Este trabalho foi apoiado por uma bolsa do programa de pesquisa Monsanto milho Rootworm conhecimento, conceder número AG/1005 (ao MDL e YC) e fundos de start-up de MDL da NCSU. FC foi apoiado por subsídios do programa de pesquisa de conhecimento para repele de milho da Monsanto (AG/1005) e o National Science Foundation, conceda número MCB-1244772 (MDL). Os autores declaram não interesses concorrentes. FC e MDL concebido e desenhado os experimentos; FC, PW e SP realizaram os experimentos; FC e MDL analisaram os resultados; e FC, NG e MDL escreveram o manuscrito. Agradecemos a Teresa O'Leary, William Klobasa e Stephanie Gorski por sua assistência especializada em triagem WCR. Agradecemos também o Dr. Wade French (USDA-ARS, Laboratório Central de pesquisa agrícola do Norte, Brookings, SD) para remessas de ovos para estabelecer colônias de laboratório e proporcionando a criação de protocolos.

1. nível de colônia de criação dos adultos WCR
<ol><li>Obter uma quantidade suficiente de adultos WCR (500-1.000) de um laboratório confiável de empresa ou de pesquisa (ver Tabela de materiais para obter um exemplo) e coloque em uma gaiola de3 30 cm. Nota: O uso de uma cepa não-diapausing é altamente recomendado.</li>
	<li>Preparar a dieta artificial WCR, seguindo o protocolo do fabricante e despeje uma camada de 1 cm de espessura em um recipiente de 38 oz (ver Tabela de materiais). Após a mistura, armazene dieta não utilizada a 4 ° C por até 2 meses.</li>
	<li>Colocar uma placa de Petri (100 x 15 mm) com dieta adulta (10-15 g) em uma gaiola de3 30 cm. Adicione mais dieta quando a comida é baixo ou seco.</li>
	<li>Colocar um balão (300 mL) com água, coberta com uma bola de algodão, que é usada para segurar no lugar de um rolo de algodão (6 "x 3/8"), para servir como uma fonte de água. Mude a água quando ele é executado baixo.</li>
	<li>Manter WCR a 26 ° C, com 60% de humidade e um 14:10 luz do ciclo em um inseto incubadora de criação.</li>
</ol>2. alinhamento e colheita de embriões
<ol><li>Fazer uma câmara de coleta de ovos usando ágar de 1% em água estéril 100 x 15 mm2 placa de Petri. Armazenar a 4 ° C, após o ágar solidifies.</li>
	<li>Para coletar ovos recém-estabelecidas, coloque uma camada de papel de filtro, seguido de 4 camadas de gaze (cada corte ao tamanho) na superfície do ágar (figura 1A). Nota: Gaze pode ser reutilizado, depois de ser lavado em água sanitária e esterilizada, mas não é necessário fazer isso para a primeira utilização. Papel de filtro não deve ser reutilizado e não precisa ser esterilizado.</li>
	<li>Coloque a placa de coleção de ovo na gaiola WCR mais tarde no dia possível (final do dia de trabalho) e cobrir com uma tenda de papel alumínio. Deixe durante a noite. Nota: Tendo as luzes de 10:00 a postura de ovos 12 A.M. (14 h) atrasos, facilitando assim a recolha dos ovos mais jovens para o microinjection sem a necessidade de pessoal de laboratório colocar a câmara de recolha na gaiola à noite.</li>
	<li>Remova a câmara de recolha de ovos em torno de 8 ou 09:00</li>
	<li>Use fórceps para pegar 1 camada de gaze em tempo e lugar num copo (500 mL) de água. Lave os ovos de gaze agitando suavemente (figura 1B e 1C).</li>
	<li>Use uma pipeta de bulbo para transferir os ovos para outro copo (500 mL) de água. Repita 2-3 x para limpar os ovos.</li>
	<li>Use a pipeta de bulbo para transferir ovos em papel de filtro (Figura 1), minimizando a quantidade de transferência de água.</li>
	<li>Corte papel filtro preto em tiras tão largas como uma lâmina de vidro e a fita firmemente para o slide para certificar-se o papel fixa plana (Figura 2A). Nota: Papel filtro preto ajuda a melhorar a visibilidade sob o microscópio, reduzindo a quantidade de luz refletida.</li>
	<li>Aplicar a cola não-tóxica (ver Tabela de materiais para obter um exemplo) para o papel de filtro em linhas finas (Figura 2B). Manter as linhas de cola mais fino do que o diâmetro de um ovo de modo a evitar os ovos revestidos em cola.</li>
	<li>Use um pincel fino para mover suavemente WCR os ovos um a um de seu papel de filtro para a linha de cola. Para colocar os ovos na cola antes de secar para fora e tentar manter distância de pelo menos um ovo entre cada ovo.</li>
	<li>Maximize a eficiência de injeção, colocando várias linhas de ovos no slide um papel de filtro (Figura 3).</li>
	<li>Depois que todos os ovos para fora o papel de filtro, espere até que toda a cola secar antes de microinjeção. Nota: Para transformação do germline e genoma CRISPR/Cas9-mediada edição, injete todos os embriões do meio-dia (12:00) para que o embrião mais velho é velho não mais de 19 h. No entanto, para RNAi, não há nenhuma limitação de tempo porque, em WCR, dsRNA pode mover entre as células. Na verdade, para RNAi, embriões de envelhecimento podem resultar em melhores taxas de sobrevivência.</li>
</ol>3. preparação de DNAs plasmideo e agulhas de injeção
<ol><li>Prepare-se alta qualidade supercoiled Plasmídeo usando um comercial livre de endotoxinas kit (veja a Tabela de materiais para obter um exemplo).</li>
	<li>Para preparar a solução de injeção, verifique a concentração de cada plasmídeo e então combinar auxiliar do plasmídeo (concentração final de 250 ng / µ l), doador do plasmídeo (concentração final de 750 ng / µ l) e buffer de vermelho de fenol (20% de concentração final). Vórtice brevemente a solução e centrifugar para 3 min na velocidade máxima para precipitar partículas que podem entupir a agulha.</li>
	<li>Puxe agulhas de vidro de borosilicato (O.D. 1 mm, identificação 0,58 mm, comprimento de 10 cm) usando um extrator de micropipeta tipo flamejante/marrom. Para armazenamento, secure agulhas puxadas na fita adesiva dupla-face em uma placa de Petri ou outro recipiente transparente. Nota: Para o sistema extrator de micropipeta, as configurações usadas foram: calor = 335, FIL = 4, VEL = 40, DEL = 200, PUL = 100.</li>
	<li>Aterramento a agulha de injeção, usando um microcarregador de ponta (ver Tabela de materiais) para pipetar 0,5 - 1,0 µ l para baixo dentro da agulha, perto da extremidade pontiaguda. Remova as bolhas da ponta da agulha se ocorrer qualquer um.</li>
	<li>Introduza o suporte da agulha, a agulha de injeção.</li>
	<li>Abra a ponta da agulha suavemente rompendo com um belo par de pinças, ou suavemente tocar/arrastando a ponta no slide lamela ou vidro. Nota: O objetivo é ter a menor abertura que ainda permite a combinação de injeção para sair (aberturas menores geralmente requerem maior pressão de injeção). Agulhas chanfradas não exigem este passo desde já estão abertas.</li>
</ol>4. microinjection e cuidados pós-injeção
<ol><li>Usando um pincel fino, cuidadosamente molhe a superfície de 1-3 ovos com água estéril, introduza a agulha e injetar a solução. Injete cada ovo antes que seque a superfície. Olha para a aparência uma pequena quantidade de cor vermelha dentro dos ovos durante a injeção para indicar a microinjeção de sucesso. Esmagar ou remover todos os ovos que está danificado, vazio, ou caso contrário não podem ser injetados. Nota: Embora a pressão de injeção varia de acordo com o tamanho de furo de agulha, 10-13 psi é um bom ponto de partida.</li>
	<li>Depois que todos os ovos são injetados, remova a lâmina de vidro o papel de filtro.</li>
	<li>Coloque o papel de filtro sobre a superfície de um prato de 1% de ágar (descrito acima).</li>
	<li>Use filme plástico parafina para selar o prato e coloque em um inseto de 26 ° C criação de incubadora durante 12 dias. Nota: O filme de parafina ajudará a evitar a contaminação cruzada do molde entre pratos. No entanto, qualquer outro tratamento para evitar mofo e crescimento bacteriano poderia reduzir a sobrevivência dos embriões injetados.</li>
</ol>5. cultivo e incubação de embriões
<ol><li>Comece a verificar injeção após 12 dias de embriões para larvas recém-eclodidas e continuar diariamente para as próximas 2 semanas.</li>
	<li>Transferência de larvas, usando um pincel fino, para uma caixa de criação com milho germinado e solo. Nota: Molde pode crescer no prato agar, mas as larvas eclodem ainda na maioria dos casos. Procure larvas cuidadosamente dentro do molde para pegá-los todos. Algumas larvas podem passar para a tampa do prato e podem ficar preso a condensação.</li>
	<li>Larvas a 26 ° C, seguindo o padrão WCR criação métodos25de trás.</li>
</ol>

