Name: injection of an. stephensi embryos to generate malaria-resistant mosquitoes
Uploaded: 2007-07-04T00:00:00
Description: Watch this Scientific Journal Video about Injection of An. stephensi Embryos to Generate Malaria-resistant Mosquitoes at JoVE.com

<p class="jove_title"> Preparação antes da microinjeção: </p><ol><li> Mosquitos alimentam de sangue: para injeção de segunda a quarta-feira fêmeas alimentam na sexta-feira anterior. Para injetar quinta-feira e sexta-feira, as fêmeas se alimentam de segunda-feira da mesma semana. <br /><br /></li><li> Prepare as agulhas de quartzo usando o programa 2 e tampão isotônica (veja Materiais). <br /><br /></li><li> &quot;Deitado tubo&quot; para os embriões é o frasco de cultura regulares Drosophila. Algodão molhado no fundo, com um disco d...

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<td>Injection Quartz needles</td>
<td>&nbsp;</td>
<td>Sutter Instrument</td>
<td>&nbsp;</td>
<td>O.D.- 1.0 mm, I.D.-0.70, 10 cm length, are prepared by drawing capillary tubing into a fine 3-8 um with a shaft of approximately 100-300 mm micropipette puller. </td>
</tr>
<tr>
<td>Quartz puller</td>
<td>Tool</td>
<td>Sutter Instrument Co</td>
<td>P-2000</td>
<td>Set-up: Heat-275, Fil-3, Vel-38, Del-250, Pull-141</td>
</tr>
<tr>
<td>EndoFree plasmid kit</td>
<td>&nbsp;</td>
<td>Quiagen</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Isopropanol</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Millex-GV column</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>SLGV RO4 NL</td>
<td>for DNA cleaning</td>
</tr>
<tr>
<td>Construct DNA</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>0.5 mg/ml </td>
</tr>
<tr>
<td>Helper plasmid</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>0.3 mg/ml </td>
</tr>
<tr>
<td>Injection solution</td>
<td>Buffer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>1x solution: 5mM KCl, 0.1mM sodium phosphate, ph 6. 8</td>
</tr>
<tr>
<td>Isotonic buffer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>see recipe in the protocol section</td>
</tr>
<tr>
<td>Dessication oil</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Halocarbon oil 700:Halocarbon oil 27(1:1). Mix well. Prepare in advance of microinjection.</td>
</tr>
<tr>
<td>Blood</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>to feed mosquitos</td>
</tr>
<tr>
<td>Anopheles stephensi</td>
<td>Animal</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Mosquitos, male and female.</td>
</tr>
<tr>
<td>Drosophila culture vial</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>for egg laying</td>
</tr>
<tr>
<td>Petri dishes</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>with isotonic buffer, for embryo hatching</td>
</tr>
<tr>
<td>Fine paintbrush</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Sable # 0000</td>
</tr>
<tr>
<td>3MM Whatman paper</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Forceps #5</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>double sided tape</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Microloader</td>
<td>&nbsp;</td>
<td>Eppendorf</td>
<td>5242 956.003</td>
<td>&nbsp;</td>
<tr>
<td>Transjector</td>
<td>&nbsp;</td>
<td>Eppendorf</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Microscope</td>
<td>&nbsp;</td>
<td>Leica</td>
<td>&nbsp;</td>
<td>with moving stage and  micromanipulator, set at 10x magnification </td>
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injection of an. stephensi embryos to generate malaria-resistant mosquitoes

<p class="jove_content">A introdução de genes exógenos em genomas de mosquitos exige técnicas de microinjeção sob medida para as espécies de interesse específicos. Este protocolo de vídeo demonstra um método utilizado pelo laboratório de James para microinject constrói DNA em embriões de Anopheles stephensi para a geração de mosquitos transformada. Técnicas de preparação agulhas microinjeção, coleta e preparação de embriões e na execução da microinjeção são ilustradas.</p>

Watch this Scientific Journal Video about Injection of An. stephensi Embryos to Generate Malaria-resistant Mosquitoes at JoVE.com

Injection of An. stephensi Embryos to Generate Malaria-resistant Mosquitoes

<p class="jove_content">Mosquitos Anopheles stephensi são vetores da malária e da Índia que habitam em toda a Ásia. Este vídeo demonstra a técnica para a realização de microinjeções desta espécie com transgenes que conferem resistência à malária para o mosquito. Grande parte da metodologia demonstrada neste vídeo é aplicável às técnicas de microinjeção de outras espécies de mosquito.</p>

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