Preparation in advance of microinjection
<ol>
<li>Blood feed mosquitoes: for injection from Monday to Friday feed females on the previous Thursday.</li>
<li>Prepare glass needles using program 3 or 2 (see Materials).</li>
<li>Laying tube for the embryos is Drosophila culture vial. Wet cotton wool in the bottom, with a wet disk of filter paper covering it.</li>
<li>Prepare plastic cover slip by sticking double-side tape to one end. Trim tape to cover slip so that it ends at edge of cover slip.</li>
<li>Prepare oil for desiccation (see Materials)</li>
<li>150 ml plastic beaker with a wet cotton wool in the bottom, covered with a wet disk of filter paper (80mm Whatman) to keep embryos after injection.</li>
</ol>
Set up of forced laying
<ol>
<li>Collect 6-10 blood fed females with the use of an aspirator and transfer them to the Drosophila culture vial with wet cotton and filter paper.</li>
<li>Put the mosquitoes back into insectory conditions in the dark, and allow to lay eggs for 1h and 15min.</li>
<li>Let adults fly into the cage and remove the filter paper disk with embryos.</li>
<li>To line up eggs, do so under dissecting scope. Using fine forceps No. 5 or a fine paintbrush, pick gray to darkish-grey embryos and arrange in line on square of 3MM Whatman paper soaked with water. All embryos must be in the same orientation, as injection has to be at the posterior pole. The anterior end of the embryo is slightly wider than the posterior. Dry the filter paper with embryos by pressing dry filter paper to it (wet things won't stick to tape). To transfer the eggs, invert the slide containing the double-sided tape and gently press against the eggs. The posterior end of the eggs have to be very close to the edge of the double sided tape.</li>
<li>Desiccate the embryos about 1 min. at room temperature. They start to dimple slightly as they dry.</li>
<li>Cover desiccated embryos with halocarbon oil to prevent further desiccation.</li>
</ol>
Microinjection of the embryos
<ol>
<li>The most important aspect of good injection is the quality of the needle (see Materials).</li>
<li>Fill a needle with the DNA solution to be injected by using a microloader. Very little injection solution is needed: 1 to 2 &mu;l.</li>
<li>Connect the needle to the Transjector, which controls the injection time (Manu) and pressure (600), and the backpressure (250). Microinjection is performed using a microscope with a moving stage (Leica) at x 10 magnification and micromanipulator. If necessary, a raised microscope stage may be prepare by stacking 4-5 microscope slides. Place the cover slip carrying the embryos onto raised stage. Inject embryos horizontally to prevent tearing of the endochorion; penetration should be at the posterior pole. For injection, I keep the needle stationary and move the stage of the microscope.</li>
<li>Inject 0.2-0.5 nl of solution, corresponding to approximately 1-5% of the embryo volume. Control the injection volume by adjusting the injection pressure and time. The slightly bowed, desiccated embryos should regain their turgid appearance following injection.</li>
<li>Pick embryos from the cover slip using fine forceps or a fine brush, and place them in a plastic beaker with wet cotton and wet filter paper. Cover beaker with tissue paper. Keep in place with elastic band around the rim and return to insectary conditions for 4 days.</li>
</ol>
Post-injection
<ol>
<li>Four days after, carefully place embryo paper, embryo-side down, on the water in large plastic box with a small amount of food. Embryos hatch over a very long period: don't throw out the culture until at least 4-5 weeks post-injection.</li>
</ol>
Notes
DNA for injection
Plasmid DNA solution for injection was isolated using EndoFree plasmid kit. Construct and Helper plasmid mix together in right concentration, precipitated with izopropanol. The pellet was washed with 70% ethanol before resuspending in injection buffer. Before injection, clean DNA using Millex-GV column.

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>1x Injection solution</td>
<td>Buffer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>5mM KCl, 0.1mM sodium phosphate, ph 6. 8</td>
</tr>
<tr>
<td>Construct plasmid</td>
<td>DNA</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>0.5 mg/ml. </td>
</tr>
<tr>
<td>Helper plasmid</td>
<td>DNA</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>0.3 mg/ml </td>
</tr>
<tr>
<td>EndoFree Plasmid kit</td>
<td>Kit</td>
<td>Qiagen</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>To clean DNA.</td>
</tr>
<tr>
<td>Glass puller</td>
<td>Instrument</td>
<td>Sutter Instrument Co</td>
<td>Model P-87</td>
<td>Heat-815, Pull-10, Vel-10, Time-100.
Heat-700, Pull-30, Vel-36, Time-50
</td>
</tr>
<tr>
<td>Quartz puller</td>
<td>Instrument</td>
<td>Sutter Instrument Co</td>
<td>Model P-2000</td>
<td>Heat-275, Fil-3, Vel-38, Del-250, Pull-141</td>
</tr>
<tr>
<td>Injection glass needles</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Prepared by drawing capillary tubing into a fine 3-8 um with a shaft of approximately 100-300 um micropipette puller. When the needle is new, the tip is sealed and usually breaks in the first or second injection or the glass needle tip can be beveled using a Brown type micro-pipette beveller (Sutter Instrument).</td>
</tr>
<tr>
<td>Aedes aegypti</td>
<td>Animal</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Male and female mosquitos, to obtain embryos.</td>
</tr>
<tr>
<td>Drosophila culture vial</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>For mosquito egg-laying.</td>
</tr>
<tr>
<td>Halocarbon oil</td>
<td>Reagent</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Oil for desiccation: Halocarbon oil 700:Halocarbon oil 27(1:1)</td>
</tr>
<tr>
<td>Forceps No5</td>
<td>Tool</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Fine paintbrush</td>
<td>&nbsp;</td>
<td>Sable</td>
<td>No. 0000</td>
<td>&nbsp;</td>
<tr>
<td>Whatman paper</td>
<td>Tool</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Microloader</td>
<td>Tool</td>
<td>Eppendorf</td>
<td>5242 956.003</td>
<td>&nbsp;</td>
<tr>
<td>Transjector</td>
<td>Tool</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.</td>
</tr>
<tr>
<td>3MM Whatman paper</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>		</tbody>
		</table>
		</div>
			<div>
					</div>

