Este trabajo fue financiado por una beca del programa de investigación de Monsanto maíz Rootworm conocimiento, conceder fondos de puesta en marcha y número AG/1005 (para MDL y YC) a MDL de NCSU. FC fue apoyado por becas del programa de investigación de Monsanto maíz Rootworm conocimiento (AG/1005) y la National Science Foundation, grant número MCB-1244772 (para MDL). Los autores declaran a no hay intereses contrapuestos. FC y MDL concibieron y diseñado los experimentos; FC, PW y SP realizan los experimentos; MDL y analizaron los resultados; y FC, NG y MDL escribieron el manuscrito. Agradecemos a Teresa O'Leary, William Klobasa y Stephanie Gorski por su ayuda experto en investigación de la región del gran Caribe. También agradecemos a Dr. Wade French (USDA-ARS, Laboratorio Central de investigación agrícola del norte, Brookings, SD) para los envíos de huevos para establecer colonias de laboratorio y proporcionar protocolos de cría.

1. nivel de la colonia de cría de adultos de la región del gran Caribe
<ol><li>Obtener una cantidad suficiente de los adultos de la región del gran Caribe (500-1.000) de un laboratorio de investigación o empresa confiable (véase Tabla de materiales por ejemplo) y colocar en una jaula de 30 cm3 . Nota: Se recomienda uso de una cepa no diapausa.</li>
	<li>Preparar la dieta artificial WCR siguiendo el protocolo del fabricante y vierta una 1 cm de espesor capa en un recipiente de 38 oz (véase Tabla de materiales). Después de mezclar, guarde dieta sin usar a 4 ° C hasta por 2 meses.</li>
	<li>Poner un plato de Petri (100 x 15 mm) con dieta adultos (10-15 g) en una jaula de 30 cm3 . Añadir más dieta cuando la comida sea baja o seca.</li>
	<li>Coloque un matraz (300 mL) con agua, cubierto con una bola de algodón, que se utiliza para mantener en su lugar un trozo de algodón (6 "x 3/8"), para servir como una fuente de agua. Cambie el agua cuando está baja.</li>
	<li>Mantener la región del gran Caribe a 26 ° C con una humedad de 60% y un 14:10 luz del ciclo de un insecto que se cría la incubadora.</li>
</ol>2. alineación y la colección embrionaria
<ol><li>Hacer una cámara de recogida del huevo utilizando agar al 1% en agua estéril de 100 x 15 mm2 caja de Petri. Almacenar a 4 ° C después de que el agar se solidifica.</li>
	<li>Para recoger los huevos recién puestos, coloque una sola capa de papel de filtro, seguido por 4 capas de Gasa (cada corte a tamaño) en la superficie del agar (figura 1A). Nota: Estopilla se puede reutilizar, después de ser lavado en lejía y esterilizado, pero no es necesario hacer esto para el primer uso. Papel de filtro no debe ser reutilizado y no necesita ser esterilizado.</li>
	<li>Coloque la placa de colección de huevo en jaula de la región del gran Caribe como tarde en el día como sea posible (final de la jornada de trabajo) y cubrir con una carpa de papel de estaño. Dejar durante la noche. Nota: Tener las luces encendidas de 10:00 a la ponedora de retrasos de 12 de la mañana (14 h), facilitando así la recolección de huevos más jóvenes para microinyección sin necesidad de personal de laboratorio colocar cámara de recogida en jaula por la noche.</li>
	<li>Retire la cámara de colección de huevo alrededor de 8 o 9:00</li>
	<li>Use pinzas para recoger 1 capa de estopilla en tiempo y lugar en un vaso de precipitados (500 mL) de agua. Lavar huevos de estopilla agitando suavemente (figura 1B y 1C).</li>
	<li>Use una pipeta de bulbo para transferir huevos otro vaso de precipitados (500 mL) de agua. Repetir 2-3 x para limpiar los huevos.</li>
	<li>Utilizar la pipeta de bulbo para transferir huevos en papel de filtro (figura 1) y reducir al mínimo la cantidad de remanente de agua.</li>
	<li>Cortar papel filtro negro tiras tan amplia como una diapositiva de cristal y cinta firmemente al portaobjetos para asegurarse de que el libro pone plana (figura 2A). Nota: Papel filtro negro ayuda a mejorar la visibilidad en el microscopio al reducir la cantidad de luz reflejada.</li>
	<li>Aplicar pegamento no tóxico (véase Tabla de materiales por ejemplo) para el papel de filtro en las líneas finas (figura 2B). Mantener las líneas de cola más delgada que el diámetro de un huevo para evitar que los huevos con pegamento.</li>
	<li>Utilizar un pincel fino para mover suavemente huevos WCR uno por uno desde su papel de filtro a la línea de pegamento. Trate de poner los huevos sobre el pegamento antes de se seca y mantener la distancia de por lo menos un huevo entre cada huevo.</li>
