Somos gratos aos Drs.. Dinesh Kumar-SP e Yule Liu para TRV-VIGS vetores, e as Dras. Chuck Kenerley, Terry Wheeler, Jim Starr e Bayer CropScience para o fornecimento de sementes de algodão. Este trabalho foi financiado pela NSF para LS e NIH para PH

1. Crescer mudas de algodão <ol><li> Semear as sementes do algodão herbáceo (Gossypium hirsutum) variedade Fibermax 832, PhytoGen 425RF, PhytoGen 480WR e Deltapine 90 em vasos (7 cm de diâmetro) contendo Metro Mix 700 (SunGR, Beavile, WA). </li><li> Manter os vasos em uma bandeja coberta com uma cúpula de plástico a 23 ° C, 120 μE m -2 S luz -1, com um fotoperíodo 12 horas horas light/12 escuros em sala de crescimento. </li><li> Remova a cúpula quando dois cotilédones têm surgido. </li><li> Aproximadamente duas semanas depois, use as plantas com dois cotilédones totalmente expandidos para o ensaio VIGS. Nesta fase, as folhas verdade ainda não surgiram (Figura 1). </li></ol> 2. Construir vector VIGS carregando GrCLA1 ​​gene <ol><li> Amplificar o Cloroplastos alterados 1 gene de algodão (GrCLA1) por PCR com primers GrCLA1-F, 5&#39;-GCCCTTTGTGCATCTTC-3 &#39;e GrCLA1-R, 5&#39;-CTCTAGGGGCATTGAAG-3&#39; a partir de uma biblioteca de cDNA de G. tecidos raimondii folha. </li><li> Digerir os produtos de PCR GrCLA1 ​​com EcoRI e KpnI e inserir em pYL156 (pTRV-RNA2) vetor por ligadura. </li><li> Tela os clones em placas LB ágar contendo 50 mcg / mL de canamicina. Verifique se as construções de digestão por enzimas de restrição e seqüenciamento. </li><li> Transformar o plasmídeo em Agrobacterium tumefaciens GV3101 por eletroporação e recuperar em meio líquido LB a 28 ° C. Selecione o transformantes em placas com LB + canamicina (50 mg / mL) + Gentamicina (25 mg / mL). As bactérias podem ser armazenados em glicerol 25% a -80 ° C para o uso a longo prazo. </li></ol> 3. Executar VIGS inoculação <ol><li> Três dias antes da inoculação, raia stocks glicerol congelados de Agrobacterium tumefaciens carregando pYL192 (TRV): RNA1, pYL156 (TRV): RNA2 (vetor sozinho) e pYL156 (TRV): RNA2-GrCLA1 ​​em placas de LB ágar contendo 50 mcg / mL de canamicina e 25 mg / mL de gentamicina. Incubar as placas a 28 ° C por 24 horas. </li><li> Dois dias antes da infiltração VIGS, escolher uma única colônia de cada construção das placas acima e inocular-lo em 5 mL de meio LB suplementado com 50 mg / mL de canamicina e 25 mcg / ml de Gentamicina; Incubar a cultura bacteriana em 28 ° C para a noite em um tambor de rolo a uma velocidade de 50 rpm. </li><li> Transferir a cultura acima para um frasco com 50 ml de meio LB suplementado com 50 mg / mL de canamicina e 25 mcg / ml de gentamicina, acrescido de 10 mM MES (2 - (4 morfolino) etano-sulfônico) e 20 mM acetosyringone. Crescer a cultura a 28 ° C para a noite em uma coqueteleira com uma velocidade de 50 rpm. </li><li> No dia seguinte, girar as células agro-bacteriana a 4000 rpm por 5 minutos; re-suspender a cultura no buffer de infiltração contendo 10 mM MgCl 2, 10 mM MES e 200 acetosyringone mM. Ajustar o OD 600 da cultura para 1,5. </li><li> Deixe a cultura no banco em temperatura ambiente por 3 horas. </li><li> Antes de infiltração Agrobacterial, soco a parte de baixo cotilédones de plantas de algodão com uma agulha G 25 sem perfurar através dos cotilédones. Um ou dois furos (depende da maciez do tecido) foram perfurados em cada seção do cotilédone (Figura 2). </li><li> Mix suspensão cultura Agrobacterial de pYL192 (TRV): RNA1 e pYL156 (TRV): RNA2 ou pYL156 (TRV): RNA2-GrCLA1 ​​na proporção de 1:1; mão infiltrar a mistura do lado de cotilédones através dos sites ferindo usando a 1 mL seringa sem agulha (Figura 3). </li><li> Cobrir as plantas com uma cúpula de plástico e deixe as plantas se infiltraram em temperatura ambiente sob condições de luz fraca para pernoite. </li><li> Transferência de plantas para sala de crescimento com a temperatura de 23 ° C, 120 μE m -2 S luz -1 com um ciclo de 12 horas horas de luz / 12 escuro. </li></ol> Nota: VIGS funciona quando as plantas estão sob ou 23 ° C ou em estufa (25-28 ° C). Quando a temperatura é relativamente baixa (23-25 ​​° C), é mais fácil para inoculação e fenótipo é mais uniforme em comparação com maior (28-30 ° C) ou temperatura oscilou. Cobrindo as plantas com uma cúpula também faz VIGS mais eficiente. <ol start="10"><li> Examine o fenótipo silenciar a 7 ~ infiltração pós 8 dias. As folhas de plantas verdade silenciada por pYL156 (TRV): RNA2-GrCLA1 ​​começou a exibir o fenótipo albino (Figura 4). As plantas infiltradas com transporte Agrobacterium pYL156 (TRV): RNA2 servir como controle. </li><li> Verificar a eficiência silenciamento do gene ao examinar o nível de expressão de genes endógenos com transcrição reversa reação em cadeia da polimerase (RT-PCR) com RNA isolado de controle de plantas de algodão e silenciados. </li></ol> Nota: As plantas VIGS-ed devem ser mantidos sob quarentena e condições contidas plantas devem ser devidamente esterilizado e descartado após os ensaios como TRV tem uma ampla gama de hospedeiros e é um patógeno de notificação obrigatória. 4.Resultados representativos: Aproximadamente duas semanas após a mão-de infiltração com mistura Agrobacterial, o fenótipo albino causada por GrCLA1 ​​silenciamento foi claramente observado nas folhas verdadeiras. A eficiência silenciar atinge quase 100% em múltiplas experiências com mais de 50 plantas. As plantas silenciadas exibir um fenótipo uniformemente distribuída e muito forte em novos albino desenvolvimento folhas com um mosaico nas folhas mais velho (Figura 4). <img src="/files/ftp_upload/2938/2938fig1.jpg" alt="Figura 1" /> Figura 1. Um mudas de algodão em cerca de duas semanas de estágio de idade com dois cotilédones totalmente expandidos utilizado para infiltração Agrobacterial. <img src="/files/ftp_upload/2938/2938fig2.jpg" alt="Figura 2" /> Figura 2. Perfuração de pequenos orifícios na parte inferior do cotilédones de plantas de algodão com uma agulha de 25 G para facilitar a infiltração Agrobacterial. <img src="/files/ftp_upload/2938/2938fig3.jpg" alt="Figura 3" /> Figura 3. Infiltração Mão de mistura Agrobacterial na cotilédones de algodão através dos sites ferindo usando uma seringa sem agulha <img src="/files/ftp_upload/2938/2938fig4.jpg" alt="Figura 4" /> Figura 4. Fenótipo Albino apareceu na VIGS silenciados plantas de algodão. Quatro cultivares são mostrados. 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>

