Os autores Obrigado Greyson Butler e Margaret Barrett para ajudar na realização dos experimentos, Judy Stone para aconselhamento sobre homogeneização de grão e Lynn Hannum para aconselhamento sobre fluorometry. Este trabalho foi financiado pela National Science Foundation (IOB 0443676), por um prêmio de desenvolvimento institucional (ideia) do Instituto Nacional de General Medical Ciências, dos institutos nacionais de saúde, sob número de concessão P20GM0103423 e por concessões do a divisão de Colby College de ciências naturais.

1. preparação do efetor e Gene repórter constrói
<ol><li>Construa construções efetoras que empregam um promotor constitutivo (por exemplo, milho ubiquitin, UBI) a expressão de unidade de um frame de leitura aberto desejado. Por exemplo, construa um plasmídeo que contém UBI::TaABF1 a expressão constitutiva forte de unidade de TaABF15.</li>
	<li>Construa construções de repórter que incluem o promotor, cuja actividade está a ser medido, colocados a montante de um frame de leitura aberto, tais como GUS. Por exemplo, construa um plasmídeo que contém HVA1::GUS para medir a expressão do promotor HVA1 2,5.</li>
	<li>Construir uma construção de controle interno (por exemplo, UBI::luciferase).</li>
	<li>Usando uma coluna de ligação de sílica protocolo (consulte Tabela de materiais), preparar o plasmídeo de alta qualidade para cada uma das construções de gene necessário. Usando espectroscopia ultravioleta (veja a Tabela de materiais), cuidadosamente medir a concentração de cada preparação do plasmídeo.</li>
</ol>2. preparação de grãos de cevada para bombardeio
<ol><li>Configurar uma planilha (ver Figura 1 para obter um exemplo) indicando o plasmídeo que será bombardeado, os tratamentos hormonais adequados e outras informações relevantes para cada tratamento que farão parte do experimento.</li>
	<li>Usando uma placa de corte e uma lâmina de barbear estéril, cortar os embriões a partir de grãos de cevada Himalaya (40 para cada tratamento incluído no experimento). Exclua os grãos que são descoloridos ou substancialmente menor do que a média.</li>
	<li>Coloque todos os grãos de embryoless em um tubo de 50 mL. Adicione 40 mL de água e pedra por 10 minutos.</li>
	<li>Despeje a água com cuidado (para evitar a perda de grãos) e adicionar 40 mL de água sanitária 10%. Rocha por 20 minutos.</li>
	<li>Deite fora a água sanitária e adicionar 40 mL de água estéril. Pedra por 5 minutos. Repeti esta lavagem de água estéril quatro vezes mais.</li>
	<li>Adicionar 40 mL de solução de absorver (succinato de sódio de 20 mM, 20 mM de cloreto de cálcio, pH 5,0) e 40 μL de solução cloranfenicol (10 mg/mL).</li>
	<li>Transferência da solução (com cloranfenicol) absorver a maioria do tubo em uma placa de Vermiculite (ver Tabela de materiais). Depois transferi os grãos para a superfície do papel filtro molhado na placa de vermiculita. Espalhe os grãos uniformemente. Despeje em suficiente imbibing solução para manter os grãos parcialmente (mas não completamente) submerso. Coloque a tampa na parte superior da placa de vermiculita.</li>
	<li>Incube os grãos de embryoless a 24 ° C, durante cerca de 48 h com iluminação fluorescente. Adicione solução absorvendo adicionais conforme necessário.</li>
	<li>Abrir-se um prato de petri de plástico 90mm e despeje 20 mL da solução de absorver para o prato em si e 20 mL a tampa invertida. Adicione 20 μL de cloranfenicol (10 mg/mL) a cada um. Esterilize dois pares de pinças de ponta fina, mergulhando-os em álcool.</li>
	<li>Coloque alguns grãos de embryoless na tampa da caixa de Petri. Usando a pinça, remova suavemente a camada de sementes (transparente) de cada grão. Ao remover o revestimento de semente do lado liso do grão, não danificam células subjacentes (aleurona). Transfira o grão descascado para a placa de Petri contendo solução absorvendo.</li>
	<li>Depois de descascar o revestimento de semente de todos os grãos, devolvê-los à placa de vermiculita. Incube os grãos descascados embryoless a 24 ° C por 16-20 horas.</li>
	<li>Imediatamente antes de usar os grãos para bombardeio, remova a umidade da superfície sacudindo-os entre papéis de filtro em um prato de petri.</li>
</ol>3. preparação do ADN do plasmídeo para bombardeio
<ol><li>Rotule um tubo de 1,5 mL para cada tratamento que será incluído no experimento de bombardeio. A cada tubo, adicionar 2,5 µ g de UBI:: plasmídeo de controle interno deluciferase , 2,5 µ g de um plasmídeo do repórter GUS e a quantidade desejada (normalmente 0,025 µ g de 2,5 µ g) de um plasmídeo effector. Adicione água para trazer o volume total de 5 µ l.</li>
	<li>Prepare-se 1,6 µm ouro microcarriers (ver Tabela de materiais) seguinte publicado protocolos6.</li>
	<li>Para cada tratamento, adicione 50 µ l de suspensão microcarriers no tubo de 1,5 mL contendo Plasmídeo. Revesti o microcarriers com o plasmídeo de acordo com o protocolos6 do fabricante.</li>
	<li>Na etapa final, quando os plasmídeo-revestido microcarriers são suspensos em 48 µ l 100% de etanol, pipetar 8 μL da suspensão microcarriers para cada um dos cinco macrocarriers e deixe-os ao ar seco.</li>
</ol>4. partícula bombardeio de grãos de cevada Embryoless
<ol><li>Configurar o aparelho de bombardeio de partículas (ver Tabela de materiais)6.</li>
	<li>Coloque um disco de ruptura de 1550 psi na retenção PAC e montá-lo no instrumento.</li>