	<li>Maximizar la eficiencia de la inyección mediante la colocación de múltiples líneas de huevos en la diapositiva de un papel de filtro (figura 3).</li>
	<li>Después de todos los huevos se ponen hacia fuera en el papel de filtro, espere hasta que el pegamento se seque antes de microinyección. Nota: Para la transformación de la línea germinal y para la edición genómica CRISPR/Cas9-mediada, inyectar todos los embriones al mediodía (12:00) para que el embrión más viejo es viejo no más de 19 h. Sin embargo, para RNAi, hay ninguna limitación de tiempo porque en RGC, dsRNA puede moverse entre células. De hecho, para RNAi, embriones envejecimiento podrían resultar en mejores tasas de supervivencia.</li>
</ol>3. preparación de plásmido DNAs y agujas de inyección
<ol><li>Preparación de ADN plasmídico superenrollado de alta calidad mediante un comercial libre de endotoxina kit (véase Tabla de materiales por ejemplo).</li>
	<li>Para preparar la solución de la inyección, Compruebe la concentración de cada plásmido y luego combinar plásmido helper (concentración final de 250 ng/μL), plásmido donante (concentración final de 750 ng/μL) y buffer de rojo de fenol (concentración final de 20%). Mezclar la solución brevemente y centrifugar durante 3 minutos en velocidad máxima para precipitar partículas que podrían obstruir la aguja.</li>
	<li>Tire las agujas de vidrio borosilicato (O.D. 1 mm I.D. 0,58 mm, longitud 10 cm) usando un extractor de micropipeta tipo llamas/marrón. Para el almacenamiento, asegurar agujas tiradas sobre la cinta adhesiva de doble cara en una placa de Petri u otro recipiente transparente. Nota: Para el sistema de tirador de la micropipeta, los valores utilizados fueron: calor = 335, FIL = 4, VEL = 40, DEL = 200, PUL = 100.</li>
	<li>Relleno la aguja de inyección utilizando un cargador de micro punta (véase Tabla de materiales) de pipetear 0,5 - 1,0 μL abajo dentro de la aguja, cerca de la punta cónica. Elimine las burbujas de la punta de la aguja si se produce alguna.</li>
	<li>Inserte la aguja en el portaagujas.</li>
	<li>Abra la punta de la aguja rompiendo suavemente con un buen par de pinzas, o tocar/arrastrando suavemente la punta sobre el portaobjetos cubreobjetos o vidrio. Nota: La meta es que la abertura más pequeña que permite la mezcla de inyección volver a salir (las aberturas más pequeñas generalmente requieren mayor presión de inyección). Agujas biseladas no requieren este paso puesto que ya están abiertas.</li>
</ol>4. microinyección y el cuidado después de la inyección
<ol><li>Con un pincel fino, humedezca la superficie de 1-3 huevos con agua estéril con cuidado Inserte la aguja e inyectar la solución. Inyectar cada huevo antes de que se seque la superficie. Busque la aparición una pequeña cantidad de color rojo dentro de huevos durante la inyección para indicar que la exitosa microinyección. Aplastar o eliminar los huevos que parecen dañados, vacío, o de lo contrario no inyectar. Nota: Aunque la presión de la inyección varía dependiendo de tamaño de alesaje de la aguja, 10-13 psi es un buen punto de partida.</li>
	<li>Después se inyectan todos los huevos, quitar el papel de filtro de lo portaobjetos de vidrio.</li>
	<li>Coloque el papel de filtro sobre la superficie de un plato de agar al 1% (ver arriba).</li>
	<li>Use película de parafina plástico para sellar el plato y poner en un insecto de 26 ° C la incubadora de cría durante 12 días. Nota: La película de parafina le ayudará a evitar la contaminación del molde entre platos. Sin embargo, cualquier otro tratamiento para prevenir el crecimiento de bacterias y moho podría reducir la supervivencia de los embriones inyectados.</li>
</ol>5. cultivo e incubación de embriones
<ol><li>Iniciar control de embriones 12 días después de la inyección para las larvas recién nacidas y continuar diariamente durante 2 semanas.</li>
	<li>Transferencia de larvas, utilizando un pincel fino, a una caja de cría con germinado de maíz y el suelo. Nota: Molde podría crecer en el plato de agar, pero las larvas eclosionan aún en la mayoría de los casos. Busque larvas cuidadosamente en el molde para cogerlos todos. Algunas larvas pueden pasar a la tapa del plato y pueden atascarse en la condensación.</li>
	<li>Posterior de las larvas a 26 ° C siguiendo el estándar WCR cría métodos25.</li>
</ol>