Não há conflitos de interesse declarados.

VIGS foi provado ser uma ferramenta poderosa na análise genômica funcional por transitoriamente derrubando a expressão de genes endógenos. Neste estudo, desenvolvemos um VIGS Agrobacterium mediada pela utilização de um vetor binário TRV-based. O algodão CLA1 gene (GrCLA1) foi desenvolvido como um marcador visual para monitorar a eficiência silenciar. Temos consistentemente obteve 100% de eficiência silenciamento gênico, demonstrado pelo fenótipo albino aparecendo nas folhas verdadeiras em t...

<table border="1"><tbody><tr><th> Nome dos reagentes e equipamentos </th><th> Companhia </th><th> Catálogo / número de série </th><th> Comentários </th></tr><tr><td> Rolo de tambor </td><td> Glas-Col, LLC. </td><td> 099A TC108 </td><td> Agro-bactéria cultura </td></tr><tr><td> Incubadora </td><td> Sheldon Manufacturing, Inc. </td><td> 01046209 </td><td> Agro-bactéria cultura </td></tr><tr><td> UV / Vis espectrofotômetro </td><td> Beckman Coulter </td><td> Modelo: DU530 </td><td> Medição OD </td></tr><tr><td> Gene Pulser </td><td> BioRad </td><td> Modelo: 1652076; Serial: 154BR3880 </td><td> Eletroporação </td></tr><tr><td> Pulsador Controlador </td><td> BioRad </td><td> Modelo: 1652098; Serial: 232BR4833 </td><td> Eletroporação </td></tr><tr><td> Micropulser Eletroporação cuvete </td><td> BioRad </td><td> 165-2081 </td><td> Eletroporação </td></tr><tr><td> 1 Seringa ml </td><td> BD Biosciences </td><td> 30962 </td><td> Inoculação de bactéria agro- </td></tr><tr><td> Metro Mix 700 </td><td> SUNGR </td><td> SKU # 553001 </td><td> Mudas em crescimento </td></tr><tr><td> Terra Cotta pot </td><td> TOPlastics </td><td> GPS 3001B2 </td><td> Mudas em crescimento </td></tr><tr><td> Monohidratada MES </td><td> USB </td><td> 18886 </td><td> Buffer de infiltração </td></tr><tr><td> Acetosyringone </td><td> Sigma </td><td> D134406 </td><td> Buffer de infiltração </td></tr></tbody></table>

