Estamos muy agradecidos a los Dres. SP Dinesh-Kumar y Liu Yule para TRV-VIGS vectores, y los Dres. Chuck Kenerley, Wheeler Terry, Starr y Jim Bayer CropScience para el suministro de semillas de algodón. Este trabajo fue financiado por la NSF para LS y los NIH para PH

1. Crecer las plántulas de algodón <ol><li> Sembrar las semillas del algodón (Gossypium hirsutum) variedad FiberMax 832, Phytogen 425RF, 480WR Phytogen y Deltapine 90 en macetas (7 cm de diámetro) que contienen la mezcla Metro 700 (SunGR, Beavile, WA). </li><li> Mantenga los recipientes en una bandeja cubierta con una cúpula de plástico a 23 ° C, 120 μE m -2 S -1 luz, con un fotoperíodo de 12 horas light/12 horas oscuras en una sala de crecimiento. </li><li> Retire la terminal cuando dos cotiledones han surgido. </li><li> Aproximadamente dos semanas después, el uso de las plantas con dos cotiledones completamente abiertos para el ensayo de VIGS. En esta etapa, las hojas verdaderas, no han salido a la luz (Figura 1). </li></ol> 2. Construir VIGS vector portador del gen GrCLA1 <ol><li> Amplificar el gen de una Alterados Cloroplastos de algodón (GrCLA1) por PCR con cebadores GrCLA1-F, 5&#39;-GCCCTTTGTGCATCTTC-3 &#39;y GrCLA1 ​​R-, 5&#39;-CTCTAGGGGCATTGAAG-3&#39; de una biblioteca de cDNA de G. raimondii hoja de tejidos. </li><li> Digerir los productos PCR de GrCLA1 ​​con EcoRI y KpnI y se insertan en pYL156 (pTRV-ARN2) vector de la ligadura. </li><li> Pantalla de los clones en placas de agar LB que contenía 50 mg / ml de kanamicina. Verificar las construcciones por la digestión con enzimas de restricción y secuenciación. </li><li> Transformar el plásmido en Agrobacterium tumefaciens GV3101 mediante electroporación y se recuperan en medio LB líquido a 28 ° C. Seleccionar los transformantes en placas con LB + kanamicina (50 mg / ml) + gentamicina (25 mg / mL). Las bacterias se pueden almacenar en un 25% de glicerol a -80 ° C para el uso a largo plazo. </li></ol> 3. VIGS realizar la inoculación <ol><li> Tres días antes de la inoculación, las acciones de glicerol racha de congelado de Agrobacterium tumefaciens llevar pYL192 (TRV): ARN1, pYL156 (TRV): ARN2 (vector por sí solo) y pYL156 (TRV): ARN2-GrCLA1 ​​en placas de agar LB que contenía 50 mg / ml de kanamicina y 25 mg / ml de gentamicina. Las placas se incuban a 28 ° C durante 24 horas. </li><li> Dos días antes de la infiltración VIGS, elegir una sola colonia para construir cada uno de los platos por encima y por inocular en 5 ml de medio LB suplementado con 50 mg / ml de kanamicina y 25 mg / ml de gentamicina; Mantener el cultivo de bacterias a 28 ° C para pasar la noche en un tambor a una velocidad de 50 rpm. </li><li> La transferencia de la cultura por encima de un frasco con 50 ml de medio LB suplementado con 50 mg / ml de kanamicina y 25 mg / ml de gentamicina, además de 10 mM MES (2 - (4 morfolino)-etano sulfónico) y 20 acetosiringona M. Mantener el cultivo a 28 ° C durante la noche en una coctelera con una velocidad de 50 rpm. </li><li> Al día siguiente, vuelta a las células agro-bacteriana a 4000 rpm durante 5 minutos, volver a suspender la cultura en el buffer de la infiltración que contiene 10 mM de MgCl2, 10 mM MES y 200 acetosiringona M. Ajuste el OD 600 de la cultura a 1,5. </li><li> Salir de la cultura en el banquillo a temperatura ambiente durante 3 horas. </li><li> Antes de la infiltración Agrobacterial, ponche de la parte inferior de los cotiledones de las plantas de algodón con una aguja de 25 G sin perforar a través de los cotiledones. Uno o dos agujeros (depende de la suavidad de los tejidos) fueron perforados en cada sección de los cotiledones (Figura 2). </li><li> Mezcla de cultivo en suspensión de Agrobacterial pYL192 (TRV): ARN1 y pYL156 (TRV): ARN2 o pYL156 (TRV): ARN2-GrCLA1 ​​en una proporción de 1:1; mano infiltrarse en la mezcla de la parte inferior de los cotiledones a través de los sitios de heridas con un 1 ml jeringa sin aguja (Figura 3). </li><li> Cubrir las plantas con una cúpula de plástico y dejar que las plantas se infiltraron a temperatura ambiente bajo condiciones de luz tenue de la noche. </li><li> Transferencia de las plantas a una sala de crecimiento con la temperatura de 23 ° C, 120 μE m -2 S -1 luz con un ciclo de 12 horas horas de luz / oscuridad 12. </li></ol> Nota: VIGS funciona cuando las plantas están ya sea 23 ° C o en un invernadero (25-28 ° C). Cuando la temperatura es relativamente baja (23 a 25 ° C), es más fácil para la inoculación y el fenotipo es más uniforme en comparación con los más altos (28-30 ° C) o la temperatura fluctuó. Cubriendo las plantas con una cúpula también hace VIGS más eficiente. <ol start="10"><li> Examine el fenotipo de silenciar a las 7 ~ 8 días después de la infiltración. Las hojas de las plantas de verdad silenciada por pYL156 (TRV): ARN2-GrCLA1 ​​comenzó a mostrar el fenotipo albino (Figura 4). Las plantas se infiltraron con la realización de agrobacterias pYL156 (TRV): ARN2 servir como un control. </li><li> Verificar la eficiencia de silenciamiento de genes mediante el examen de nivel de expresión de genes endógenos usando la polimerasa de transcripción inversa reacción en cadena (RT-PCR) con ARN aislado de control y silenció las plantas de algodón. </li></ol> Nota: Las plantas VIGS de opinión deben mantenerse bajo condiciones de cuarentena contenidos y las plantas deben estar debidamente esterilizado en autoclave y desecharse después de los ensayos como TRV cuenta con una amplia gama de huéspedes y es un patógeno de declaración obligatoria. 4.Los resultados representativos: Aproximadamente dos semanas después de la mano con la mezcla de infiltración Agrobacterial, el fenotipo albino causada por GrCLA1 ​​silenciamiento se observó claramente en las hojas verdaderas. La eficiencia de silenciamiento alcanza casi el 100% en varios experimentos con más de 50 plantas. Las plantas presentan un fenotipo silenciado uniformemente distribuidos y muy fuerte albino en el desarrollo de nuevas hojas con un mosaico en las hojas mayores (Figura 4). <img src="/files/ftp_upload/2938/2938fig1.jpg" alt="Figura 1" /> Figura 1. Un brote de algodón alrededor de la etapa de dos semanas de edad con dos cotiledones completamente abiertos utilizados para la infiltración Agrobacterial. <img src="/files/ftp_upload/2938/2938fig2.jpg" alt="Figura 2" /> Figura 2. Perforación de pequeños agujeros en la parte inferior de los cotiledones de las plantas de algodón con una aguja G 25 para facilitar la infiltración Agrobacterial. <img src="/files/ftp_upload/2938/2938fig3.jpg" alt="Figura 3" /> Figura 3. Infiltración de la mano de la mezcla de Agrobacterial en los cotiledones de algodón a través de los sitios de heridas con una jeringa sin aguja <img src="/files/ftp_upload/2938/2938fig4.jpg" alt="Figura 4" /> Figura 4. Fenotipo Albino apareció en VIGS silenciado las plantas de algodón. Cuatro cultivares se muestran. A) FiberMax 832, B) Phytogen 480WR; C) Phytogen 425RF; D) Deltapine 90.