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Embora microinjection de WCR com dsRNAs com a finalidade de RNAi tem sido relatado9, este é o primeiro protocolo para estabelecer as melhores práticas para microinjecting precellular embriões WCR, um processo crítico para a realização da linha germinal transformação e/ou CRISPR/Cas9 genoma edição nesta espécie. Sucesso microinjeção de embriões WCR é dependente de muitos fatores, como eficiência de transformação. Discutidas a seguir estão algumas das principais questões impactan...

Repele do milho ocidental (RPT), Diabrotica virgifera virgifera, é uma praga importante do milho1. Curiosamente, WCR aparece superar o controle mede mais rapidamente do que as pragas mais agrícolas, desde que eles não só se adaptar fisiologicamente mas também comportamentalmente2,3,4,5, 6. na última década, o RNA de interferência (RNAi), uma poderosa ferramenta de genômica funcional, foi investigado como um método de controle potencial para WCR7,8e também tem sido usado como um meio para o estudo de função do gene9. No entanto, enquanto RNAi é frequentemente realizada por microinjeção de RNA double-stranded (dsRNA) em outras espécies, baseado na injeção de RNAi é raro em WCR. Na verdade, existem apenas alguns relatos de RNAi através de microinjeção de dsRNA em WCR9. A razão é que WCR pode atingir alto-níveis de gene "knockdown" através de ingestão de dsRNA7,10, mesmo permitindo o estudo dos efeitos embrionários alimentando dsRNA para a mãe11. Enquanto este método torna WCR um excelente assunto para análise genômica funcional via RNAi,-retardou o progresso no desenvolvimento de metodologias para o embrionário microinjection nesta espécie.
Apesar do poder de RNAi, existem alguns inconvenientes. Por exemplo, nem todos os genes respondem igualmente a RNAi. Esta variabilidade pode fazer a interpretação dos resultados de um ensaio de genómica funcional mais difícil. Além disso, RNAi é transitória e não gera mutações hereditárias. Por outro lado, a transformação da linha germinal, outra ferramenta genômica funcional, pode gerar mutações hereditárias através de inserção de um elemento marcado transponíveis no genoma12,13. Isto faz a transformação do germline uma excelente ferramenta para a criação de cepas mutantes para uso em estudos de genéticos a longo prazo. Transformação também pode ser usada para resgatar uma mutação, entregando uma cópia funcional do gene de um genoma mutante14. Além disso, a transformação do germline é a pedra angular de uma grande variedade de técnicas de genéticas moleculares. Além de bater para fora e/ou resgatar a função do gene, germline transformação pode ser usada para realçador de armadilhagem15, gene armadilhagem16, expressão ectópica baseada em Gal417e mutagênese genoma-largo18.
Mais recentemente, foram desenvolvidas ferramentas que permitem a edição sofisticada do genoma. Essas ferramentas incluem transcrição Activator-Like Effector Nucleases (TALEN) e o sistema CRISPR/Cas9-nuclease19,20. A vantagem dessas novas ferramentas é que eles oferecem a capacidade de induzir uma quebra de DNA dupla-hélice em quase qualquer local dentro de quase qualquer genoma para pesquisadores. Uma vez que induzida, estas quebras podem ser reparadas através das extremidades não-homóloga juntar-se, que podem apresentar deleções ou inserções no gene alvo, ou através de reparação homologia-dirigido, que na presença de uma construção projetada, pode catalisar a substituição de genes inteiras ou regiões genéticas21,22. No entanto, esses métodos exigem microinjeção de DNA, RNA e/ou proteína em embriões muito jovens.
Importante, ao contrário do dsRNAs usado para RNAi, os ácidos nucleicos e proteínas usadas para germline transformação e edição de genoma não podem facilmente cruzar as membranas celulares. Portanto, microinjeção de DNAs plasmideo, mRNAs e/ou proteínas deve ser efectuada durante a fase de Drosophila sincicial (ou seja, antes do embrião inseto cellularizes). Isso faz com que o timing de injeções um fator crítico. Por exemplo, em Drosophila melanogaster, embriões precisam ser injetado dentro de duas horas depois de ovo colocar12. Portanto, desenvolvimento de um protocolo bem sucedido microinjeção embrionárias para WCR deve levar em conta as melhores condições para a colocação de óvulo feminino, assim como o melhor método para coletar uma quantidade suficiente de embriões precellular.
Um esforço para trazer uma ampla gama de ferramentas de genéticas moleculares sobre Biologia WCR requer desenvolver métodos de recolha e microinjecting precellular embriões WCR. Aqui nós fornecemos instruções detalhadas, juntamente com dicas e truques, para ajudar os outros a usar técnicas baseadas em transformação na pesquisa WCR. Além de estender as tecnologias transgênicas para WCR para uso em estudos de genômicos funcionais, estas técnicas também permitem poderosas novas estratégias de controle de pragas tais como gene unidade23,24 a ser testado neste economicamente importante pragas.