agrobacterium-mediated virus-induced gene silencing assay in cotton

Algodão (Gossypium hirsutum) é uma das culturas mais importantes do mundo. Esforços consideráveis ​​têm sido feitos sobre melhoramento molecular de novas variedades. A análise de genes em larga escala funcional em algodão tem sido ficado para trás a maioria das espécies de plantas modernas, provavelmente devido a seu grande tamanho do genoma, o gene duplicação e poliploidia, ciclo de crescimento longo e recalcitrância à transformação genética 1. Para facilitar o estudo funcional de alto rendimento genética / genômica em algodão, tentamos desenvolver rápida e eficiente ensaios transitórios para avaliar as funções dos genes do algodão. Virus-Induced silenciamento gênico (VIGS) é uma poderosa técnica que foi desenvolvida com base no host silenciamento pós-transcricional Gene (PTGS) para reprimir a proliferação viral 2,3. Mediada por Agrobacterium VIGS tem sido aplicado com sucesso em uma ampla gama de espécies como dicotiledôneas Solanaceae, Arabidopsis e espécies de leguminosas, e as espécies monocotiledôneas incluindo trigo, cevada e milho, por vários estudos genômicos funcionais 3,4. Como esta abordagem rápida e eficiente evita transformação de plantas e supera redundância funcional, é particularmente atraente e adequado para o estudo do genoma funcional de espécies de culturas como o algodão não passíveis de transformação. Neste estudo, relatamos o protocolo detalhado de Agrobacterium mediada sistema VIGS em algodão. Entre os vários vetores virais VIGS, o vírus chocalho tabaco (TRV) invade uma ampla gama de hospedeiros e é capaz de espalhar vigorosamente em toda a planta ainda produzir sintomas leves na hosts5. Para monitorar a eficiência silenciamento, GrCLA1, um gene homólogo de Cloroplastos Arabidopsis alterados um gene (AtCLA1) em algodão, foi clonado e inserido no vetor binário VIGS pYL156. CLA1 gene está envolvida no desenvolvimento do cloroplasto 6, e estudos anteriores demonstraram que perda de função de AtCLA1 resultou em um fenótipo albino em folhas verdadeiras 7, proporcionando um excelente marcador visual para silenciar eficiência. Em aproximadamente duas semanas infiltração Agrobacterium post, o fenótipo albino começaram a aparecer nas folhas verdadeiras, com eficiência de 100% em silenciar todos os experimentos replicados. O silenciamento da expressão do gene endógeno também foi confirmada por RT-PCR. Significativamente, silenciando poderia potently ocorrer em todas as cultivares testadas, incluindo diversas variedades comercialmente cultivadas em Texas. Essa rápida e eficiente mediada por Agrobacterium ensaio VIGS fornece uma ferramenta muito poderosa para a análise em larga escala rápida de funções dos genes no genoma nível em algodão.

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

Apresentamos o protocolo detalhado para Agrobacterium mediada induzida pelo vírus ensaio de silenciamento de genes (VIGS) em algodão. O vírus chocalho tabaco (TRV) derivados de vetores VIGS foram mobilizados para induzir silenciamento de RNA de algodão GrCLA1, Cloroplastos alterados um gene. O fenótipo albino causada pelo silenciamento GrCLA1 ​​foi observada na fase de mudas dentro de 2 semanas após a inoculação.

Agrobacterium-Mediated Virus-Induced Assay silenciamento gênico Em Cotton

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 visualizações.  Texas A&M University. Apresentamos o protocolo detalhado para Agrobacterium mediada induzida pelo vírus ensaio de silenciamento de genes (VIGS) em algodão. O vírus chocalho tabaco (TRV) derivados de vetores VIGS foram mobilizados para induzir silenciamento de RNA de algodão GrCLA1, Cloroplastos alterados um gene. O fenótipo albino causada pelo silenciamento GrCLA1 ​​foi observada na fase de mudas dentro de 2 semanas após a inoculação.

Vídeo: 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.

Induzida por vírus silenciamento gênico (VIGS) em Nicotiana benthamiana Tomate e

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

Biologia

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.

Imagens ao vivo de cálcio celular Combinado com siRNA silenciamento gênico mediado Identifica Ca 2 + Canais de vazamento na membrana ER e seus mecanismos de regulação

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.

Indução de hipóxia e Testes em Cultura de Células

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.

Isolamento de Ribossomos ligados a polipeptídeos nascentes<em&gt; In vitro</em&gt; Identificar Sites Pausa translacionais Junto mRNA

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.

Usando Seqüência de Detenção SECM como uma ferramenta para isolar Ribossomos Polipeptídeos encadernados

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.

Otimização e Utilização de<em&gt; Agrobacterium</em&gt; Mediada produção de proteína transiente em<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.

A quitosana / RNA de interferência Nanoparticle Mediated silenciamento gênico na Doença Vector Mosquito Larvas

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.

Meiotic Avaliação de fuso em oócitos mouse por silenciamento mediado por siRNA

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.

Silenciando a faísca: Edição do genoma CRISPR/Cas9 em peixes fraca elétricos

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-Transformação de embrião imaturo mediado de linhas de milho recalcitrante usando genes morfogênicos

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