Name: microinjection of western corn rootworm, diabrotica virgifera virgifera, embryos for germline transformation, or crispr/cas9 genome editing
Uploaded: 2018-04-27T14:30:00
Description: 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

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 m...

<ol>
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G., Carroll, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+chromosomal+cleavage+and+mutagenesis+in+Drosophila+using+zinc-finger+nucleases.">Targeted chromosomal cleavage and mutagenesis in Drosophila using zinc-finger nucleases.</a> Genetics. 161 (3), 1169-1175 (2002).</li><li>Jeggo, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+breakage+and+repair.">DNA breakage and repair.</a> Adv Genet. 38, 185-218 (1998).</li><li>Gantz, V. M., Bier, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome+editing.+The+mutagenic+chain+reaction:+a+method+for+converting+heterozygous+to+homozygous+mutations.">Genome editing. 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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 ...

The overall goal of this Microinjection protocol is to modify the Western Corn Rootworm genome. This method can help answer key questions regarding Western Corn Rootworm biology and their ability to quickly overcome pest control methods such as BT Toxin and RANI. Besides Western Corn Rootworms, this method can also be applied to similar species, such as Southern and Northern Rootworms.I first had the idea for this method when I was asked to help with injection based RANI in Western Corn Rootworm back in the 90's. Generally, individuals new to this method will struggle. Because rearing Western Corn Rootworms in the laboratory is labor intensive.And survival rates remain low using current technology. Obtain between 500 and 1, 000 non-diapausing rootworm adults from a reliable company or research laboratory. And house them at 30 cubic centimeter cages.Store the standard rootworm artificial diet in food storage containers about one centimeter deep. Unused diet can be stored at four degrees Celsius for up to two months. To feed the rootworm, load 100 centimeter Petri dishes with 10 to 15 grams of diet.Load one dish of food into each rootworm cage. And replenish it when the supply runs low or the diet dries out. Also keep a 300 milliliter water reservoir, covered by a cotton ball, in each cage to serve as a water source.Change the water when it runs low. Maintain the caged rootworm at 26 degrees Celsius with 60%humidity and a 14/10 light cycle in an insect rearing incubator. Set the lights off time later in the day such as at midnight so eggs are laid before morning work hours.First, collect the embryos. For an egg collection surface, load a 100 millimeter Petri dish with 30 milliliters of 1%agar and water. Next, place a single layer of filter paper on the agar, followed by four layers of cheese cloth.Each layer should be cut to size. Next, as late in the day as possible, place the egg collection plate into the rootworm cage and cover it with a tinfoil tent. Then on the following morning, remove the egg collection chamber.Use forceps to pick up one layer of cheese cloth at a time and place into 500 milliliter beaker loaded with water. Wash the eggs off of the cheese cloth by swirling in the water. Then, use a bulb pipet to transfer the eggs to another beaker of water as a wash step.Perform two or three washes in this manner. Then, deposit the cleaned eggs onto filter paper with as little water as possible. Next, prepare a slide by taping a piece of black filter paper flat to the slide to improve contrast.Then drizzle fine lines of non-toxic glue onto the paper that will be used to secure the eggs. Keep the glue below the girth of an egg. While the glue is wet, use a fine brush to place the eggs onto the glue line.Keep at least one egg's distance between each egg. Prepare multiple lines of eggs per slide. The eggs should now be injected within 30 minutes.Now, combine the helper plasmid, donor plasmid, and phenol red buffer. Use high quality, super coiled plasmids made with an endotoxin-free commercial kit. Fortex briefly to mix.And then spin down particulates for three minutes at maximum speed. Next, backfill pulled borosilicate glass needles with 0.5 to 1 microliters of injection solution. If any bubbles form, be sure to remove them.Then, attach the injection needle to the micromanipulator. Now, if necessary, under the microscope, gently break off the sealed needle tip using a fine pair of forceps or by gently touching the tip into a glass barrier. Try to make the smallest possible opening that releases injection solution.Before injecting the rootworm eggs, use a fine paintbrush to carefully wet the surface of one to three prepared eggs with sterile water. Then, inject the wetted embryos. Gently insert the needle into the middle of the embryo, and inject a small volume of solution.Start with using 10 to 13 psi of pressure and adjust the pressure as needed. Dry embryos has a distinct round shape, by pre-wetting each embryo with water will change their shape and soften the eggshell thus allowing the needle to easily penetrate. A successfully injected embryo will have a small amount of red color.If an egg was damaged, looks peculiar, or cannot be injected then crush it or remove it from the slide. After all eggs are injected, move the filter paper with the eggs on it to a 1%agar dish. Then, seal the dish with paraffin film and incubate it at 26 degrees Celsius until larvae hatch.12 days after injection, start checking for larvae everyday for the next two weeks. Transfer the larvae using a fine brush to a rearing box with sprouted corn and soil to rear them under standard conditions. Germline transformation of rootworm was attempted by co-injecting a transposase carrying plasmid with a transposon carrying plasmid into pre-cellular embryos.In the 26 survivors, each EFP expression was used to screen for donor element insertion. Although thoracent progeny were identified, screening rootworm embryos with the intense light source proved to be lethal. The next round of germline transformation utilized a red florescent protein gene driven by a constitutively active promoter from Drosophila melanogaster.Survival rates improved, but only one DS red positive larvae was recorded. Next, embryos were micro-injected with buffer alone or with helper and thorescently marked donor DNA plasmids. A similar ratio of embryos hatched when injected with plasmids or buffer alone.And interestingly, only about half of un-injected embryos hatched. This put the lethality of manipulating the embryos for the injection as high as performing the injection. After watching this video, you should have a good understanding of how to micro inject Western Corn Rootwall embryos.Once mastered two to three hundred embryos can be injected per hour using this technique. This method is indebted to techniques developed for germline transformation of Drosophila melanogaster which serve as templates applicable to many other species. Following this procedure, other methods like CRISPR/Cas9 based genome editing can be performed to make targeted mutations in Western Corn Rootwall's genome.In order to answer specific questions about genome function.

Research

JoVE Journal

Bioengineering

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

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Bioengenharia

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