<ol>
	<li>Chen, Z. J., Scheffler, B. E., Dennis, E., Triplett, B. A., Zhang, T., Guo, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toward+sequencing+cotton+(Gossypium)+genomes.">Toward sequencing cotton (Gossypium) genomes.</a> Plant Physiol. 145, 1303-1310 (2007).</li><li>Dinesh-Kumar, S. P., Anandalakshmi, R., Marathe, R., Schiff, M., Liu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Virus-induced+gene+silencing.">Virus-induced gene silencing.</a> Methods Mol Biol. 236, 287-294 (2003).</li><li>Hamilton, A. J., Baulcombe, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+species+of+small+antisense+RNA+in+posttranscriptional+gene+silencing+in+plants.">A species of small antisense RNA in posttranscriptional gene silencing in plants.</a> Science. 286, 950-952 (1999).</li><li>Burch-Smith, T. M., Anderson, J. C., Martin, G. B., Dinesh-Kumar, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applications+and+advantages+of+virus-induced+gene+silencing+for+gene+function+studies+in+plants.">Applications and advantages of virus-induced gene silencing for gene function studies in plants.</a> Plant J. 39, 734-746 (2004).</li><li>Liu, Y., Schiff, M., Dinesh-Kumar, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Virus-induced+gene+silencing+in+tomato.">Virus-induced gene silencing in tomato.</a> Plant J. 31, 777-786 (2002).</li><li>Estevez, J. M., Cantero, A., Romero, C., Kawaide, H., Jimenez, L. F., Kuzuyama, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+the+expression+of+CLA1,+a+gene+that+encodes+the+1-deoxyxylulose+5-phosphate+synthase+of+the+2-C-methyl-D-erythritol-4-phosphate+pathway+in+Arabidopsis.">Analysis of the expression of CLA1, a gene that encodes the 1-deoxyxylulose 5-phosphate synthase of the 2-C-methyl-D-erythritol-4-phosphate pathway in Arabidopsis.</a> Plant Physiol. 124, 95-104 (2000).</li><li>Mandel, M. A., Feldmann, K. A., Herrera-Estrella, L., Rocha-Sosa, M., Leon, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CLA1,+a+novel+gene+required+for+chloroplast+development,+is+highly+conserved+in+evolution.">CLA1, a novel gene required for chloroplast development, is highly conserved in evolution.</a> Plant J. 9, 649-658 (1996).</li><li>Gao, X., Wheeler, T., Li, Z., Kenerley, C., He, P., Shan, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Silencing+GhNDR1+and+GhMKK2+compromised+cotton+resistance+to+Verticillium+wilt.">Silencing GhNDR1 and GhMKK2 compromised cotton resistance to Verticillium wilt.</a> Plant J. 66, 293-305 (2011).</li></ol>