<table border="0" cellpadding="0" cellspacing="0" width="1155">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Qualitative Filter Paper (Black)</td>
			<td style="width:189px;">Ahlstrom</td>
			<td style="width:287px;">8613-0900</td>
			<td style="width:339px;">For egg microinjection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1 oz Containers&#160;</td>
			<td style="width:189px;">Anny&#39;s Plastic Tableware</td>
			<td>ASET101</td>
			<td>Egg container</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Drosophila Agar Type II</td>
			<td style="width:189px;">Apex</td>
			<td style="width:287px;">66-103</td>
			<td>Substrate for WCR egg-laying dish</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Featherweight Forceps&#160;</td>
			<td>Bioquip</td>
			<td style="width:287px;">4748</td>
			<td>Handling larvae and pupae&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Clorox Regular-Bleach1</td>
			<td style="width:189px;">Clorox</td>
			<td style="width:287px;">44600-30770</td>
			<td>Wash corn and dishes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trucker&#39;s Favorite Yellow</td>
			<td>Coor Farm Supply</td>
			<td>502</td>
			<td>Corn for feeding WCR</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Microinjection System (Homemade)</td>
			<td style="width:189px;">NA</td>
			<td style="width:287px;">Parts &#38; instructions available upon request</td>
			<td>Controls injection pressure (0-20 psi)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Elmer&#39;s Non-toxic Glue</td>
			<td>Elmer&#39;s Products, Inc.</td>
			<td style="width:287px;">E304</td>
			<td>Glue eggs on filter paper</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">epTIPS Microloader Tips</td>
			<td style="width:189px;">Eppendorf</td>
			<td style="width:287px;">C2554691</td>
			<td>Backfilling needle loading tips</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Falcon Tissue Culture Dishes</td>
			<td style="width:189px;">Falcon</td>
			<td>25383-103</td>
			<td>Sprouting corn /150 x 25 mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fisherbrand&#160;Quantitative-Grade Filter Paper Circles</td>
			<td style="width:189px;">Fisher Scientific</td>
			<td>S47576C</td>
			<td>Making agar dish for egg-lay/9cm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Sparkleen</td>
			<td style="width:189px;">Fisher Scientific</td>
			<td style="width:287px;">04-320-4</td>
			<td>Wash dishes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Plain Microscope Slides</td>
			<td style="width:189px;">Fisher Scientific</td>
			<td style="width:287px;">12-549-3</td>
			<td>Holding filter paper and eggs for microinjection</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Western Corn Rootworm w/o Pollen Substitute</td>
			<td style="width:189px;">Frontier Agricultural Sciences&#160;</td>
			<td>F9766B</td>
			<td>WCR adult artificial diet</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cotton Balls, Large</td>
			<td>Genesee Scientific</td>
			<td>51-101</td>
			<td>Close the flask</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Globe Scientific 3.0 mL Small Bulb Transfer Pipettes</td>
			<td>Globe Scientific</td>
			<td>137035</td>
			<td>Collect and transfer eggs</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Leica M165 FC Fluorescence Stereomicroscope&#160;</td>
			<td style="width:189px;">Leica</td>
			<td>M165 FC&#160;</td>
			<td>WCR screening</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DsRed Filter Set for Fluorescence Stereomicroscope</td>
			<td style="width:189px;">Leica</td>
			<td>DSR</td>
			<td>Excitation filter: 510-560 nm, emission filter: 590-650 nm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EGFP filter</td>
			<td style="width:189px;">Leica</td>
			<td>GFP2</td>
			<td>Excitation filter: 440&#8211;520 nm, emission filter: 510 nm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BugDorm</td>
			<td>MegaView Science</td>
			<td>DP1000_5P</td>
			<td>Cage for adult colony</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Miniature Paint Brush</td>
			<td style="width:189px;">MyArtscape</td>
			<td>MAS-102-MINI</td>
			<td>Liner 2/0&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Joystick Micromanipulator</td>
			<td style="width:189px;">Narishige</td>
			<td style="width:287px;">MN-151</td>
			<td>Micromanipulator and needle holder</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:343px;">Microscope XY Stage</td>
			<td style="width:189px;">Olympus</td>
			<td style="width:287px;">265515</td>
			<td style="width:339px;">Microscope stage (adapted for use with stereoscope)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Grade 90 Cheesecloth</td>
			<td style="width:189px;">Online Fabric Store</td>
			<td>CHEE90</td>
			<td>For egg-lay</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Plastic paraffin film&#160;</td>
			<td style="width:189px;">Pechiney Plastic Packaging</td>
			<td>PM-996</td>
			<td>Seal agar dish after microinjection/Roll size 4 in. x 125 ft</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Percival Incubator</td>
			<td style="width:189px;">Percival&#160;</td>
			<td style="width:287px;">I41VLH3C8</td>
			<td>WCR growing chamber (insect rearing incubator)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Narrow Mouth Erlenmeyer Flasks</td>
			<td style="width:189px;">VWR</td>
			<td>4980-300-PK</td>
			<td>Water container for adult colony&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Griffin Low Form Beakers</td>
			<td style="width:189px;">VWR</td>
			<td>1000-600-PK</td>
			<td>For egg wash</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Braided Cotton Rolls</td>
			<td>Richmond Dental Cotton Co.</td>
			<td>605-3599</td>
			<td>Use in water supply for adult colony&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Petri Dishes (35x10mm)</td>
			<td>SIGMA</td>
			<td>CLS430588-500EA</td>
			<td>For diet plates</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phenol Red</td>
			<td>Sigma</td>
			<td style="width:287px;">143-78-8</td>
			<td>Microinjection buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Laser-based Micropipiette Puller</td>
			<td>Sutter Instrument</td>
			<td>P-2000/G</td>
			<td>Needle puller/Heat = 335, FIL = 4, VEL = 40, DEL = 200, PUL = 100</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Premium Topsoil</td>
			<td>The Scotts Company</td>
			<td style="width:287px;">71130758</td>
			<td>Soil for cron growing</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Reloc Zippit 2 Mil Zipper Bags</td>
			<td>United States Plastic Corporation</td>
			<td>48342</td>
			<td>Bag agar dish after microinjection/5x7</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Petri Dishes (100x15m)</td>
			<td style="width:189px;">VWR</td>
			<td style="width:287px;">89038-968</td>
			<td>Making agar dish for egg-lay/ 100 x 15 mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">6 oz Containers</td>
			<td style="width:189px;">Webstaurantstore</td>
			<td>128E506</td>
			<td>WCR single pair mating chamber&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">38 oz Containers</td>
			<td style="width:189px;">Webstaurantstore</td>
			<td>128NC888</td>
			<td>Larvae rearing box/38 oz</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ChoiceHD 16 oz. Microwavable Translucent Plastic Deli Container</td>
			<td style="width:189px;">Webstaurantstore</td>
			<td>128HRD16</td>
			<td>Larvae rearing box/16 oz</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Microinjection Scope&#160;</td>
			<td style="width:189px;">Wild Heerbrugg</td>
			<td style="width:287px;">WILD-M8</td>
			<td style="width:339px;">Microinjection scope outfited with an XY stage</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Standard Glass Capillaries</td>
			<td>World Precision Instruments</td>
			<td>1B100F-4</td>
			<td>Microinjection needles</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:343px;">Adult Western Corn Rootworms</td>
			<td style="width:189px;">North Centeral Aqricultural reasearch Laboratory</td>
			<td style="width:287px;">Non-diapase strain</td>
			<td>Request from Dr. Bryan Wade French&#39;s lab</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Plasmid DNA Midi Kit</td>
			<td style="width:189px;">Qiagen</td>
			<td>12143</td>
			<td>Purification of injection-ready plasmid DNAs</td>
		</tr>
	</tbody>
</table>

