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 vues.  University of California, Irvine (UCI). Dans cette vidéo, Nijole Jasinskiene démontre la méthodologie employée pour générer transgéniques moustiques Aedes aegypti, vecteurs de la dengue. Les techniques pour la préparation correcte des aiguilles de microinjection, desséchants embryons, et effectuant la microinjection sont démontrés.

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

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

The introduction of exogenous genes into the genomes of mosquitoes requires microinjection techniques tailored to the specific species of interest. This video protocol demonstrates a method used by the James laboratory to microinject DNA constructs into Anopheles stephensi embryos for the generation of transformed mosquitoes. Techniques for preparing microinjection needles, collecting and preparing embryos and performing the microinjection are illustrated.

Anopheles stephensi mosquitoes are vectors for malaria inhabiting India and throughout Asia. This video demonstrates the technique for performing microinjections of this species with transgenes that will confer resistance to the malaria to the mosquito. Much of the methodology demonstrated in this video is applicable to microinjection techniques of other mosquito species.

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Protocol for Dengue Infections in Mosquitoes (A. aegypti) and Infection Phenotype Determination

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Protocol for Dengue Infections in Mosquitoes A. aegypti and Infection Phenotype Determination

Once a gene is identified as potentially refractory for the dengue virus, it must be evaluated for it's role in preventing viral infections within the mosquito. This protocol illustrates how the extent of dengue infections of mosquitoes can be assayed. The techniques for growing up the virus in culture, membrane feeding mosquitoes human blood, and assaying viral titers in the mosquito midgut are demonstrated.

The purpose of this procedure is to infect the Aedes mosquito with dengue virus in a laboratory condition and examine the infection level and dynamic of ...

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The mosquito midgut and salivary glands are key entry and exit points for pathogens such as Plasmodium parasites and Dengue viruses. This video protocol demonstrates dissection techniques for removal of the midgut and salivary glands from Aedes aegypti mosquitoes. 



The mosquito midgut and salivary glands are key entry and exit points for vector pathogens like Plasmodium falciparum and the dengue virus. This video demonstrates the dissection techniques for removing the midgut and salivary glands from Aedes aegypti mosquitoes.

The mosquito midgut and salivary glands are key entry and exit points for pathogens such as Plasmodium parasites and Dengue viruses. This video protocol ...

Building a Better Mosquito: Identifying the Genes Enabling Malaria and Dengue Fever Resistance in A. gambiae and A. aegypti Mosquitoes

In this interview, George Dimopoulos focuses on the physiological mechanisms used by mosquitoes to combat Plasmodium falciparum and dengue virus infections.   Explanation is given for how key refractory genes, those genes conferring resistance to vector pathogens, are identified in the mosquito and how this knowledge can be used to generate transgenic mosquitoes that are unable to carry the malaria parasite or dengue virus.

In this interview, George Dimopoulos focuses on the physiological mechanisms used by mosquitoes to combat Plasmodium falciparum and dengue virus infections. Explanation is given for how key refractory genes, those genes conferring resistance to vector pathogens, are identified in the mosquito and how this knowledge can be used to generate transgenic mosquitoes that are unable to carry the malaria parasite or dengue virus.

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Generation of Stable Transgenic C. elegans Using Microinjection

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.

This video demonstrates the technique of microinjection into the gonad of C. elegans to create transgenic animals.

Transgenic Caenorhabditis elegans can be readily created via microinjection of a DNA plasmid solution into the gonad 1.  The plasmid DNA rearranges to ...

Microinjection of Zebrafish Embryos to Analyze Gene Function

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.

This video shows how morpholino or mRNA can be injected into zebrafish embryos at the one-cell stage to decrease or increase the level of specific gene products during subsequent development.

One of the advantages of studying zebrafish is the ease and speed of manipulating protein levels in the embryo.  Morpholinos, which are synthetic ...

Microinjection of Medaka Embryos for use as a Model Genetic Organism

In this video, we demonstrate the technique of microinjection into one-cell stage medaka embryos. Medaka is a small egg-laying freshwater fish that allows both genetic and embryological analyses and is one of the vertebrate model organisms in which genome-wide phenotype-driven mutant screens were carried out 1, as in zebrafish and the mouse. Divergence of functional overlap of related genes between medaka and zebrafish allows identification of novel phenotypes that are unidentifiable in a single species 2, thus medaka and zebrafish are complementary for genetic dissection of vertebrate genome functions.
To take advantage of medaka fish whose embryos are transparent and develop externally, microinjection is an essential technique to inject cell-tracers for labeling cells, mRNAs or anti-sense oligonucleotides for over-expressing and knocking-down genes of interest, and DNAs for making transgenic lines.