	<li>Coloque um macrocarrier que contém a combinação desejada de plasmídeo (juntamente com uma tela de parar) o assembly de lançamento de microcarrier. Introduza o conjunto na ranhura superior da câmara de bombardeio. Definir a distância entre o disco de ruptura, mantendo a tampa e a tampa de cobertura macrocarrier à distância padrão (6 mm) e coloque o suporte de tela de parar na posição do meio (padrão), permitindo uma distância de viagem macrocarrier de 11 mm (Figura 2A).</li>
	<li>Arrume 8 grãos embryoless em um padrão radial apertado (com as extremidades mais finas, apontando para dentro) em um círculo de papel de filtro que está pregado no chão para a tampa de um prato de petri de 90 mm (ver Figura 2B).</li>
	<li>Coloque o prato com os grãos dentro da câmara de bombardeio, a uma distância de 6 cm a partir da tela de parar.</li>
	<li>Evacuar a câmara de bombardeio para 95 kPa e então bombardeia a amostra.</li>
	<li>Depois de lançar o vácuo, transferi os grãos bombardeados para um prato de petri 60mm etiquetados contendo 4 mL de solução absorvendo contendo 1 mg/ml de cloranfenicol.</li>
	<li>Repita até que todos os bombardeamentos foram concluídos. Incube (em uma plataforma de agitação a 100 rpm) a 24 ° C por 24 horas.</li>
</ol>5. extração de proteína solúvel de grãos bombardeados
<ol><li>Usando a pinça, remova os 8 grãos bombardeou um prato de petri 60mm e exterminá-los seco sobre papel absorvente. Divida os grãos em dois tubos etiquetados 2,0 mL (4 grãos por tubo) cada um contendo 800 μL tampão de moagem (100mm at fosfato pH 7,2, 5 mM DTT, 10 µ g/mL leupeptin, 20% de glicerol) e um grânulo de aço inoxidável de 5mm. Repita este processo para todos os de grãos bombardeados.</li>
	<li>Coloque os dois racks de homogeneizador do grânulo (até 48) tubos de 2,0 mL (ver Tabela de materiais). Monte as prateleiras do homogenizador.</li>
	<li>Agite as amostras em 30 Hz durante 3 minutos.</li>
	<li>Retire os cestos homogenizador do homogenizador e colocá-los no gelo (5 min) para re-fixe as amostras.</li>
	<li>Monte os racks volta do homogenizador - na orientação oposta. Agite as amostras novamente a 30 Hz durante 3 minutos.</li>
	<li>Legal os racks no gelo, em seguida, repita passos 5.3 a 5.5. Isto irá adicionar um total de 12 minutos de agitação.</li>
	<li>Centrifugue os extratos de grão a 16.000 x g a 4 ° C por 10 minutos.</li>
	<li>
Imediatamente após a centrifugação, decantar os sobrenadantes claros em um segundo conjunto de tubos do microcentrifuge. Dar a cada tubo um vórtice rápido. Armazene os tubos em gelo.</li>
	<li>Depois de transferir o sobrenadante, recupere as esferas de aço de pelotas para que eles podem ser lavados e reutilizados.</li>
</ol>6. medida da atividade de Luciferase de extratos de grão
<ol><li>Para cada tubo de extracto de grãos a ser analisada, prepare um tubo de ensaio de vidro 12 x 75 mm contendo 200 μL de mistura de ensaio Luciferase (45 mM Tris sulfato de pH 7,7, 15 mM MgCl2, 20 mM DTT, 1,5 mM EDTA, Luciferina de 1 mM, 1 mM ATP).</li>
	<li>Adicione 100 μL do primeiro extracto de grãos para o primeiro tubo de ensaio. Vórtice rapidamente para misturar bem. Imediatamente , coloque o tubo no suporte do tubo do luminómetro e feche a porta. Quando a medição estiver concluída, retire o tubo do luminómetro – deixando a porta aberta para a colocação do tubo próxima.</li>
	<li>Repita este processo até que todas as amostras foram medidas.</li>
</ol>7. medição de GUS atividade de extratos de grão
<ol><li>Adicione 200 μL de tampão de ensaio de GUS (50mm at fosfato pH 7,2, 2,5 mM 4-methylumbelliferyl - D-glicuronídeo, 10 mM DTT, 2 mM EDTA, 10 µ g/mL leupeptin, 20% de metanol, 0,02% de azida de sódio) para cada poço de uma placa bem 96.</li>
	<li>Colocar 50 μL de cada extrato de grão para a correspondente bem. Misture com a ponta após a adição. Sele o topo com película de vedação.</li>
	<li>Incube a placa bem 96 a 37 ° C, no escuro por 20 horas.</li>
	<li>Centrifugue a placa bem 96 a 4.000 x g a 4 ° C durante 10 minutos e coloque-o no gelo.</li>
	<li>Adicione 250 μL de 0,2 M Na2CO3 para cada poço de uma placa bem preto 96.</li>
	<li>Adicionar 6,25 μL de cada mistura de ensaio centrifugada GUS para a correspondente bem (contendo 0,2 M Na2CO3) do negro 96 placa bem. Misture com a ponta após a adição. Inclua poços com 6,25 μL de 10 μM 40 μM e 100 μM 4-methylumbelliferone como padrões.</li>
	<li>Coloque a placa preta em um dados de placa e ler a fluorescência de methylumbelliferone. Fazer as leituras em um comprimento de onda de excitação de 360 nm e um comprimento de onda de emissão de 460 nm. Defina o ganho do instrumento tal que um bem contendo 6,25 μL de 40 μM 4-methylumbelliferone e 250 μL de 0,2 M Na2CO3 dão uma leitura de 1000 unidades de fluorescência.</li>
</ol>8. análise de dados
<ol><li>Insira os valores para a luciferase e ensaios de GUS em uma planilha. Exclua da consideração qualquer amostra com uma leitura do luciferase inferior a luminescência relativa de 20.000 unidades de s-1, já que indica que as construções do gene não foram entregue com êxito para as células de aleurona.</li>
	<li>Para cada grão bombardeado com sucesso o extrato (representando um grupo de quatro grãos embryoless), calcular a expressão de GUS normalizada a partir dos dados brutos de GUS e luciferase como segue: normalizado GUS = [(expressão de GUS - GUS em branco) x (2.000.000)] / (luciferase expressão – luciferase em branco). Nota: Isto normaliza a quantidade de atividade de GUS, o que poderia ter sido observado em um bombardeio que deu uma leitura de luciferase de 2.000.000.</li>
	<li>Relatar o nível relativo de expressão de uma construção de repórter especial na presença de construções de efetor ou tratamentos hormonais, sendo testados, comparando o nível de expressão na ausência de efetores ou hormônios.</li>
</ol>