microinjection of western corn rootworm, diabrotica virgifera virgifera, embryos for germline transformation, or crispr/cas9 genome editing

Repele o milho (RPT) é uma importante praga do milho e é conhecida por sua capacidade de rapidamente se adaptar às estratégias de manejo de pragas. Embora o RNA de interferência (RNAi) provou para ser uma poderosa ferramenta para estudar Biologia WCR, tem suas limitações. Especificamente, RNAi em si é transitório (ou seja, não resulta em longo prazo mendeliana do fenótipo associado), e exige saber a sequência de DNA do gene alvo. Este último pode ser um fator limitante se o fenótipo de interesse é controlado por genes mal conservados, ou mesmo romance, porque identificar alvos úteis ia ser um desafio, se não impossível. Portanto, o número de ferramentas caixa de ferramentas genômicas do WCR deve ser expandido pelo desenvolvimento de métodos que pode ser usada para criar cepas mutantes estáveis e permitir pesquisas independentes de sequência do genoma WCR. Neste documento, detalhamos os métodos usados para coletar e microinjeção precellular WCR os embriões com ácidos nucleicos. Enquanto os protocolos descritos visam a criação de transgénico WCR, edição de CRISPR/Cas9-genoma poderia também ser realizada utilizando os mesmos protocolos, com a única diferença é a composição da solução injetada os embriões.

Uma consideração importante ao desenvolver este protocolo foi palco do desenvolvimento embrionário no momento da microinjeção. Especificamente, o germline transformação requer microinjeção de DNAs antes celularização embrionária. Isso ocorre porque o DNAs facilmente não podem atravessar as membranas celulares. Tenta visualizar os estágios do desenvolvimento embrionário WCR, usando uma mancha nuclear não tiveram sucesso porque dechorionation de embriões WCR essencialmente ...