No hay conflictos de interés declarado.

VIGS ha demostrado ser una poderosa herramienta en el análisis de la genómica funcional de forma transitoria derribar la expresión de genes endógenos. En este estudio, hemos desarrollado un VIGS mediada por Agrobacterium mediante la utilización de un vector binario TRV-basado. El algodón CLA1 (GrCLA1) gen fue desarrollado como una marca visual para monitorear la eficiencia de silenciamiento. Siempre hemos obtenido el 100% de eficiencia de silenciamiento génico, como lo demuestra el fenotipo albino que ap...

<table border="1"><tbody><tr><th> Nombre de los reactivos y equipos </th><th> Empresa </th><th> Catálogo / número de serie </th><th> Comentarios </th></tr><tr><td> Rodillos de tambor </td><td> Glas-Col, LLC. </td><td> 099A TC108 </td><td> Agro-bacteria cultura </td></tr><tr><td> Incubadora </td><td> Sheldon Manufacturing, Inc. </td><td> 01046209 </td><td> Agro-bacteria cultura </td></tr><tr><td> UV / Vis espectrofotómetro </td><td> Beckman Coulter </td><td> Modelo: DU530 </td><td> La medición de OD </td></tr><tr><td> Gene Emisores </td><td> BioRad </td><td> Modelo: 1652076, de serie: 154BR3880 </td><td> Electroporación </td></tr><tr><td> Generador de impulsos del controlador </td><td> BioRad </td><td> Modelo: 1652098, de serie: 232BR4833 </td><td> Electroporación </td></tr><tr><td> Micropulser cubeta de electroporación </td><td> BioRad </td><td> 165-2081 </td><td> Electroporación </td></tr><tr><td> 1 ml jeringa </td><td> BD Biosciences </td><td> 30962 </td><td> La inoculación de las agro-bacteria </td></tr><tr><td> Metro Mix 700 </td><td> SUNGR </td><td> SKU # 553001 </td><td> Plántulas de crecimiento </td></tr><tr><td> Terra Cotta olla </td><td> TOPlastics </td><td> GPS 3001B2 </td><td> Plántulas de crecimiento </td></tr><tr><td> MES monohidrato </td><td> USB </td><td> 18886 </td><td> La infiltración de búfer </td></tr><tr><td> Acetosiringona </td><td> Sigma </td><td> D134406 </td><td> La infiltración de búfer </td></tr></tbody></table>