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Os autores não têm nada para divulgar.

A introdução do gene effector constrói através do bombardeamento de partículas, seguido pela expressão transiente de construções de repórter é uma estratégia valiosa para dissecar o papel dos actores particulares na GA/ABA sinalização e no gene resultante hormônio-regulada expressão. No entanto, os protocolos existentes para a realização de tais experiências em cevada aleurona células2,3,4,<sup c...

A camada de aleurona de cevada é um sistema bem estabelecido de modelo para o estudo da expressão gênica de hormônio-regulada em plantas1. Em particular, um número de genes necessários para a germinação ou desenvolvimento inicial de plântulas é ativado por Giberelina (GA), enquanto os genes associados com respostas de estresse são ativados pelo ácido abscísico (ABA). O GA e ABA vias de sinalização estão interligados, como a expressão de alguns genes GA-ativado é inibida pela ABA e vice-versa1.
Uma estratégia valiosa para a compreensão que do papel dos actores particulares na GA/ABA sinalização foi a introdução das construções de gene effector através do bombardeamento de partículas, seguido pela expressão transiente de construções de repórter que permitem que o efeito resultante na expressão do gene a jusante para ser determinado. O uso de genes repórter como GUS (β-glucuronidase) ou Luciferase permite a medição quantitativa e sensível da expressão do gene especificamente dentro das células que receberam a construção effector. Por exemplo, a introdução de uma construção effector codificação o fator de transcrição TaABF15,6 estabeleceu que genes induzida por ABA como HVA1 são induzidos por TaABF1, enquanto genes GA-induzido, como Amy32b são reprimidos. Bombardeio de partículas como uma estratégia experimental serviu por vários laboratórios para investigar os diversos aspectos de sinalização GA/ABA. Esse trabalho levou à identificação de elementos do promotor importantes para a ativação de GA-induzido2 e genes induzida por ABA3e a descoberta de proteínas quinases4 e fatores de transcrição5 que regulam o expressão destes genes.
O existente protocolos2,3,4,5,6 para o bombardeamento de partículas e mensuração subsequente da expressão do gene transiente são bastante trabalhoso, como cada conjunto de bombardeados mal de grãos é homogeneizado à mão em um almofariz e um pilão e os ensaios enzimáticos são realizados individualmente. Este manuscrito relata um protocolo melhorado que parcialmente automatiza e agiliza a etapa de homogeneização e o GUS ensaios para permitir que a taxa de transferência significativamente mais, permitindo um maior número de tratamentos a ser testado no mesmo experimento, e/ou a inclusão de mais repetições para cada tratamento obter mais resultados estatisticamente robustos. Resultados representativos são mostrados para a expressão de construções HVA1 e Amy32b do repórter, regulados pelo factor de transcrição TaABF1, bem como pelo GA, ABA e outras moléculas reguladoras.