Watch this Scientific Journal Video about Microinjection of Western Corn Rootworm, Diabrotica virgifera virgifera, Embryos for Germline Transformation, or CRISPR/Cas9 Genome Editing at JoVE.com

Microinjection of Western Corn Rootworm, Diabrotica virgifera virgifera, Embryos for Germline Transformation, or CRISPR/Cas9 Genome Editing

Microinjeção de Western milho repele, Diabrotica virgifera virgifera, embriões de transformação da linha germinal, ou edição de genoma CRISPR/Cas9

Aqui nós apresentamos protocolos para coletando e microinjecting precellular milho ocidental embriões repele com a finalidade de realizar ensaios funcionais-genômica como germline transformação e edição de CRISPR/Cas9-genoma.

The western corn rootworm (WCR) is an important pest of corn and is well known for its ability to rapidly adapt to pest management strategies. Although ...

microinjection-western-corn-rootworm-diabrotica-virgifera-virgifera

Research

JoVE Journal

Bioengineering

Microinjection of Western Corn Rootworm, Diabrotica virgifera virgifera, Embryos for Germline Transformation, or CRISPR/Cas9 Genome Editing

7.3K visualizações.  North Carolina State University. Aqui nós apresentamos protocolos para coletando e microinjecting precellular milho ocidental embriões repele com a finalidade de realizar ensaios funcionais-genômica como germline transformação e edição de CRISPR/Cas9-genoma.

Vídeo: Microinjection of Western Corn Rootworm, Diabrotica virgifera virgifera, Embryos for Germline Transformation, or CRISPR/Cas9 Genome Editing

Viral Nanoparticles for In vivo Tumor Imaging

The use of nanomaterials has the potential to revolutionize materials science and medicine. Currently, a number of different nanoparticles are being investigated for applications in imaging and therapy. Viral nanoparticles (VNPs) derived from plants can be regarded as self-assembled bionanomaterials with defined sizes and shapes. Plant viruses under investigation in the Steinmetz lab include icosahedral particles formed by Cowpea mosaic virus (CPMV) and Brome mosaic virus (BMV), both of which are 30 nm in diameter. We are also developing rod-shaped and filamentous structures derived from the following plant viruses: Tobacco mosaic virus (TMV), which forms rigid rods with dimensions of 300 nm by 18 nm, and Potato virus X (PVX), which form filamentous particles 515 nm in length and 13 nm in width (the reader is referred to refs. 1 and 2 for further information on VNPs).
From a materials scientist's point of view, VNPs are attractive building blocks for several reasons: the particles are monodisperse, can be produced with ease on large scale in planta, are exceptionally stable, and biocompatible. Also, VNPs are "programmable" units, which can be specifically engineered using genetic modification or chemical bioconjugation methods 3. The structure of VNPs is known to atomic resolution, and modifications can be carried out with spatial precision at the atomic level4, a level of control that cannot be achieved using synthetic nanomaterials with current state-of-the-art technologies.
In this paper, we describe the propagation of CPMV, PVX, TMV, and BMV in Vigna ungiuculata and Nicotiana benthamiana plants. Extraction and purification protocols for each VNP are given. Methods for characterization of purified and chemically-labeled VNPs are described. In this study, we focus on chemical labeling of VNPs with fluorophores (e.g. Alexa Fluor 647) and polyethylene glycol (PEG). The dyes facilitate tracking and detection of the VNPs 5-10, and PEG reduces immunogenicity of the proteinaceous nanoparticles while enhancing their pharmacokinetics 8,11. We demonstrate tumor homing of PEGylated VNPs using a mouse xenograft tumor model. A combination of fluorescence imaging of tissues ex vivo using Maestro Imaging System, fluorescence quantification in homogenized tissues, and confocal microscopy is used to study biodistribution. VNPs are cleared via the reticuloendothelial system (RES); tumor homing is achieved passively via the enhanced permeability and retention (EPR) effect12. The VNP nanotechnology is a powerful plug-and-play technology to image and treat sites of disease in vivo. We are further developing VNPs to carry drug cargos and clinically-relevant imaging moieties, as well as tissue-specific ligands to target molecular receptors overexpressed in cancer and cardiovascular disease.

Viral Nanoparticles for In vivo Tumor Imaging

Plant viral nanoparticles (VNPs) are promising platforms for applications in biomedicine. Here, we describe the procedures for plant VNP propagation, purification, characterization, and bioconjugation. Finally, we show the application of VNPs for tumor homing and imaging using a mouse xenograft model and fluorescence imaging.

Nanopartículas virais para<em&gt; Em vivo</em&gt; Imaging Tumor

The use of nanomaterials has the potential to revolutionize materials science and medicine. Currently, a number of different nanoparticles are being ...

Bioengenharia

Environmentally-controlled Microtensile Testing of Mechanically-adaptive Polymer Nanocomposites for ex vivo Characterization

Implantable microdevices are gaining significant attention for several biomedical applications1-4. Such devices have been made from a range of materials, each offering its own advantages and shortcomings5,6. Most prominently, due to the microscale device dimensions, a high modulus is required to facilitate implantation into living tissue. Conversely, the stiffness of the device should match the surrounding tissue to minimize induced local strain7-9. Therefore, we recently developed a new class of bio-inspired materials to meet these requirements by responding to environmental stimuli with a change in mechanical properties10-14. Specifically, our poly(vinyl acetate)-based nanocomposite (PVAc-NC) displays a reduction in stiffness when exposed to water and elevated temperatures (e.g. body temperature). Unfortunately, few methods exist to quantify the stiffness of materials in vivo15, and mechanical testing outside of the physiological environment often requires large samples inappropriate for implantation. Further, stimuli-responsive materials may quickly recover their initial stiffness after explantation. Therefore, we have developed a method by which the mechanical properties of implanted microsamples can be measured ex vivo, with simulated physiological conditions maintained using moisture and temperature control13,16,17. 
To this end, a custom microtensile tester was designed to accommodate microscale samples13,17 with widely-varying Young's moduli (range of 10 MPa to 5 GPa). As our interests are in the application of PVAc-NC as a biologically-adaptable neural probe substrate, a tool capable of mechanical characterization of samples at the microscale was necessary. This tool was adapted to provide humidity and temperature control, which minimized sample drying and cooling17. As a result, the mechanical characteristics of the explanted sample closely reflect those of the sample just prior to explantation.
The overall goal of this method is to quantitatively assess the in vivo mechanical properties, specifically the Young's modulus, of stimuli-responsive, mechanically-adaptive polymer-based materials. This is accomplished by first establishing the environmental conditions that will minimize a change in sample mechanical properties after explantation without contributing to a reduction in stiffness independent of that resulting from implantation. Samples are then prepared for implantation, handling, and testing (Figure 1A). Each sample is implanted into the cerebral cortex of rats, which is represented here as an explanted rat brain, for a specified duration (Figure 1B). At this point, the sample is explanted and immediately loaded into the microtensile tester, and then subjected to tensile testing (Figure 1C). Subsequent data analysis provides insight into the mechanical behavior of these innovative materials in the environment of the cerebral cortex.