microinjection of a. aegypti embryos to obtain transgenic mosquitoes

In this video, Nijole Jasinskiene demonstrates the methodology employed to generate transgenic Aedes aegypti mosquitoes, which are vectors for dengue fever.  The techniques for correctly preparing microinjection needles, dessicating embryos, and performing microinjection are demonstrated.

Watch this Scientific Journal Video about Microinjection of A. aegypti Embryos to Obtain Transgenic Mosquitoes at JoVE.com

Microinjection of A. aegypti Embryos to Obtain Transgenic Mosquitoes

In this video, Nijole Jasinskiene demonstrates the methodology employed to generate transgenic Aedes aegypti mosquitoes, which are vectors for dengue fever. The techniques for correctly preparing microinjection needles, dessicating embryos, and performing microinjection are demonstrated.

In this video, Nijole Jasinskiene demonstrates the methodology employed to generate transgenic Aedes aegypti mosquitoes, which are vectors for dengue ...

microinjection-of-a-aegypti-embryos-to-obtain-transgenic-mosquitoes

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JoVE Journal

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15.2K vistas.  University of California, Irvine (UCI). En este video, Nijole Jasinskiene demuestra la metodología utilizada para generar transgénicos mosquito Aedes aegypti, que son vectores de la fiebre del dengue. Las técnicas para la correcta preparación de las agujas de microinyección, desecación embriones, y la realización de la microinyección se demuestran.

Video: Microinjection of A. aegypti Embryos to Obtain Transgenic Mosquitoes

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Transgenic Caenorhabditis elegans can be readily created via microinjection of a DNA plasmid solution into the gonad 1. The plasmid DNA rearranges to form extrachromosomal concatamers that are stably inherited, though not with the same efficiency as actual chromosomes 2. A gene of interest is co-injected with an obvious phenotypic marker, such as rol-6 or GFP, to allow selection of transgenic animals under a dissecting microscope. The exogenous gene may be expressed from its native promoter for cellular localization studies. Alternatively, the transgene can be driven by a different tissue-specific promoter to assess the role of the gene product in that particular cell or tissue. This technique efficiently drives gene expression in all tissues of C. elegans except for the germline or early embryo 3. Creation of transgenic animals is widely utilized for a range of experimental paradigms. This video demonstrates the microinjection procedure to generate transgenic worms. Furthermore, selection and maintenance of stable transgenic C. elegans lines is described.

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One of the advantages of studying zebrafish is the ease and speed of manipulating protein levels in the embryo.  Morpholinos, which are synthetic oligonucleotides with antisense complementarity to target RNAs, can be added to the embryo to reduce the expression of a particular gene product.  Conversely, processed mRNA can be added to the embryo to increase levels of a gene product.  The vehicle for adding either mRNA or morpholino to an embryo is microinjection.  Microinjection is efficient and rapid, allowing for the injection of hundreds of embryos per hour.  This video shows all the steps involved in microinjection.  Briefly, eggs are collected immediately after being laid and lined up against a microscope slide in a Petri dish.  Next, a fine-tipped needle loaded with injection material is connected to a microinjector and an air source, and the microinjector controls are adjusted to produce a desirable injection volume.  Finally, the needle is plunged into the embryo's yolk and the morpholino or mRNA is expelled.

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RNAi-mediated Gene Knockdown and In Vivo Diuresis Assay in Adult Female Aedes aegypti Mosquitoes

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Generation of Transgenic Hydra by Embryo Microinjection

Stably transgenic Hydra are made by microinjection of plasmid DNA into embryos followed by random genomic integration and asexual propagation to establish a uniform line. Transgenic Hydra are used to track cell movements, overexpress genes, study promoter function, or knock down gene expression using RNAi.

As a member of the phylum Cnidaria, the sister group to all bilaterians, Hydra can shed light on fundamental biological processes shared among ...

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