<table><tbody><tr><td>GeneElute HP plasmid Maxiprep kit</td><td>Sigma</td><td>NA0310-1KT</td><td></td></tr><tr><td>UV-vis spectrophotometer</td><td>Nanodrop</td><td>ND-1000</td><td></td></tr><tr><td>Himalaya barley grains</td><td>/</td><td>/</td><td>A variety of hulless barley (store in the dark at 4&amp;deg; C)</td></tr><tr><td>sodium succinate</td><td>Sigma</td><td>S2378</td><td>Reagent for Imbibing Solution</td></tr><tr><td>calcium chloride (dihydrate)</td><td>Fisher</td><td>C79-500</td><td>Reagent for Imbibing Solution</td></tr><tr><td>Imbibing Solution</td><td>home made</td><td>/</td><td>20 mM sodium succinate, 20 mM calcium chloride, pH 5.0. Sterilize by autoclaving before use.</td></tr><tr><td>chloramphenicol</td><td>Sigma</td><td>C0378</td><td>Prepare a 10 mg/mL stock solution in 70% ethanol.</td></tr><tr><td>vermiculite</td><td>Fisher</td><td>NC0430369</td><td>Used for vermiculite plates.</td></tr><tr><td>filter paper circles (90 mm)</td><td>Whatman</td><td>1001 090</td><td>Used for vermiculite and for pre-bombardment grain preparation</td></tr><tr><td>Vermiculite Plates</td><td>home made</td><td>/</td><td>Add 50 mL of vermiculite to a glass petri dish. Place a 90 mm paper circle on top of the vermiculite. Autoclave.</td></tr><tr><td>forceps (fine pointed)</td><td>Fisher</td><td>13-812-42</td><td>Used for removing seed coat from barley grains.</td></tr><tr><td>forceps (ultra fine point)</td><td>Fisher</td><td>12-000-122</td><td>Used for removing seed coat from barley grains.</td></tr><tr><td>gold microcarriers (1.6 &amp;mu;m)</td><td>BioRad</td><td>1652264</td><td></td></tr><tr><td>macrocarriers</td><td>BioRad</td><td>1652335</td><td></td></tr><tr><td>calcium chloride (dihydrate)</td><td>Fisher</td><td>C79-500</td><td>Prepare a 2.5 M stock solution and store 1 mL aliquots at -20&amp;deg; C.</td></tr><tr><td>spermidine</td><td>Sigma</td><td>S0266</td><td>Prepare a 100 mM stock solution and store 500 &amp;mu;L aliquots at -20&amp;deg; C (use within 2 months).</td></tr><tr><td>rupture discs (1550 psi)</td><td>BioRad</td><td>1652331</td><td></td></tr><tr><td>stopping screens</td><td>BioRad</td><td>1652336</td><td></td></tr><tr><td>macrocarrier holders</td><td>BioRad</td><td>1652322</td><td></td></tr><tr><td>Biolistic particle delivery system</td><td>BioRad</td><td>PDS-1000/He</td><td></td></tr><tr><td>sodium phosphate monobasic monohydrate</td><td>Sigma</td><td>S9638</td><td>Reagent for 1M sodium phosphate pH 7.2</td></tr><tr><td>sodium phosphate dibasic</td><td>Sigma</td><td>S9763</td><td>Reagent for 1M sodium phosphate pH 7.2</td></tr><tr><td>1M sodium phosphate pH 7.2</td><td>home made</td><td>/</td><td>Combine 6.9 g of sodium phosphate monobasic monohydrate with 7.1 g of sodium phosphate dibasic. Add water to 100 mL. Add NaOH to get pH 7.2.</td></tr><tr><td>dithiothrietol (DTT)</td><td>Sigma</td><td>43819</td><td>Dissolve in water to 1 M. Store at -20&amp;deg; in 1 mL aliquots.</td></tr><tr><td>leupeptin</td><td>Sigma</td><td>L2884</td><td>Dissolve in water to 10 mg/mL. Store at -20&amp;deg; C.</td></tr><tr><td>glycerol</td><td>Sigma</td><td>G5516</td><td>Prepare a 50% solution in water.</td></tr><tr><td>Grinding Buffer</td><td>home made</td><td>/</td><td>Combine 10 mL of 1 M sodium phosphate pH 7.2, 500 &amp;mu;L of 1 M DTT, 100 &amp;mu;L of 10 mg/mL leupeptin, and 40 mL of 50% glycerol. Add water to 100 mL.</td></tr><tr><td>stainlesss steel beads (5 mm)</td><td>Qiagen</td><td>69989</td><td></td></tr><tr><td>2.0 mL tubes</td><td>Eppendorf</td><td>22363352</td><td>This specific model of tube is recommended for use with the homogenizer.</td></tr><tr><td>bead homogenizer (TissueLyser)</td><td>Qiagen</td><td>85210</td><td></td></tr><tr><td>12mm x 75 mm glass test tubes</td><td>Fisher</td><td></td><td></td></tr><tr><td>luciferin</td><td>Goldbio</td><td>LUCK-100</td><td>Prepare a 25 mM stock solution and store 1 mL aliquots at -20&amp;deg; C.</td></tr><tr><td>ATP</td><td>Sigma</td><td>A7699</td><td>Prepare a 100 mM stock solution and store 250 &amp;mu;L aliquots at -20&amp;deg;&amp;nbsp;C.</td></tr><tr><td>Tris base</td><td>Sigma</td><td>T1503</td><td>Reagent for 1M Tris sulfate pH 7.7.</td></tr><tr><td>sulfuric acid</td><td>Sigma</td><td>258105</td><td>Reagent for 1M Tris sulfate pH 7.7.</td></tr><tr><td>1M Tris sulfate pH 7.7</td><td>home made</td><td>/</td><td>Dissolve 12.1 g Tris base in 100 mL of water. Adjust pH to 7.7 with sulfuric acid.</td></tr><tr><td>magnesium chloride</td><td>Sigma</td><td>M9397</td><td>Dissolve in water to 2 M.</td></tr><tr><td>Luciferase Assay Buffer (LAB)</td><td>home made</td><td>/</td><td>Combine 3 mL of 1 M Tris sulfate pH 7.7, 500 &amp;mu;L of 2 M magnesium chloride, 1 mL of 1 M DTT, and 200 &amp;mu;L of 0.5 M EDTA. Add water to 50&amp;nbsp;mL.</td></tr><tr><td>Luciferase Assay Mixture</td><td>home made</td><td>/</td><td>Combine 15 mL of LAB, 800 &amp;mu;L of 25 mM luciferin, 200 &amp;mu;L of 100 mM ATP, and 4 mL of water. This makes enough assay mixture (20&amp;nbsp;mL) for 100 luciferase assays.</td></tr><tr><td>luminometer (Sirius)</td><td>Berthold</td><td>/</td><td></td></tr><tr><td>4-methylumbelliferyl-&amp;beta;-D-glucuronide (MUG)</td><td>Goldbio</td><td>MUG1</td><td>Dissolve in DMSO to 100 mM.</td></tr><tr><td>sodium azide</td><td>Sigma</td><td>S8032</td><td>Prepare a 2% stock solution in water and store 1 mL aliquots at -20&amp;deg; C.</td></tr><tr><td>96 well plates (standard)</td><td>Fisher</td><td>12565501</td><td></td></tr><tr><td>GUS assay buffer</td><td>home made</td><td>/</td><td>Combine 2.5 mL of MUG, 5 mL of 1 M sodium phosphate pH 7.2, 400 &amp;mu;L of 0.5 M EDTA, 1 mL of 1 M DTT, 100 &amp;mu;L of 10 mg/ml leupeptin, 20 mL of methanol, and 1 mL of 2% sodium azide. Add water to 100 mL.</td></tr><tr><td>TempPlate sealing film</td><td>USA Scientific</td><td>2921-1000</td><td></td></tr><tr><td>96 well plates (black)</td><td>Costar</td><td>3916</td><td></td></tr><tr><td>sodium carbonate</td><td>Sigma</td><td>S7795</td><td>Prepare a 200 mM solution in water.</td></tr><tr><td>4-methylumbelliferone</td><td>Sigma</td><td>M1381</td><td>Prepare a 100 &amp;mu;M solution in water. Freeze 1 mL aliquots at -20&amp;deg;&amp;nbsp;C.</td></tr><tr><td>microplate fluouresence reader</td><td>Bio-Tek</td><td>FLX-800</td><td></td></tr></tbody></table>