Environmentally-controlled Microtensile Testing of Mechanically-adaptive Polymer Nanocomposites for ex vivo Characterization

A method is discussed by which the in vivo mechanical behavior of stimuli-responsive materials is monitored as a function of time. Samples are tested ex vivo using a microtensile tester with environmental controls to simulate the physiological environment. This work further promotes understanding the in vivo behavior of our material.

Teste de microtração ambientalmente controlado mecanicamente adaptativas nanocompósitos poliméricos para<em&gt; Ex vivo</em&gt; Caracterização

Implantable microdevices are gaining significant attention for several biomedical applications1-4. Such devices have been made from a range of materials, ...

Microinjection Wound Assay and In vivo Localization of Epidermal Wound Response Reporters in Drosophila Embryos.

The Drosophila embryo develops a robust epidermal layer that serves both to protect the internal cells from a harsh external environment as well as to maintain cellular homeostasis. Puncture injury with glass needles provides a direct method to trigger a rapid epidermal wound response that activates wound transcriptional reporters, which can be visualized by a localized reporter signal in living embryos or larvae. Puncture or laser injury also provides signals that promote the recruitment of hemocytes to the wound site. Surprisingly, severe (through and through) puncture injury in late stage embryos only rarely disrupts normal embryonic development, as greater than 90% of such wounded embryos survive to adulthood when embryos are injected in an oil medium that minimizes immediate leakage of hemolymph from puncture sites. The wound procedure does require micromanipulation of the Drosophila embryos, including manual alignment of the embryos on agar plates and transfer of the aligned embryos to microscope slides. The Drosophila epidermal wound response assay provides a quick system to test the genetic requirements of a variety of biological functions that promote wound healing, as well as a way to screen for potential chemical compounds that promote wound healing. The short life cycle and easy culturing routine make Drosophila a powerful model organism. Drosophila clean wound healing appears to coordinate the epidermal regenerative response, with the innate immune response, in ways that are still under investigation, which provides an excellent system to find conserved regulatory mechanisms common to Drosophila and mammalian epidermal wounding.

Microinjection Wound Assay and In vivo Localization of Epidermal Wound Response Reporters in Drosophila Embryos.

The embryonic epidermis of very late stage Drosophila embryos provides an in vivo system for rapid puncture wound response analysis and can be combined with genetic manipulations or chemical microinjection treatments to advance studies in wound healing for translation into mammalian models.

Microinjeção Ferida Ensaio e<em&gt; In vivo</em&gt; Localização de Epidermal Wound resposta Jornalistas em<em&gt; Drosophila</em&gt; Os embriões.

The Drosophila embryo develops a robust epidermal layer that serves both to protect the internal cells from a harsh external environment as well as to ...

Efficient Generation and Editing of Feeder-free IPSCs from Human Pancreatic Cells Using the CRISPR-Cas9 System

Embryonic and induced pluripotent stem cells can self-renew and differentiate into multiple cell types of the body. The pluripotent cells are thus coveted for research in regenerative medicine and are currently in clinical trials for eye diseases, diabetes, heart diseases, and other disorders. The potential to differentiate into specialized cell types coupled with the recent advances in genome editing technologies including the CRISPR/Cas system have provided additional opportunities for tailoring the genome of iPSC for varied applications including disease modeling, gene therapy, and biasing pathways of differentiation, to name a few. Among the available editing technologies, the CRISPR/Cas9 from Streptococcus pyogenes has emerged as a tool of choice for site-specific editing of the eukaryotic genome. The CRISPRs are easily accessible, inexpensive, and highly efficient in engineering targeted edits. The system requires a Cas9 nuclease and a guide sequence (20-mer) specific to the genomic target abutting a 3-nucleotide &#34;NGG&#34; protospacer-adjacent-motif (PAM) for targeting Cas9 to the desired genomic locus, alongside a universal Cas9 binding tracer RNA (together called single guide RNA or sgRNA). Here we present a step-by-step protocol for efficient generation of feeder-independent and footprint-free iPSC and describe methodologies for genome editing of iPSC using the Cas9 ribonucleoprotein (RNP) complexes. The genome editing protocol is effective and can be easily multiplexed by pre-complexing sgRNAs for more than one target with the Cas9 protein and simultaneously delivering into the cells. Finally, we describe a simplified approach for identification and characterization of iPSCs with desired edits. Taken together, the outlined strategies are expected to streamline generation and editing of iPSC for manifold applications.

This protocol describes in detail the generation of footprint-free induced pluripotent stem cells (iPSCs) from human pancreatic cells in feeder-free conditions, followed by editing using CRISPR/Cas9 ribonucleoproteins and characterization of the modified single-cell clones.

Eficiente geração e edição de IPSCs livre de alimentador de células pancreáticas humanas utilizando o sistema CRISPR-Cas9

Embryonic and induced pluripotent stem cells can self-renew and differentiate into multiple cell types of the body. The pluripotent cells are thus coveted ...