agrobacterium-mediated virus-induced gene silencing assay in cotton

Algodón (Gossypium hirsutum) es uno de los cultivos más importantes del mundo. Los esfuerzos se han hecho considerables en la reproducción molecular de nuevas variedades. El análisis genético a gran escala funcional de algodón se ha quedado atrás la mayor parte de las especies de plantas modernas, probablemente debido a su gran tamaño de la duplicación del genoma, los genes y la poliploidía, largo ciclo de crecimiento y la obstinación a la transformación genética 1. Para facilitar el estudio funcional de alto rendimiento genética / genómica en algodón, tratamos de desarrollar ensayos transitoria rápida y eficiente para evaluar las funciones de genes de algodón. Inducida por el virus de silenciamiento génico (VIGS) es una poderosa técnica que fue desarrollada con base en el host post-transcripcional silenciamiento génico (PTGS) para reprimir la proliferación viral 2,3. Mediada por Agrobacterium VIGS ha sido aplicado con éxito en una amplia gama de especies dicotiledóneas como Solanaceae, Arabidopsis y otras especies de leguminosas y especies monocotiledóneas como la cebada, el trigo y el maíz, por diversos estudios de genómica funcional 3,4. Como este método rápido y eficaz evita la transformación de plantas y supera la redundancia funcional, es particularmente atractiva y adecuada para el estudio de genómica funcional en las especies de cultivos como el algodón no es susceptible de transformación. En este estudio, se presenta el protocolo detallado de Agrobacterium VIGS sistema en el algodón. Entre los varios vectores VIGS viral, el virus del cascabel del tabaco (TRV) invade una amplia gama de huéspedes y es capaz de propagarse con fuerza a lo largo de toda la planta sin embargo, producen síntomas leves en la hosts5. Para controlar la eficacia de silenciamiento, GrCLA1, un gen homólogo de Arabidopsis Cloroplastos Alterados un gen (AtCLA1) en el algodón, ha sido clonado y se inserta en el vector binario VIGS pYL156. CLA1 gen está involucrado en el desarrollo del cloroplasto 6, y estudios previos han demostrado que pérdida de función de AtCLA1 dio lugar a un fenotipo albino de 7 hojas verdaderas, proporcionando un excelente marcador visual para silenciar la eficiencia. En aproximadamente dos semanas después de la infiltración de Agrobacterium, el fenotipo albino comenzaron a aparecer en las hojas verdaderas, con una eficiencia del 100% en silenciar a todos los experimentos replicados. El silenciamiento de la expresión de genes endógenos también fue confirmado por RT-PCR análisis. Significativamente, el silenciamiento potente podría ocurrir en todas las variedades que hemos probado, incluyendo varias variedades cultivadas comercialmente en Texas. Esta rápida y eficiente mediada por Agrobacterium VIGS ensayo proporciona una herramienta muy poderosa para un rápido análisis a gran escala de las funciones de los genes en el genoma de nivel en el algodón.

Watch this Scientific Journal Video about Agrobacterium-Mediated Virus-Induced Gene Silencing Assay In Cotton at JoVE.com

Agrobacterium-Mediated Virus-Induced Gene Silencing Assay In Cotton

Se presenta el protocolo detallado para la mediada por Agrobacterium inducida por el virus de silenciamiento génico (VIGS) de ensayo en el algodón. El virus del cascabel del tabaco (TRV) vectores derivados de VIGS fueron desplegados para inducir el silenciamiento de ARN de algodón GrCLA1, Alterados Cloroplastos un gen. El fenotipo albino causadas por el silenciamiento GrCLA1 ​​se observó en el estado de plántula dentro de 2 semanas después de la inoculación.

Mediada por Agrobacterium inducida por el virus Gene ensayo silenciamiento en algodón

Cotton (Gossypium hirsutum) is one of the most important crops worldwide. Considerable efforts have been made on molecular breeding of new varieties.  The ...

agrobacterium-mediated-virus-induced-gene-silencing-assay-in-cotton

Research

JoVE Journal

Biology

24.2K vistas.  Texas A&M University. Se presenta el protocolo detallado para la mediada por Agrobacterium inducida por el virus de silenciamiento génico (VIGS) de ensayo en el algodón. El virus del cascabel del tabaco (TRV) vectores derivados de VIGS fueron desplegados para inducir el silenciamiento de ARN de algodón GrCLA1, Alterados Cloroplastos un gen. El fenotipo albino causadas por el silenciamiento GrCLA1 ​​se observó en el estado de plántula dentro de 2 semanas después de la inoculación.

Video: Agrobacterium-Mediated Virus-Induced Gene Silencing Assay In Cotton

Virus-induced Gene Silencing (VIGS) in Nicotiana benthamiana and Tomato

RNA interference (RNAi) is a highly specific gene-silencing phenomenon triggered by dsRNA1. This silencing mechanism uses two major classes of RNA regulators: microRNAs, which are produced from non-protein coding genes and short interfering RNAs (siRNAs). Plants use RNAi to control transposons and to exert tight control over developmental processes such as flower organ formation and leaf development2,3,4. Plants also use RNAi to defend themselves against infection by viruses. Consequently, many viruses have evolved suppressors of gene silencing to allow their successful colonization of their host5.
Virus-induced gene silencing (VIGS) is a method that takes advantage of the plant RNAi-mediated antiviral defense mechanism. In plants infected with unmodified viruses the mechanism is specifically targeted against the viral genome. However, with virus vectors carrying sequences derived from host genes, the process can be additionally targeted against the corresponding host mRNAs. VIGS has been adapted for high-throughput functional genomics in plants by using the plant pathogen Agrobacterium tumefaciens to deliver, via its Ti plasmid, a recombinant virus carrying the entire or part of the gene sequence targeted for silencing. Systemic virus spread and the endogenous plant RNAi machinery take care of the rest. dsRNAs corresponding to the target gene are produced and then cleaved by the ribonuclease Dicer into siRNAs of 21 to 24 nucleotides in length. These siRNAs ultimately guide the RNA-induced silencing complex (RISC) to degrade the target transcript2.
Different vectors have been employed in VIGS and one of the most frequently used is based on tobacco rattle virus (TRV). TRV is a bipartite virus and, as such, two different A. tumefaciens strains are used for VIGS. One carries pTRV1, which encodes the replication and movement viral functions while the other, pTRV2, harbors the coat protein and the sequence used for VIGS6,7. Inoculation of Nicotiana benthamiana and tomato seedlings with a mixture of both strains results in gene silencing. Silencing of the endogenous phytoene desaturase (PDS) gene, which causes photobleaching, is used as a control for VIGS efficiency. It should be noted, however, that silencing in tomato is usually less efficient than in N. benthamiana. RNA transcript abundance of the gene of interest should always be measured to ensure that the target gene has efficiently been down-regulated. Nevertheless, heterologous gene sequences from N. benthamiana can be used to silence their respective orthologs in tomato and vice versa8.