measuring gene expression in bombarded barley aleurone layers with increased throughput

A camada de aleurona de grãos de cevada é um importante modelo sistema para expressão gênica hormônio-regulada em plantas. Nas células de aleurona, genes necessários para a germinação ou desenvolvimento inicial de plântulas são ativados por Giberelina (GA), enquanto os genes associados com respostas de estresse são ativados pelo ácido abscísico (ABA). Os mecanismos de sinalização GA e ABA podem ser interrogados por introduzir construções de gene repórter em células de aleurona através do bombardeamento de partículas, com a expressão transitória resultante medida através de ensaios enzimáticos. Um protocolo melhorado é relatado que parcialmente automatiza e agiliza a etapa de homogeneização de grão e os ensaios enzimáticos, permitindo que a taxa de transferência significativamente mais do que os métodos existentes. Homogeneização das amostras de grãos é realizada usando um homogeneizador de automatizado, e ensaios de GUS (β-glucuronidase) são realizados através de um sistema de placa de 96 poços. Resultados representativos, usando o protocolo sugerem que a atividade da fosfolipase D pode desempenhar um papel importante na ativação da expressão do gene HVA1 pela ABA, através do fator de transcrição TaABF1.

A técnica descrita aqui pode ser usada para introduzir qualquer teste de construção de gene em células de aleurona de grãos de cevada. O nível de expressão do gene de teste pode então ser convenientemente medidos (Figura 2). O protocolo de taxa de transferência superior descrito aqui grandemente aumenta a eficiência da moagem de sementes e etapas de ensaio enzima. Este método tem sido usado para avaliar a capacidade da transcrição fator TaABF1<su...

Watch this Scientific Journal Video about Measuring Gene Expression in Bombarded Barley Aleurone Layers with Increased Throughput at JoVE.com

Measuring Gene Expression in Bombarded Barley Aleurone Layers with Increased Throughput

Um protocolo melhorado é apresentado para a medição transitória da expressão do gene do repórter construções nas células de aleurona de cevada após o bombardeio de partículas. A combinação de grãos automatizado moagem com ensaios enzimáticos de placa de 96 poços fornece alto throughput para o procedimento.

Expressão gênica em camadas de aleurona cevada bombardeado com maior Throughput de medição

The aleurone layer of barley grains is an important model system for hormone-regulated gene expression in plants. In aleurone cells, genes required for ...

measuring-gene-expression-bombarded-barley-aleurone-layers-with

Universidad de Cartagena UDEC

Research

JoVE Journal

Genetics

6.4K visualizações.  Colby College. Um protocolo melhorado é apresentado para a medição transitória da expressão do gene do repórter construções nas células de aleurona de cevada após o bombardeio de partículas. A combinação de grãos automatizado moagem com ensaios enzimáticos de placa de 96 poços fornece alto throughput para o procedimento.