Production of Genetically Engineered Golden Syrian Hamsters by Pronuclear Injection of the CRISPR/Cas9 Complex

The pronuclear (PN) injection technique was first established in mice to introduce foreign genetic materials into the pronuclei of one-cell stage embryos. The introduced genetic material may integrate into the embryonic genome and generate transgenic animals with foreign genetic information following transfer of the injected embryos to foster mothers. Following the success in mice, PN injection has been applied successfully in many other animal species. Recently, PN injection has been successfully employed to introduce reagents with gene-modifying activities, such as the CRISPR/Cas9 system, to achieve site-specific genetic modifications in several laboratory and farm animal species. In addition to mastering the special set of microinjection skills to produce genetically modified animals by PN injection, researchers must understand the reproduction physiology and behavior of the target species, because each species presents unique challenges. For example, golden Syrian hamster embryos have unique handling requirements in vitro such that PN injection techniques were not possible in this species until recent breakthroughs by our group. With our species-modified PN injection protocol, we have succeeded in producing several gene knockout (KO) and knockin (KI) hamsters, which have been used successfully to model human diseases. Here we describe the PN injection procedure for delivering the CRISPR/Cas9 complex to the zygotes of the hamster, the embryo handling conditions, embryo transfer procedures, and husbandry required to produce genetically modified hamsters.

Pronuclear (PN) injection of the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein-9 nuclease (CRISPR/Cas9) system is a highly efficient method for producing genetically engineered golden Syrian hamsters. Herein, we describe the detailed PN injection protocol for the production of gene knockout hamsters with the CRISPR/Cas9 system.

Produção de geneticamente modificados Hamsters sírios dourados por injeção pró-nuclear do complexo CRISPR/Cas9

The pronuclear (PN) injection technique was first established in mice to introduce foreign genetic materials into the pronuclei of one-cell stage embryos. ...

Ultra-long Read Sequencing for Whole Genomic DNA Analysis

Third generation single-molecule DNA sequencing technologies offer significantly longer read length that can facilitate the assembly of complex genomes and analysis of complex structural variants. Nanopore platforms perform single-molecule sequencing by directly measuring the current changes mediated by DNA passage through the pores and can generate hundreds of kilobase (kb) reads with minimal capital cost. This platform has been adopted by many researchers for a variety of applications. Achieving longer sequencing read lengths is the most critical factor to leverage the value of nanopore sequencing platforms. To generate ultra-long reads, special consideration is required to avoid DNA breakages and gain efficiency to generate productive sequencing templates. Here, we provide the detailed protocol of ultra-long DNA sequencing including high molecular weight (HMW) DNA extraction from fresh or frozen cells, library construction by mechanical shearing or transposase fragmentation, and sequencing on a nanopore device. From 20-25 &#181;g of HMW DNA, the method can achieve N50 read length of 50-70 kb with mechanical shearing and N50 of 90-100 kb read length with transposase mediated fragmentation. The protocol can be applied to DNA extracted from mammalian cells to perform whole genome sequencing for the detection of structural variants and genome assembly. Additional improvements on the DNA extraction and enzymatic reactions will further increase the read length and expand its utility.

Long-read sequences greatly facilitate the assembly of complex genomes and characterization of structural variation. We describe a method to generate ultra-long sequences by nanopore-based sequencing platforms. The approach adopts an optimized DNA extraction followed by modified library preparations to generate hundreds of kilobase reads with moderate coverage from human cells.

Sequenciamento de leitura ultralonga para análise de DNA genômico completo

Third generation single-molecule DNA sequencing technologies offer significantly longer read length that can facilitate the assembly of complex genomes ...

CRISPR/Cas12a Multiplex Genome Editing of Saccharomyces cerevisiae and the Creation of Yeast Pixel Art

High efficiency, ease of use and versatility of the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system has facilitated advanced genetic modification of Saccharomyces cerevisiae, a model organism and workhorse in industrial biotechnology. CRISPR-associated protein 12a (Cas12a), an RNA-guided endonuclease with features distinguishable from Cas9 is applied in this work, further extending the molecular toolbox for genome editing purposes. A benefit of the CRISPR/Cas12a system is that it can be used in multiplex genome editing with multiple guide RNAs expressed from a single transcriptional unit (single CRISPR RNA (crRNA) array). We present a protocol for multiplex integration of multiple heterologous genes into independent loci of the S. cerevisiae genome using the CRISPR/Cas12a system with multiple crRNAs expressed from a single crRNA array construct. The proposed method exploits the ability of S. cerevisiae to perform in vivo recombination of DNA fragments to assemble the single crRNA array into a plasmid that can be used for transformant selection, as well as the assembly of donor DNA sequences that integrate into the genome at intended positions. Cas12a is pre-expressed constitutively, facilitating cleavage of the S.&#160;cerevisiae genome at the intended positions upon expression of the single crRNA array. The protocol includes the design and construction of a single crRNA array and donor DNA expression cassettes, and exploits an integration approach making use of unique 50-bp DNA connectors sequences and separate integration flank DNA sequences, which simplifies experimental design through standardization and modularization and extends the range of applications. Finally, we demonstrate a straightforward technique for creating yeast pixel art with an acoustic liquid handler using differently colored carotenoid producing yeast strains that were constructed.

CRISPR/Cas12a Multiplex Genome Editing of Saccharomyces cerevisiae and the Creation of Yeast Pixel Art

The CRISPR/Cas12a system in combination with a single crRNA array enables efficient multiplex editing of the S.&#160;cerevisiae genome at multiple loci simultaneously. This is demonstrated by constructing carotenoid producing yeast strains which are subsequently used to create yeast pixel art.

Edição do genoma de CRISPR/Cas12a multiplex de Saccharomyces cerevisiae e a criação da arte do pixel do fermento

High efficiency, ease of use and versatility of the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) ...