Virus-induced Gene Silencing VIGS in Nicotiana benthamiana and Tomato

Description of a virus-induced gene silencing (VIGS) method for knock-down of gene expression in Nicotiana benthamiana and tomato.

Inducida por el virus de silenciamiento génico (VIGS) en Nicotiana benthamiana Y tomate

RNA interference (RNAi) is a highly specific gene-silencing phenomenon triggered by dsRNA1. This silencing mechanism uses two major classes of RNA ...

Investigación

Biología

Live Cell Calcium Imaging Combined with siRNA Mediated Gene Silencing Identifies Ca2+ Leak Channels in the ER Membrane and their Regulatory Mechanisms

In mammalian cells, the endoplasmic reticulum (ER) plays a key role in protein biogenesis as well as in calcium signalling1. The heterotrimeric Sec61 complex in the ER membrane provides an aqueous path for newly-synthesized polypeptides into the lumen of the ER. Recent work from various laboratories suggested that this heterotrimeric complex may also form transient Ca2+ leak channels2-8. The key observation for this notion was that release of nascent polypeptides from the ribosome and Sec61 complex by puromycin leads to transient release of Ca2+ from the ER. Furthermore, it had been observed in vitro that the ER luminal protein BiP is involved in preventing ion permeability at the level of the Sec61 complex9,10. We have established an experimental system that allows us to directly address the role of the Sec61 complex as potential Ca2+ leak channel and to characterize its putative regulatory mechanisms11-13. This system combines siRNA mediated gene silencing and live cell Ca2+ imaging13. Cells are treated with siRNAs that are directed against the coding and untranslated region (UTR), respectively, of the SEC61A1 gene or a negative control siRNA. In complementation analysis, the cells are co-transfected with an IRES-GFP vector that allows the siRNA-resistant expression of the wildtype SEC61A1 gene. Then the cells are loaded with the ratiometric Ca2+-indicator FURA-2 to monitor simultaneously changes in the cytosolic Ca2+ concentration in a number of cells via a fluorescence microscope. The continuous measurement of cytosolic Ca2+ also allows the evaluation of the impact of various agents, such as puromycin, small molecule inhibitors, and thapsigargin on Ca2+ leakage. This experimental system gives us the unique opportunities to i) evaluate the contribution of different ER membrane proteins to passive Ca2+ efflux from the ER in various cell types, ii) characterize the proteins and mechanisms that limit this passive Ca2+ efflux, and iii) study the effects of disease linked mutations in the relevant components.

Live Cell Calcium Imaging Combined with siRNA Mediated Gene Silencing Identifies Ca2+ Leak Channels in the ER Membrane and their Regulatory Mechanisms

The endoplasmic reticulum plays a key role in protein biogenesis and in calcium homeostasis. We have established an experimental system that allows us to address the role of Ca2+ leak channels and to characterize their putative regulatory mechanisms. This system involves siRNA mediated gene silencing and live cell Ca2+ imaging.

Imágenes en vivo del calcio celular combinado con el silenciamiento de genes mediada por siRNA Identifica Ca 2 + Canales de fugas en la membrana del ER y sus mecanismos de regulación

In mammalian cells, the endoplasmic reticulum (ER) plays a key role in protein biogenesis as well as in calcium signalling1. The heterotrimeric Sec61 ...

Induction and Testing of Hypoxia in Cell Culture

Hypoxia is defined as the reduction or lack of oxygen in organs, tissues, or cells. This decrease of oxygen tension can be due to a reduced supply in oxygen (causes include insufficient blood vessel network, defective blood vessel, and anemia) or to an increased consumption of oxygen relative to the supply (caused by a sudden higher cell proliferation rate). Hypoxia can be physiologic or pathologic such as in solid cancers 1-3, rheumatoid arthritis, atherosclerosis etc&#x2026; Each tissues and cells have a different ability to adapt to this new condition. During hypoxia, hypoxia inducible factor alpha (HIF) is stabilized and regulates various genes such as those involved in angiogenesis or transport of oxygen 4. The stabilization of this protein is a hallmark of hypoxia, therefore detecting HIF is routinely used to screen for hypoxia 5-7. 
In this article, we propose two simple methods to induce hypoxia in mammalian cell cultures and simple tests to evaluate the hypoxic status of these cells.