Vídeo: Measuring Gene Expression in Bombarded Barley Aleurone Layers with Increased Throughput

Measuring mRNA Levels Over Time During the Yeast S. cerevisiae Hypoxic Response

Complex changes in gene expression typically mediate a large portion of a cellular response. Each gene may change expression with unique kinetics as the gene is regulated by the particular timing of one of many stimuli, signaling pathways or secondary effects. In order to capture the entire gene expression response to hypoxia in the yeast S. cerevisiae, RNA-seq analysis was used to monitor the mRNA levels of all genes at specific times after exposure to hypoxia. Hypoxia was established by growing cells in ~100% N2 gas. Importantly, unlike other hypoxic studies, ergosterol and unsaturated fatty acids were not added to the media because these metabolites affect gene expression. Time points were chosen in the range of 0 - 4 h after hypoxia because that period captures the major changes in gene expression. At each time point, mid-log hypoxic cells were quickly filtered and frozen, limiting exposure to O2 and concomitant changes in gene expression. Total RNA was extracted from cells and used to enrich for mRNA, which was then converted to cDNA. From this cDNA, multiplex libraries were created and eight or more samples were sequenced in one lane of a next-generation sequencer. A post-sequencing pipeline is described, which includes quality base trimming, read mapping and determining the number of reads per gene. DESeq2 within the R statistical environment was used to identify genes that change significantly at any one of the hypoxic time points. Analysis of three biological replicates revealed high reproducibility, genes of differing kinetics and a large number of expected O2-regulated genes. These methods can be used to study how the cells of various organisms respond to hypoxia over time and adapted to study gene expression during other cellular responses.

Measuring mRNA Levels Over Time During the Yeast S. cerevisiae Hypoxic Response

Here, we present a protocol using RNA-seq to monitor mRNA levels over time during the hypoxic response of S. cerevisiae cells. This method can be adapted to analyze gene expression during any cellular response.

Medição de níveis de mRNA ao longo do tempo durante a levedura s. cerevisiae hipóxica resposta

Complex changes in gene expression typically mediate a large portion of a cellular response. Each gene may change expression with unique kinetics as the ...

Genética

Saccharomyces cerevisiae Metabolic Labeling with 4-thiouracil and the Quantification of Newly Synthesized mRNA As a Proxy for RNA Polymerase II Activity

Global defects in RNA polymerase II transcription might be overlooked by transcriptomic studies analyzing steady-state RNA. Indeed, the global decrease in mRNA synthesis has been shown to be compensated by a simultaneous decrease in mRNA degradation to restore normal steady-state levels. Hence, the genome-wide quantification of mRNA synthesis, independently from mRNA decay, is the best direct reflection of RNA polymerase II transcriptional activity. Here, we discuss a method using non-perturbing metabolic labeling of nascent RNAs in Saccharomyces cerevisiae (S. cerevisiae). Specifically, the cells are cultured for 6 min with a uracil analog, 4-thiouracil, and the labeled newly transcribed RNAs are purified and quantified to determine the synthesis rates of all individual mRNA. Moreover, using labeled Schizosaccharomyces pombe cells as internal standard allows comparing mRNA synthesis in different S. cerevisiae strains. Using this protocol and fitting the data with a dynamic kinetic model, the corresponding mRNA decay rates can be determined.

Saccharomyces cerevisiae Metabolic Labeling with 4-thiouracil and the Quantification of Newly Synthesized mRNA As a Proxy for RNA Polymerase II Activity

The protocol described here is based on the genome-wide quantification of newly synthesized mRNA purified from yeast cells labeled with 4-thiouracil. This method allows to measure mRNA synthesis uncoupled from mRNA decay and, thus, provides an accurate measurement of RNA polymerase II transcription.

Saccharomyces cerevisiae Metabólica de rotulagem com 4-thiouracil e a quantificação dos recém sintetizado mRNA como um Proxy para a RNA polimerase II atividade

Global defects in RNA polymerase II transcription might be overlooked by transcriptomic studies analyzing steady-state RNA. Indeed, the global decrease in ...

Isolation of High Purity Tissues from Developing Barley Seeds

Understanding the mechanisms regulating the development of cereal seeds is essential for plant breeding and increasing yield. However, the analysis of cereal seeds is challenging owing to the minute size, the liquid character of some tissues, and the tight inter-tissue connections. Here, we demonstrate a detailed protocol for dissection of the embryo, endosperm, and seed maternal tissues at early, middle, and late stages of barley seed development. The protocol is based on a manual tissue dissection using fine-pointed tools and a binocular microscope, followed by ploidy analysis-based purity control. Seed maternal tissues and embryos are diploid, while the endosperm is triploid tissue. This allows the monitoring of sample purity using flow cytometry. Additional measurements revealed the high quality of RNA isolated from such samples and their usability for high-sensitivity analysis. In conclusion, this protocol describes how to practically dissect pure tissues from developing grains of cultivated barley and potentially also other cereals.

Here we present a protocol for high purity manual isolation and quality control of embryo, endosperm and seed maternal tissues during entire barley seed development.

Understanding the mechanisms regulating the development of cereal seeds is essential for plant breeding and increasing yield. However, the analysis of ...