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Los autores no tienen nada que revelar.

Aunque microinyección de WCR con dsRNAs para RNAi ha reportado9, este es el primer protocolo para establecer las mejores prácticas para microinjecting precellular embriones de la región del gran Caribe, un proceso crítico para llevar a cabo la transformación del germline o CRISPR/Cas9 genoma de esta especie. Exitosa microinyección de embriones WCR es dependiente en muchos factores, como es la eficiencia de transformación. A continuación algunos de los principales problemas afectan el resul...

Rootworm occidental del maíz (RGC), Diabrotica virgifera virgifera, es una plaga importante del maíz1. Curiosamente, WCR parece superar el control de las medidas más rápidamente que las plagas agrícolas más ya que ellos no sólo adaptan fisiológicamente sino también su comportamiento2,3,4,5, 6. en la última década, ARN de interferencia (RNAi), una poderosa herramienta genómica funcional, ha sido investigada como un posible método de control RCM7,8y también se ha utilizado como un medio para el estudio de la función del gen9. Sin embargo, mientras que el ARNi es realizada con frecuencia por microinyección de ARN bicatenario (dsRNA) en otras especies, ARNi basada en inyección es raro en RGC. De hecho, hay solamente algunos informes de RNAi vía microinyección de dsARN en RGC9. La razón es que RCM puede alcanzar niveles altos de gene precipitación a través de la ingestión de dsARN7,10, permitiendo incluso el estudio de efectos embrionarios de dsRNA de alimentación a la madre11. Mientras que este método hace WCR un excelente tema para análisis genómico funcional mediante RNAi, ha ralentizado el progreso en el desarrollo de metodologías para la microinyección embrionario en esta especie.
A pesar del poder de RNAi, existen algunas desventajas. Por ejemplo, no todos los genes responden igualmente a ARNi. Esta variabilidad puede hacer interpretación de los resultados de un análisis de genómica funcional más difícil. Además, ARNi es transitoria y no genera mutaciones heredables. Por otro lado, la transformación del germline, otra herramienta genómica funcional, puede generar mutaciones heredables mediante inserción de un elemento marcado transponibles en el genoma12,13. Esto hace del germline transformación una excelente herramienta para la creación de estirpes mutantes para el uso en los estudios genéticos a largo plazo. Transformación puede utilizarse también para rescatar una mutación mediante la entrega de una copia funcional del gen a un genoma mutante14. Por otra parte, la transformación del germline es la piedra angular de una gran variedad de técnicas de genética molecular. Además de noquear o rescatar la función del gene, transformación de la línea germinal puede utilizarse para potenciador captura15, gen captura16, Gal4-basado de la expresión ectópica17y genoma mutagénesis18.
