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"> Préparation à l&#39;avance de la micro-injection: </p><ol><li> Moustiques nourrissent de sang: pour l&#39;injection du lundi au mercredi femelles se nourrissent le vendredi précédent. Pour injecter jeudi et vendredi, les femelles se nourrissent le lundi de la même semaine. <br /><br /></li><li> Préparer les aiguilles à quartz utilisant le programme 2 et tampon isotonique (voir matériaux). <br /><br /></li><li> &quot;Pose du tube&quot; pour les embryons est le flacon de culture régulière drosophile. Coton humide ...

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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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<tr>
<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">L&#39;introduction de gènes exogènes dans les génomes des moustiques nécessite des techniques de micro-injection adaptés aux espèces d&#39;intérêt particulier. Ce protocole vidéo montre une méthode utilisée par le laboratoire de James microinject constructions d&#39;ADN dans des embryons stephensi Anopheles pour la génération de moustiques transformé. Les techniques de préparation des aiguilles de microinjection, la collecte et la préparation des embryons et l&#39;exécution des micro-injection sont illustrés.</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">Moustiques Anopheles stephensi sont des vecteurs de la malaria qui habitent en Inde et en Asie. Cette vidéo montre la technique pour effectuer des micro-injections de cette espèce avec des transgènes qui confèrent une résistance à la malaria au moustique. Une grande partie de la méthodologie démontrée dans cette vidéo est applicable à des techniques de micro-injection d&#39;autres espèces de moustiques.</p>

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