Obtaining High-Quality Transcriptome Data from Cereal Seeds by a Modified Method for Gene Expression Profiling

The characterization of gene expression is dependent on RNA quality. In germinating, developing and mature cereal seeds, the extraction of high-quality RNA is often hindered by high starch and sugar content. These compounds can reduce both the yield and the quality of the extracted total RNA. The deterioration in quantity and quality of total RNA can subsequently have a significant impact on the downstream transcriptomic analyses, which may not accurately reflect the spatial and/or temporal variation in the gene expression profile of the samples being tested. In this protocol, we describe an optimized method for extraction of total RNA with sufficient quantity and quality to be used for whole transcriptome analysis of cereal grains. The described method is suitable for several downstream applications used for transcriptomic profiling of developing, germinating, and mature cereal seeds. The method of transcriptome profiling using a microarray platform is shown. This method is specifically designed for gene expression profiling of cereals with described genome sequences. The detailed procedure from microarray handling to final quality control is described. This includes cDNA synthesis, cRNA labelling, microarray hybridization, slide scanning, feature extraction, and data quality validation. The data generated by this method can be used to characterize the transcriptome of cereals during germination, in various stages of grain development, or at different biotic or abiotic stress conditions. The results presented here exemplify high-quality transcriptome data amenable for downstream bioinformatics analyses, such as the determination of differentially expressed genes (DEGs), characterisation of gene regulatory networks, and conducting transcriptome-wide association study (TWAS).

A method for transcriptome profiling of cereals is presented. The microarray-based gene expression profiling starts with the isolation of high-quality total RNA from cereal grains and continues with the generation of cDNA. After cRNA labelling and microarray hybridization, recommendations are given for signal detection and quality control.

Obtenção de dados de transcriptome de alta qualidade de sementes de cereal por um método modificado para criação de perfil de expressão genética

The characterization of gene expression is dependent on RNA quality. In germinating, developing and mature cereal seeds, the extraction of high-quality ...

Bioquímica

Global Gene Expression Analysis Using a Zebrafish Oligonucleotide Microarray Platform

Gene microarray technology permits quantitative measurement and gene expression profiling of transcript levels on a genome-wide basis. Gene microarray technology is used in numerous biological disciplines in a variety of applications including global gene expression analysis in relation to developmental stage, to a disease state, and in toxic responses. Herein, we include a demonstration of global gene expression analysis using a comprehensive zebrafish-specific oligonucleotide microarray platform. The zebrafish expression microarray platform contains 385,000 probes, 60 base pairs in length, interrogating 37,157 targets with up to 12 probes per target. For this platform, all cDNA and genomic information available for the zebrafish was collected from various genomic databases including Ensembl (http://www.ensembl.org), VEGA (http://vega.sanger.ac.uk), UCSC (http://genome.ucsc.edu), and ZFIN (http://www.zfin.org). As a result this expression array provides complete coverage of the current zebrafish transcriptome. The zebrafish expression microarray was printed by Roche NimbleGen (Madison, WI). This technical demonstration includes the fluorescent labeling of a cDNA product, hybridization of the labeled cDNA product to the microarray platform, and array scanning for signal acquisition using the one color analysis strategy.  

Gene microarrays are powerful tools in gene expression profiling at a genome-wide level. This technology has application in a variety of biological disciplines including developmental biology and toxicology. In this video, we detail a protocol for global gene expression analysis using a comprehensive oligonucleotide microarray platform for the zebrafish.

Análise da expressão global de Gene usando uma plataforma Microarray Zebrafish Oligonucleotide

Gene microarray technology permits quantitative measurement and gene expression profiling of transcript levels on a genome-wide basis. Gene microarray ...

Biologia

Robotics and Dynamic Image Analysis for Studies of Gene Expression in Plant Tissues

Gene expression in plant tissues is typically studied by destructive extraction of compounds from plant tissues for in vitro analyses. The methods presented here utilize the green fluorescent protein (gfp) gene for continual monitoring of gene expression in the same pieces of tissues, over time. The gfp gene was placed under regulatory control of different promoters and introduced into lima bean cotyledonary tissues via particle bombardment. Cotyledons were then placed on a robotic image collection system, which consisted of a fluorescence dissecting microscope with a digital camera and a 2-dimensional robotics platform custom-designed to allow secure attachment of culture dishes. Images were collected from cotyledonary tissues every hour for 100 hours to generate expression profiles for each promoter. Each collected series of 100 images was first subjected to manual image alignment using ImageReady to make certain that GFP-expressing foci were consistently retained within selected fields of analysis. Specific regions of the series measuring 300 x 400 pixels, were then selected for further analysis to provide GFP Intensity measurements using ImageJ software. Batch images were separated into the red, green and blue channels and GFP-expressing areas were identified using the threshold feature of ImageJ. After subtracting the background fluorescence (subtraction of gray values of non-expressing pixels from every pixel) in the respective red and green channels, GFP intensity was calculated by multiplying the mean grayscale value per pixel by the total number of GFP-expressing pixels in each channel, and then adding those values for both the red and green channels. GFP Intensity values were collected for all 100 time points to yield expression profiles. Variations in GFP expression profiles resulted from differences in factors such as promoter strength, presence of a silencing suppressor, or nature of the promoter. In addition to quantification of GFP intensity, the image series were also used to generate time-lapse animations using ImageReady. Time-lapse animations revealed that the clear majority of cells displayed a relatively rapid increase in GFP expression, followed by a slow decline. Some cells occasionally displayed a sudden loss of fluorescence, which may be associated with rapid cell death. Apparent transport of GFP across the membrane and cell wall to adjacent cells was also observed. Time lapse animations provided additional information that could not otherwise be obtained using GFP Intensity profiles or single time point image collections.

We report a method for introduction, tracking and quantitative analysis of GFP expression in plant cells. This method utilizes a custom-designed robotics system for semi-continuous image collection from large numbers of samples, over time. We also demonstrate the use of ImageJ and ImageReady for analysis of image series.

Robótica e Análise de Imagem Dinâmica de Estudos de Expressão Gênica em Tecidos Vegetais

Gene expression in plant tissues is typically studied by destructive extraction of compounds from plant tissues for in vitro analyses. The methods ...