Medaka and zebrafish are complementary for genetic dissection of vertebrate genome functions. This protocol highlights the key points for successful microinjection into medaka embryos, an important technique for embryological and genetic analysis using medaka and zebrafish in a laboratory.

In this video, we demonstrate the technique of microinjection into one-cell stage medaka embryos. Medaka is a small egg-laying freshwater fish that allows ...

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

This video protocol demonstrates an effective technique to knockdown a particular gene in an insect and conduct a novel bioassay to measure excretion rate. This method can be used to obtain a better understanding of the process of diuresis in insects and is especially useful in the study of diuresis in blood-feeding arthropods that are able to take up huge amounts of liquid in a single blood meal. 
This RNAi-mediated gene knockdown combined with an in vivo diuresis assay was developed by the Hansen lab to study the effects of RNAi-mediated knockdown of aquaporin genes on Aedes aegypti mosquito diuresis1. 
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RNAi-mediated Gene Knockdown and In Vivo Diuresis Assay in Adult Female Aedes aegypti Mosquitoes

In this protocol we combine RNAi-mediated gene silencing with an in vivo diuresis assay to study the effects knockdown of genes of interest has on mosquito fluid excretion.

This video protocol demonstrates an effective technique to knockdown a particular gene in an insect and conduct a novel bioassay to measure excretion ...

Generation of Transgenic Hydra by Embryo Microinjection

As a member of the phylum Cnidaria, the sister group to all bilaterians, Hydra can shed light on fundamental biological processes shared among multicellular animals. Hydra is used as a model for the study of regeneration, pattern formation, and stem cells. However, research efforts have been hampered by lack of a reliable method for gene perturbations to study molecular function. The development of transgenic methods has revitalized the study of Hydra biology1. Transgenic Hydra allow for the tracking of live cells, sorting to yield pure cell populations for biochemical analysis, manipulation of gene function by knockdown and over-expression, and analysis of promoter function. Plasmid DNA injected into early stage embryos randomly integrates into the genome early in development. This results in hatchlings that express transgenes in patches of tissue in one or more of the three lineages (ectodermal epithelial, endodermal epithelial, or interstitial). The success rate of obtaining a hatchling with transgenic tissue is between 10% and 20%. Asexual propagation of the transgenic hatchling is used to establish a uniformly transgenic line in a particular lineage. Generating transgenic Hydra is surprisingly simple and robust, and here we describe a protocol that can be easily implemented at low cost.

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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Aedes aegypti mosquitoes experimentally infected with Wolbachia are being utilized in programs to control the spread of arboviruses such as dengue, chikungunya and Zika. Wolbachia-infected mosquitoes can be released into the field to either reduce population sizes through incompatible matings or to transform populations with mosquitoes that are refractory to virus transmission. For these strategies to succeed, the mosquitoes released into the field from the laboratory must be competitive with native mosquitoes. However, maintaining mosquitoes in the laboratory can result in inbreeding, genetic drift and laboratory adaptation which can reduce their fitness in the field and may confound the results of experiments. To test the suitability of different Wolbachia infections for deployment in the field, it is necessary to maintain mosquitoes in a controlled laboratory environment across multiple generations. We describe a simple protocol for maintaining Ae. aegypti mosquitoes in the laboratory, which is suitable for both Wolbachia-infected and wild-type mosquitoes. The methods minimize laboratory adaptation and implement outcrossing to increase the relevance of experiments to field mosquitoes. Additionally, colonies are maintained under optimal conditions to maximize their fitness for open field releases.

Maintaining Aedes aegypti Mosquitoes Infected with Wolbachia

Aedes aegypti mosquitoes infected with Wolbachia are being released into natural populations to suppress the transmission of arboviruses. We describe methods to rear Ae. aegypti with Wolbachia infections in the laboratory for experiments and field release, taking precautions to minimize laboratory adaptation and selection.

Aedes aegypti mosquitoes experimentally infected with Wolbachia are being utilized in programs to control the spread of arboviruses such as dengue, ...