Measuring the Spin-Lattice Relaxation Magnetic Field Dependence of Hyperpolarized [1-13C]pyruvate

The fundamental limit to in vivo imaging applications of hyperpolarized 13C-enriched compounds is their finite spin-lattice relaxation times. Various factors affect the relaxation rates, such as buffer composition, solution pH, temperature, and magnetic field. In this last regard, the spin-lattice relaxation time can be measured at clinical field strengths, but at lower fields, where these compounds are dispensed from the polarizer and transported to the MRI, the relaxation is even faster and difficult to measure. To have a better understanding of the amount of magnetization lost during transport, we used fast field-cycling relaxometry, with magnetic resonance detection of 13C nuclei at ~0.75 T, to measure the nuclear magnetic resonance dispersion of the spin-lattice relaxation time of hyperpolarized [1-13C]pyruvate. Dissolution dynamic nuclear polarization was used to produce hyperpolarized samples of pyruvate at a concentration of 80 mmol/L and physiological pH (~7.8). These solutions were rapidly transferred to a fast field-cycling relaxometer so that relaxation of the sample magnetization could be measured as a function of time using a calibrated small flip angle (3&#176;-5&#176;). To map the T1 dispersion of the C-1 of pyruvate, we recorded data for different relaxation fields ranging between 0.237 mT and 0.705 T. With this information, we determined an empirical equation to estimate the spin-lattice relaxation of the hyperpolarized substrate within the mentioned range of magnetic fields. These results can be used to predict the amount of magnetization lost during transport and to improve experimental designs to minimize signal loss.

Measuring the Spin-Lattice Relaxation Magnetic Field Dependence of Hyperpolarized [1-13C]pyruvate

We present a protocol to measure the magnetic field dependence of the spin-lattice relaxation time of 13C-enriched compounds, hyperpolarized by means of dynamic nuclear polarization, using fast field-cycled relaxometry. Specifically, we have demonstrated this with [1-13C]pyruvate, but the protocol could be extended to other hyperpolarized substrates.

Medindo a dependência do campo magnético do abrandamento da girar-estrutura de Hyperpolarizado [1-13C] piruvato

The fundamental limit to in vivo imaging applications of hyperpolarized 13C-enriched compounds is their finite spin-lattice relaxation times. Various ...

Microinjection of Corn Planthopper, Peregrinus maidis, Embryos for CRISPR/Cas9 Genome Editing

The corn planthopper, Peregrinus maidis, is&#160;a pest of maize and a vector of several maize viruses. Previously published methods describe the triggering of RNA interference (RNAi) in P. maidis through microinjection of double-stranded RNAs (dsRNAs) into nymphs and adults. Despite the power of RNAi, phenotypes generated via this technique are transient and lack long-term Mendelian inheritance. Therefore, the P. maidis toolbox needs to be expanded to include functional genomic tools that would enable the production of stable mutant strains, opening the door for researchers to bring new control methods to bear on this economically important pest. However, unlike the dsRNAs used for RNAi, the components used in CRISPR/Cas9-based genome editing and germline transformation do not easily cross cell membranes. As a result, plasmid DNAs, RNAs, and/or proteins must be microinjected into embryos before the embryo cellularizes, making the timing of injection a critical factor for success. To that end, an agarose-based egg-lay method was developed to allow embryos to be harvested from P. maidis females at relatively short intervals. Herein are provided detailed protocols for collecting and microinjecting precellular P. maidis embryos with CRISPR components (Cas9 nuclease that has been complexed with guide RNAs), and results of Cas9-based gene knockout of a P. maidis eye-color gene, white, are presented. Although these protocols describe CRISPR/Cas9-genome editing in P. maidis, they can also be used for producing transgenic P. maidis via germline transformation by simply changing the composition of the injection solution.

Microinjection of Corn Planthopper, Peregrinus maidis, Embryos for CRISPR/Cas9 Genome Editing

Herein are protocols for collecting and microinjecting precellular corn planthopper embryos for the purpose of modifying their genome via CRISPR/Cas9-based genome editing or for the addition of marked transposable elements through germline transformation.

Microinjeção de cigarrinha de milho, Peregrinus maidis, Embriões para edição do genoma CRISPR/Cas9

The corn planthopper, Peregrinus maidis, is&#160;a pest of maize and a vector of several maize viruses. Previously published methods describe the ...

Liquid Chromatography Coupled to Refractive Index or Mass Spectrometric Detection for Metabolite Profiling in Lysate-based Cell-free Systems

Engineering cellular metabolism for targeted biosynthesis can require extensive design-build-test-learn (DBTL) cycles as the engineer works around the cell&#39;s survival requirements. Alternatively, carrying out DBTL cycles in cell-free environments can accelerate this process and alleviate concerns with host compatibility. A promising approach to cell-free metabolic engineering (CFME) leverages metabolically active crude cell extracts as platforms for biomanufacturing and for rapidly discovering and prototyping modified proteins and pathways. Realizing these capabilities and optimizing CFME performance requires methods to characterize the metabolome of lysate-based cell-free platforms. That is, analytical tools are necessary for monitoring improvements in targeted metabolite conversions and in elucidating alterations to metabolite flux when manipulating lysate metabolism. Here, metabolite analyses using high-performance liquid chromatography (HPLC) coupled with either optical or mass spectrometric detection were applied to characterize metabolite production and flux in E. coli S30 lysates. Specifically, this report describes the preparation of samples from CFME lysates for HPLC analyses using refractive index detection (RID) to quantify the generation of central metabolic intermediates and by-products in the conversion of low-cost substrates (i.e., glucose) to various high-value products. The analysis of metabolite conversion in CFME reactions fed with 13C-labeled glucose through reversed-phase liquid chromatography coupled to tandem mass spectrometry (MS/MS), a powerful tool for characterizing specific metabolite yields and lysate metabolic flux from starting materials, is also presented. Altogether, applying these analytical methods to CFME lysate metabolism enables the advancement of these systems as alternative platforms for executing faster or novel metabolic engineering tasks.

The protocols describe high-performance liquid chromatography methods coupled to refractive index or mass spectrometric detection for studying metabolic reactions in complex lysate-based cell-free systems.

Cromatografia líquida acoplada ao índice refrativo ou detecção espectrométrica em massa para perfil metabólito em sistemas livres de células baseadas em lysato

Engineering cellular metabolism for targeted biosynthesis can require extensive design-build-test-learn (DBTL) cycles as the engineer works around the ...

Microinjeção de Western milho repele, Diabrotica virgifera virgifera, embriões de transformação da linha germinal, ou edição de genoma CRISPR/Cas9

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