Here we propose simple methods to induce hypoxia in cell cultures and simple tests to evaluate the hypoxic status of the cultures.

Inducción y prueba de hipoxia en el cultivo celular

Hypoxia is defined as the reduction or lack of oxygen in organs, tissues, or cells.  This decrease of oxygen tension can be due to a reduced supply in ...

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the messagefor review see 1. However, it's now widely accepted that synonymous codon usage is the primary cause of non-uniform translational elongation rates1. Synonymous codons are not used with identical frequency. A bias exists in the use of synonymous codons with some codons used more frequently than others2. Codon bias is organism as well as tissue specific2,3. Moreover, frequency of codon usage is directly proportional to the concentrations of cognate tRNAs4. Thus, a frequently used codon will have higher multitude of corresponding tRNAs, which further implies that a frequent codon will be translated faster than an infrequent one. Thus, regions on mRNA enriched in rare codons (potential pause sites) will as a rule slow down ribosome movement on the message and cause accumulation of nascent peptides of the respective sizes5-8. These pause sites can have functional impact on the protein expression, mRNA stability and protein foldingfor review see 9. Indeed, it was shown that alleviation of such pause sites can alter ribosome movement on mRNA and subsequently may affect the efficiency of co-translational (in vivo) protein folding1,7,10,11. To understand the process of protein folding in vivo, in the cell, that is ultimately coupled to the process of protein synthesis it is essential to gain comprehensive insights into the impact of codon usage/tRNA content on the movement of ribosomes along mRNA during translational elongation.
Here we describe a simple technique that can be used to locate major translation pause sites for a given mRNA translated in various cell-free systems6-8. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA. The rationale is that at low-frequency codons, the increase in the residence time of the ribosomes results in increased amounts of nascent peptides of the corresponding sizes. In vitro transcribed mRNA is used for in vitro translational reactions in the presence of radioactively labeled amino acids to allow the detection of the nascent chains. In order to isolate ribosome bound nascent polypeptide complexes the translation reaction is layered on top of 30% glycerol solution followed by centrifugation. Nascent polypeptides in polysomal pellet are further treated with ribonuclease A and resolved by SDS PAGE. This technique can be potentially used for any protein and allows analysis of ribosome movement along mRNA and the detection of the major pause sites. Additionally, this protocol can be adapted to study factors and conditions that can alter ribosome movement and thus potentially can also alter the function/conformation of the protein.

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

A technique to identify translational pause sites on mRNA is described. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA, followed by the size analysis of the nascent chains using a denaturing gel electrophoresis.

El aislamiento de los ribosomas polipéptidos nacientes Bound<em&gt; In vitro</em&gt; Para la identificación de sitios de traslación pausa a lo largo del ARNm

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the ...

Using SecM Arrest Sequence as a Tool to Isolate Ribosome Bound Polypeptides

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway that polypeptide chain follows during co-translational folding to achieve its functional form is still an enigma. In order to understand this process and to determine the exact conformation of the co-translational folding intermediates, it is essential to develop techniques that allow the isolation of RNCs carrying nascent chains of predetermined sizes to allow their further structural analysis.
SecM (secretion monitor) is a 170 amino acid E. coli protein that regulates expression of the downstream SecA (secretion driving) ATPase in the secM-secA operon6. Nakatogawa and Ito originally found that a 17 amino acid long sequence (150-FSTPVWISQAQGIRAGP-166) in the C-terminal region of the SecM protein is sufficient and necessary to cause stalling of SecM elongation at Gly165, thereby producing peptidyl-glycyl-tRNA stably bound to the ribosomal P-site7-9. More importantly, it was found that this 17 amino acid long sequence can be fused to the C-terminus of virtually any full-length and/or truncated protein thus allowing the production of RNCs carrying nascent chains of predetermined sizes7. Thus, when fused or inserted into the target protein, SecM stalling sequence produces arrest of the polypeptide chain elongation and generates stable RNCs both in vivo in E. coli cells and in vitro in a cell-free system. Sucrose gradient centrifugation is further utilized to isolate RNCs.
The isolated RNCs can be used to analyze structural and functional features of the co-translational folding intermediates. Recently, this technique has been successfully used to gain insights into the structure of several ribosome bound nascent chains10,11. Here we describe the isolation of bovine Gamma-B Crystallin RNCs fused to SecM and generated in an in vitro translation system.