Más recientemente, han desarrollado herramientas que permiten la edición del genoma sofisticado. Estas herramientas incluyen la transcripción Activator-Like efectoras nucleasas (jugadores) y el sistema CRISPR/Cas9-nucleasa19,20. La ventaja de estas nuevas herramientas es que ofrecen a los investigadores la capacidad para inducir una rotura de ADN de doble hebra en casi cualquier ubicación a casi cualquier genoma. Una vez inducida, estas roturas pueden repararse a través de ambos extremos no homóloga unirse a, que pueden introducir supresiones o inserciones en el gen específica, o reparación dirigida por homología, que en presencia de una construcción de ingeniería, puede catalizar la reemplazo de genes enteros o regiones genéticas21,22. Sin embargo, estos métodos requieren microinyección de DNA, RNA o proteínas en embriones muy jóvenes.
Lo importante, a diferencia de los dsARNs utilizados para RNAi, ácidos nucleicos y proteínas utilizadas para germline transformación y la edición del genoma no pueden fácilmente cruzar las membranas celulares. Por lo tanto, microinyección de ADN plásmido, mRNAs y proteínas debe ocurrir durante la etapa del blastoderm sincitial (es decir, antes de que el embrión insecto cellularizes). Esto hace que la sincronización de las inyecciones de un factor crítico. Por ejemplo, en Drosophila melanogaster, embriones deban ser inyectado dentro de dos horas después de huevo ponga12. Por lo tanto, el desarrollo de un protocolo exitoso microinjection embrionario para RGC debe tener en cuenta las mejores condiciones para la colocación del óvulo, así como el mejor método para la recolección de suficientes cantidades de embriones precellular.
Un esfuerzo para traer una amplia gama de herramientas genéticas moleculares en biología de la región del gran Caribe requiere desarrollar métodos para la recogida y microinjecting precellular embriones de RGC. Aquí proporcionamos instrucciones detalladas, junto con consejos y sugerencias, para ayudar a otros a utilizar técnicas basadas en la transformación en la investigación de la región del gran Caribe. Además de extender Tecnologías transgénicas a RGC para su uso en estudios de genómica funcional, estas técnicas también permiten potentes nuevas estrategias de control de plagas como gen unidad23,24 a probarse en esta importancia económica plaga.

<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

El rootworm occidental del maíz (RGC) es una plaga importante del maíz y es bien conocido por su capacidad para adaptarse rápidamente a estrategias de manejo de plagas. Aunque el ARN de interferencia (ARNi) ha demostrado para ser una poderosa herramienta para estudiar la biología de la región del gran Caribe, tiene sus limitaciones. Específicamente, ARNi sí mismo es transitorio (es decir, no resulta en la herencia mendeliana a largo plazo del fenotipo asociado), y requiere conocer la secuencia de ADN del gen objetivo. Este último puede ser limitante si el fenotipo de interés está controlado por genes mal conservados, o incluso novela, porque sería difícil identificar objetivos útiles, si no imposible. Por lo tanto, debería ampliarse el número de herramientas de herramientas genómicas de RGC por el desarrollo de métodos que podrían utilizarse para crear cepas mutantes estables y activar encuestas independientes de la secuencia del genoma del RGC. En este documento, se detallan los métodos utilizados para recoger y microinject precellular embriones WCR con ácidos nucleicos. Mientras que los protocolos descritos tienen como objetivo la creación de transgénico WCR, CRISPR Cas9-genoma edición podría también realizarse utilizando los mismos protocolos, con la única diferencia es la composición de la solución inyectada en los embriones.

