthesize in adult stages. Due to the lack of nutritional reserves in orchid seeds, fungal symbionts are essential to provide substrates and enable germination. Analyzing germination stages by structural perspectives can also answer important questions regarding the fungi interaction with the seeds. Different imaging techniques can be applied to unveil fungi endophytes in plant tissues, as are proposed in this article. Freehand and thin sections of plant organs can be stained and then observed using light microscopy. A fluorochrome conjugated to wheat germ agglutinin can be applied to the fungi and co-incubated with Calcofluor White to highlight plant cell walls in confocal microscopy. In addition, the methodologies of scanning and transmission electron microscopy are detailed for mycoheterotrophic orchids, and the possibilities of applying such protocols in related plants is explored. Symbiotic germination of orchid seeds (i.e., in the presence of mycorrhizal fungi) is described in the protocol in detail, along with possibilities of preparing the structures obtained from different stages of germination for analyses with light, confocal, and electron microscopy.

This protocol aims to provide detailed procedures for collecting, fixing, and maintaining mycoheterotrophic plant samples, applying different microscopy techniques such as scanning and transmission electron microscopy, light, confocal, and fluorescence microscopy to study fungal colonization in plants tissues and seeds germinated with mycorrhizal fungi.

Técnicas de microscopía para interpretar la colonización fúngica en tejidos de plantas micoheterótrofas y germinación simbiótica de semillas

Structural botany is an indispensable perspective to fully understand the ecology, physiology, development, and evolution of plants. When researching ...