We describe here a technique that is now routinely used to isolate stably bound ribosome nascent chain complexes (RNCs). This technique takes advantage of the discovery that a 17 amino acid long SecM "arrest sequence" can halt translation elongation in a prokaryotic (E. coli) system, when inserted into (or fused to the C-terminus) of virtually any protein.

Usar secuencias de detención SecM como una herramienta para aislar Ribosome polipéptidos enlazados

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway ...

Optimization and Utilization of Agrobacterium-mediated Transient Protein Production in Nicotiana

Agrobacterium-mediated transient protein production in plants is a promising approach to produce vaccine antigens and therapeutic proteins within a short period of time. However, this technology is only just beginning to be applied to large-scale production as many technological obstacles to scale up are now being overcome. Here, we demonstrate a simple and reproducible method for industrial-scale transient protein production based on vacuum infiltration of Nicotiana plants with Agrobacteria carrying launch vectors. Optimization of Agrobacterium cultivation in AB medium allows direct dilution of the bacterial culture in Milli-Q water, simplifying the infiltration process. Among three tested species of Nicotiana, N. excelsiana (N. benthamiana &#215; N. excelsior) was selected as the most promising host due to the ease of infiltration, high level of reporter protein production, and about two-fold higher biomass production under controlled environmental conditions. Induction of Agrobacterium harboring pBID4-GFP (Tobacco mosaic virus-based) using chemicals such as acetosyringone and monosaccharide had no effect on the protein production level. Infiltrating plant under 50 to 100 mbar for 30 or 60 sec resulted in about 95% infiltration of plant leaf tissues. Infiltration with Agrobacterium laboratory strain GV3101 showed the highest protein production compared to Agrobacteria laboratory strains LBA4404 and C58C1 and wild-type Agrobacteria strains at6, at10, at77 and A4. Co-expression of a viral RNA silencing suppressor, p23 or p19, in N. benthamiana resulted in earlier accumulation and increased production (15-25%) of target protein (influenza virus hemagglutinin).

Optimization and Utilization of Agrobacterium-mediated Transient Protein Production in Nicotiana

Transient protein production in Nicotiana plants based on vacuum infiltration with Agrobacteria carrying launch vectors (Tobacco mosaic virus-based) is a rapid and economic approach to produce vaccine antigens and therapeutic proteins. We simplified the procedure and improved target accumulation by optimizing conditions of bacteria cultivation, selecting host species, and co-introducing RNA silencing suppressors.

La optimización y utilización de<em&gt; Agrobacterium</em&gt; Mediada por la producción de proteína transitoria en<em&gt; Nicotiana</em

Agrobacterium-mediated transient protein production in plants is a promising approach to produce vaccine antigens and therapeutic proteins within a short ...

Chitosan/Interfering RNA Nanoparticle Mediated Gene Silencing in Disease Vector Mosquito Larvae

Vector mosquitoes inflict more human suffering than any other organism&#8212;and kill more than one million people each year. The mosquito genome projects facilitated research in new facets of mosquito biology, including functional genetic studies in the primary African malaria vector Anopheles gambiae and the dengue and yellow fever vector Aedes aegypti. RNA interference- (RNAi-) mediated gene silencing has been used to target genes of interest in both of these disease vector mosquito species. Here, we describe a procedure for preparation of chitosan/interfering RNA nanoparticles that are combined with food and ingested by larvae. This technically straightforward, high-throughput, and relatively inexpensive methodology, which is compatible with long double stranded RNA (dsRNA) or small interfering RNA (siRNA) molecules, has been used for the successful knockdown of a number of different genes in A. gambiae and A. aegypti larvae. Following larval feedings, knockdown, which is verified through qRT-PCR or in situ hybridization, can persist at least through the late pupal stage. This methodology may be applicable to a wide variety of mosquito and other insect species, including agricultural pests, as well as other non-model organisms. In addition to its utility in the research laboratory, in the future, chitosan, an inexpensive, non-toxic and biodegradable polymer, could potentially be utilized in the field.

Here we describe a procedure for inhibiting gene function in disease vector mosquitoes through the use of chitosan/interfering RNA nanoparticles that are ingested by larvae.

El quitosano / ARN de interferencia mediada por nanopartículas silenciamiento génico en la enfermedad vectorial larvas de mosquitos

Vector mosquitoes inflict more human suffering than any other organism&#8212;and kill more than one million people each year. The mosquito genome projects ...