Una consideración importante en el desarrollo de este protocolo fue la etapa del desarrollo embrionario en el momento de microinyección. Transformación del germline requiere específicamente, microinyección de ADN antes de la cellularization embrionaria. Esto es porque el ADN no puede cruzar fácilmente las membranas celulares. Para visualizar las etapas del desarrollo embrionario de la región del gran Caribe mediante una tinción nuclear las tentativas eran fracasadas porque dechori...

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

Microinyección de Western gusano de raíz del maíz, Diabrotica virgifera virgifera, embriones para transformación de línea germinal, o edición del genoma CRISPR/Cas9

Presentamos aquí protocolos de recogida y microinjecting maíz occidental precellular embriones de gusano de la raíz con el fin de realizar análisis funcional genómica como transformación del germline CRISPR Cas9-genoma de edición.

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 vistas.  North Carolina State University. Presentamos aquí protocolos de recogida y microinjecting maíz occidental precellular embriones de gusano de la raíz con el fin de realizar análisis funcional genómica como transformación del germline CRISPR Cas9-genoma de edición.

Video: 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.

Las nanopartículas virales para<em&gt; En vivo</em&gt; Imágenes de tumores

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

Investigación

Bioingeniería

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.

Microinyección de Heridas Ensayo y<em&gt; In vivo</em&gt; Localización de la herida epidérmica Reporteros de respuesta en<em&gt; Drosophila</em&gt; Embriones.

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 generación y edición de IPSCs alimentador-libre de las células pancreáticas humanas utilizando el 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.

Producción de hámsters sirios dorados genéticamente por inyección Pronuclear del complejo 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. ...

An Ultrahigh-throughput Microfluidic Platform for Single-cell Genome Sequencing

Sequencing technologies have undergone a paradigm shift from bulk to single-cell resolution in response to an evolving understanding of the role of cellular heterogeneity in biological systems. However, single-cell sequencing of large populations has been hampered by limitations in processing genomes for sequencing. In this paper, we describe a method for single-cell genome sequencing (SiC-seq) which uses droplet microfluidics to isolate, amplify, and barcode the genomes of single cells. Cell encapsulation in microgels allows the compartmentalized purification and tagmentation of DNA, while a microfluidic merger efficiently pairs each genome with a unique single-cell oligonucleotide barcode, allowing &gt;50,000 single cells to be sequenced per run. The sequencing data is demultiplexed by barcode, generating groups of reads originating from single cells. As a high-throughput and low-bias method of single-cell sequencing, SiC-seq will enable a broader range of genomic studies targeted at diverse cell populations.

Single-cell sequencing reveals genotypic heterogeneity in biological systems, but current technologies lack the throughput necessary for the deep profiling of community composition and function. Here, we describe a microfluidic workflow for sequencing &gt;50,000 single-cell genomes from diverse cell populations.

Una plataforma de ultra rendimiento microfluidos para la secuenciación del genoma de unicelular

Sequencing technologies have undergone a paradigm shift from bulk to single-cell resolution in response to an evolving understanding of the role of ...

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.

Muy largo leer la secuencia para el análisis de la DNA de Genomic entero

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.

CRISPR/Cas12a Multiplex Genoma Edición de Saccharomyces cerevisiae y la creación de Arte de píxeles de levadura

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.

Medición de la dependencia del campo magnético de relajación Spin-Lattice de hiperpolarizado [1-13C]pyruvate

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.

Microinyección de saltamontes de maíz, Peregrinus maidis, embriones para la edición del 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.

Cromatografía líquida acoplada a la detección de índice de refracción o espectrometría de masas para perfiles de metabolitos en sistemas libres de células basados en lisato

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

Microinyección de Western gusano de raíz del maíz, Diabrotica virgifera virgifera, embriones para transformación de línea germinal, o edición del genoma CRISPR/Cas9

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