A Cell-to-cell Macromolecular Transport Assay in Planta Utilizing Biolistic Bombardment

Here, we present a simple and rapid protocol to detect and assess the extent of cell-to-cell macromolecular transport in planta. In this protocol, a fluorescently tagged-protein of interest is transiently expressed in plant tissue following biolistic delivery of its encoding DNA construct. The intra- and intercellular distribution of the tagged protein is then analyzed by confocal microscopy. We describe this technology in detail, providing step-by-step protocols to assay and evaluate the extent of symplastic protein transport in three plant species, Arabidopsis thaliana, Nicotiana benthamiana and N. tabacum (tobacco).

A Cell-to-cell Macromolecular Transport Assay in Planta Utilizing Biolistic Bombardment

Macromolecular trafficking between plant cells can be assessed by transiently expressing a fluorescently-tagged protein of interest and analyzing its intra- and intercellular distribution by confocal microscopy.

A célula-célula Assay Transporte Macromoleculares Na Planta Utilizando Bombardeio biobalística

Here, we present a simple and rapid protocol to detect and assess the extent of cell-to-cell macromolecular transport in planta. In this protocol, a ...

Competitive Genomic Screens of Barcoded Yeast Libraries

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily. The tempo of these advances is accelerating, promising greater depth and breadth. In light of these extraordinary advances, the need for fast, parallel methods to define gene function becomes ever more important. Collections of genome-wide deletion mutants in yeasts and E. coli have served as workhorses for functional characterization of gene function, but this approach is not scalable, current gene-deletion approaches require each of the thousands of genes that comprise a genome to be deleted and verified. Only after this work is complete can we pursue high-throughput phenotyping. Over the past decade, our laboratory has refined a portfolio of competitive, miniaturized, high-throughput genome-wide assays that can be performed in parallel. This parallelization is possible because of the inclusion of DNA 'tags', or 'barcodes,' into each mutant, with the barcode serving as a proxy for the mutation and one can measure the barcode abundance to assess mutant fitness. In this study, we seek to fill the gap between DNA sequence and barcoded mutant collections. To accomplish this we introduce a combined transposon disruption-barcoding approach that opens up parallel barcode assays to newly sequenced, but poorly characterized microbes. To illustrate this approach we present a new Candida albicans barcoded disruption collection and describe how both microarray-based and next generation sequencing-based platforms can be used to collect 10,000 - 1,000,000 gene-gene and drug-gene interactions in a single experiment.

We have developed comprehensive, unbiased genome-wide screens to understand gene-drug and gene-environment interactions. Methods for screening these mutant collections are presented.

Competitiva Screens Genomic de Bibliotecas Yeast Barcoded

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily.  The tempo of these advances is ...

High Throughput Yeast Strain Phenotyping with Droplet-Based RNA Sequencing

The powerful tools available to edit yeast genomes have made this microbe a valuable platform for engineering. While it is now possible to construct libraries of millions of genetically distinct strains, screening for a desired phenotype remains a significant obstacle. With existing screening techniques, there is a tradeoff between information output and throughput, with high-throughput screening typically being performed on one product of interest. Therefore, we present an approach to accelerate strain screening by adapting single cell RNA sequencing to isogenic picoliter colonies of genetically engineered yeast strains. To address the unique challenges of performing RNA sequencing on yeast cells, we culture isogenic yeast colonies within hydrogels and spheroplast prior to performing RNA sequencing. The RNA sequencing data can be used to infer yeast phenotypes and sort out engineered pathways. The scalability of our method addresses a critical obstruction in microbial engineering.

A bottleneck in the &#8216;design-build-test&#8217; cycle of microbial engineering is the speed at which we can perform functional screens of strains. We describe a high-throughput method for strain screening applied to hundreds to thousands of yeast cells per experiment that utilizes droplet-based RNA sequencing.

Fenotipagem de tensão de levedura de alta produtividade com sequenciamento de RNA baseado em gotículas

The powerful tools available to edit yeast genomes have made this microbe a valuable platform for engineering. While it is now possible to construct ...

Bioengenharia

Temporal Quantification of MAPK Induced Expression in Single Yeast Cells

The quantification of gene expression at the single cell level uncovers novel regulatory mechanisms obscured in measurements performed at the population level. Two methods based on microscopy and flow cytometry are presented to demonstrate how such data can be acquired. The expression of a fluorescent reporter induced upon activation of the high osmolarity glycerol MAPK pathway in yeast is used as an example. The specific advantages of each method are highlighted. Flow cytometry measures a large number of cells (10,000) and provides a direct measure of the dynamics of protein expression independent of the slow maturation kinetics of the fluorescent protein. Imaging of living cells by microscopy is by contrast limited to the measurement of the matured form of the reporter in fewer cells. However, the data sets generated by this technique can be extremely rich thanks to the combinations of multiple reporters and to the spatial and temporal information obtained from individual cells. The combination of these two measurement methods can deliver new insights on the regulation of protein expression by signaling pathways.

Two complementary methods based on flow cytometry and microscopy are presented which enable the quantification, at the single cell level, of the dynamics of gene expression induced by the activation of a MAPK pathway in yeast.

A quantificação temporal da expressão MAPK induzida em células de levedura Solteiro

The quantification of gene expression at the  single cell level uncovers novel regulatory mechanisms obscured in measurements  performed at the population ...