Meiotic Spindle Assessment in Mouse Oocytes by siRNA-mediated Silencing

Errors in chromosome segregation during meiotic division in gametes can lead to aneuploidy that is subsequently transmitted to the embryo upon fertilization. The resulting aneuploidy in developing embryos is recognized as a major cause of pregnancy loss and congenital birth defects such as Down&#8217;s syndrome. Accurate chromosome segregation is critically dependent on the formation of the microtubule spindle apparatus, yet this process remains poorly understood in mammalian oocytes. Intriguingly, meiotic spindle assembly differs from mitosis and is regulated, at least in part, by unique microtubule organizing centers (MTOCs). Assessment of MTOC-associated proteins can provide valuable insight into the regulatory mechanisms that govern meiotic spindle formation and organization. Here, we describe methods to isolate mouse oocytes and deplete MTOC-associated proteins using a siRNA-mediated approach to test function. In addition, we describe oocyte fixation and immunofluorescence analysis conditions to evaluate meiotic spindle formation and organization.

Here, we present a protocol for specific siRNA-mediated mRNA depletion followed by immunofluorescence analysis to evaluate meiotic spindle assembly and organization in mouse oocytes. This protocol is suitable for in vitro depletion of transcripts and functional assessment of different spindle and/or MTOC-associated factors in oocytes.

Evaluación del huso meiótico en ovocitos de ratón por silenciamiento siRNA mediada

Errors in chromosome segregation during meiotic division in gametes can lead to aneuploidy that is subsequently transmitted to the embryo upon ...

Silencing the Spark: CRISPR/Cas9 Genome Editing in Weakly Electric Fish

Electroreception and electrogenesis have changed in the evolutionary history of vertebrates. There is a striking degree of convergence in these independently derived phenotypes, which share a common genetic architecture. This is perhaps best exemplified by the numerous convergent features of gymnotiforms and mormyrids, two species-rich teleost clades that produce and detect weak electric fields and are called weakly electric fish. In the 50 years since the discovery that weakly electric fish use electricity to sense their surroundings and communicate, a growing community of scientists has gained tremendous insights into evolution of development, systems and circuits neuroscience, cellular physiology, ecology, evolutionary biology, and behavior. More recently, there has been a proliferation of genomic resources for electric fish. Use of these resources has already facilitated important insights with regards to the connection between genotype and phenotype in these species. A major obstacle to integrating genomics data with phenotypic data of weakly electric fish is a present lack of functional genomics tools. We report here a full protocol for performing CRISPR/Cas9 mutagenesis that utilizes endogenous DNA repair mechanisms in weakly electric fish. We demonstrate that this protocol is equally effective in both the mormyrid species Brienomyrus brachyistius and the gymnotiform Brachyhypopomus gauderio by using CRISPR/Cas9 to target indels and point mutations in the first exon of the sodium channel gene scn4aa. Using this protocol, embryos from both species were obtained and genotyped to confirm that the predicted mutations in the first exon of the sodium channel scn4aa were present. The knock-out success phenotype was confirmed with recordings showing reduced electric organ discharge amplitudes when compared to uninjected size-matched controls.

Here, a protocol is presented to produce and rear CRISPR/Cas9 genome knockout electric fish. Outlined in detail are the required molecular biology, breeding, and husbandry requirements for both a gymnotiform and a mormyrid, and injection techniques to produce Cas9-induced indel F0 larvae.

Silenciar la chispa: Edición del genoma CRISPR/Cas9 en peces débilmente eléctricos

Electroreception and electrogenesis have changed in the evolutionary history of vertebrates. There is a striking degree of convergence in these ...

Agrobacterium-Mediated Immature Embryo Transformation of Recalcitrant Maize Inbred Lines Using Morphogenic Genes

Demonstrated here is a detailed protocol for Agrobacterium-mediated genetic transformation of maize inbred lines using morphogenic genes Baby boom (Bbm) and Wuschel2 (Wus2). Bbm is regulated by the maize phospholipid transferase gene (Pltp) promoter, and Wus2 is under the control of a maize auxin-inducible (Axig1) promoter. An Agrobacterium strain carrying these morphogenic genes on transfer DNA (T-DNA) and extra copies of Agrobacterium virulence (vir) genes are used to infect maize immature embryo explants. Somatic embryos form on the scutella of infected embryos and can be selected by herbicide resistance and germinated into plants. A heat-activated cre/loxP recombination system built into the DNA construct allows for removal of morphogenic genes from the maize genome during an early stage of the transformation process. Transformation frequencies of approximately 14%, 4%, and 4% (numbers of independent transgenic events per 100 infected embryos) can be achieved for W22, B73, and Mo17, respectively, using this protocol.

Agrobacterium-Mediated Immature Embryo Transformation of Recalcitrant Maize Inbred Lines Using Morphogenic Genes

Plant morphogenic genes can be used to improve genetic transformation of recalcitrant genotypes. Described here is an Agrobacterium-mediated genetic transformation (QuickCorn) protocol for three important public maize inbred lines.

Agrobacterium-Transformación de embriones inmaduros mediados de líneas endogámicas de maíz recalcitrante utilizando genes morfogénicos

Demonstrated here is a detailed protocol for Agrobacterium-mediated genetic transformation of maize inbred lines using morphogenic genes Baby boom (Bbm) ...