Preparazione in anticipo di microiniezione <ol><li> Zanzare alimentazione del sangue: per iniezione da Lunedi a Venerdì femmine alimentare sul precedente Giovedi. </li><li> Preparare gli aghi di vetro utilizzando il programma di 3 o 2 (vedi Materiali). </li><li> Posa tubo per gli embrioni è fiala cultura Drosophila. Cotone idrofilo bagnato in fondo, con un disco di carta da filtro umida che lo ricopre. </li><li> Preparare antiscivolo in plastica di copertura da attaccare a doppio lato del nastro ad una estremità. Tagliare il nastro per coprire scivolare in modo che termina al bordo del vetrino. </li><li> Preparare l&#39;olio per essiccazione (vedi Materiali) </li><li> Becher di 150 ml di plastica con un batuffolo di cotone umido nella parte inferiore, coperto con un disco di carta da filtro umida (80mm Whatman) per mantenere gli embrioni dopo l&#39;iniezione. </li></ol> Messa a punto di posa forzata <ol><li> Raccogliere 6-10 sangue femmine alimentate con l&#39;uso di un aspiratore e di trasferirli al flaconcino cultura Drosophila con cotone umido e carta da filtro. </li><li> Mettere le zanzare nuovamente dentro condizioni insectory al buio, e permettono di deporre le uova per 1h e 15min. </li><li> Lasciate che gli adulti volare nella gabbia e rimuovere il disco filtro di carta con gli embrioni. </li><li> Per allineare le uova, lo fanno in ambito dissezione. Utilizzando una pinza sottile n. 5 o un pennello fine, scegliere grigio al grigio-scure embrioni e sistemateli in linea su piazza di carta Whatman 3MM inzuppato d&#39;acqua. Tutti gli embrioni devono essere con lo stesso orientamento, in quanto l&#39;iniezione deve essere al polo posteriore. L&#39;estremità anteriore dell&#39;embrione è leggermente più larga della posteriore. Asciugare la carta da filtro con gli embrioni da pressatura a secco carta da filtro ad esso (le cose bagnate non si atterrà a nastro). Per trasferire le uova, invertire la diapositiva che contiene il nastro biadesivo e premere delicatamente contro le uova. L&#39;estremità posteriore delle uova devono essere molto vicino al bordo del nastro biadesivo. </li><li> Essiccare gli embrioni di circa 1 min. a temperatura ambiente. Cominciano a fossetta un po &#39;come si asciugano. </li><li> Coprire embrioni essiccati con olio Halocarbon per prevenire l&#39;essiccamento ulteriormente. </li></ol> Microiniezione degli embrioni <ol><li> L&#39;aspetto più importante di iniezione buona è la qualità dell&#39;ago (vedi Materiali). </li><li> Riempire un ago con la soluzione di DNA da iniettare utilizzando un microloader. Molto soluzione iniettabile poco è necessario: 1 a 2 microlitri. </li><li> Collegare l&#39;ago al Transjector, che controlla il tempo di iniezione (Manu) e pressione (600), e la contropressione (250). Microiniezione viene eseguita utilizzando un microscopio con un palco in movimento (Leica) in x 10 ingrandimenti e micromanipolatore. Se necessario, una fase microscopio può essere sollevato preparare impilando vetrini da microscopio 4-5. Posizionare il vetrino porta la embrioni sul palco rialzato. Iniettare embrioni orizzontalmente per evitare la lacerazione dei endochorion; penetrazione dovrebbe essere al polo posteriore. Per l&#39;iniezione, tengo l&#39;ago fermo e spostare lo stadio del microscopio. </li><li> Iniettare 0,2-0,5 nl di soluzione, corrispondenti a circa il 1-5% del volume dell&#39;embrione. Controllare il volume di iniezione regolando la pressione di iniezione e il tempo. La leggermente piegato, gli embrioni essiccato dovrebbero riprendere la turgida dopo l&#39;iniezione aspetto. </li><li> Scegliere gli embrioni dal vetrino con una pinza sottile o un pennello fine, e metterli in un bicchiere di plastica con cotone umido e bagnato carta da filtro. Coprire bicchiere con la carta velina. Conservare in luogo con elastico intorno al cerchio e ritorno a condizioni insectary per 4 giorni. </li></ol> Post-iniezione <ol><li> Quattro giorni dopo, con attenzione embrione di carta posto, embrione rivolto verso il basso, l&#39;acqua nella grande scatola di plastica con una piccola quantità di cibo. Gli embrioni schiudono in un periodo molto lungo: non buttare via la cultura fino ad almeno 4-5 settimane dopo l&#39;iniezione. </li></ol> Note DNA per l&#39;iniezione Soluzione iniettabile DNA plasmidico è stato isolato utilizzando EndoFree kit plasmide. Costruire e mescolare Helper plasmide insieme in giusta concentrazione, precipitato con izopropanol. Il pellet è stato lavato con etanolo al 70% prima di risospendere nel buffer di iniezione. Prima dell&#39;iniezione, il DNA pulito utilizzando Millex-GV colonna.

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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 questo video, Nijole Jasinskiene dimostra la metodologia utilizzata per generare transgenici zanzare Aedes aegypti, che sono vettori per la febbre dengue. Le tecniche per la corretta preparazione di aghi microiniezione, disidratanti embrioni, e l&#39;esecuzione di microiniezione sono dimostrati.

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

Microiniezione di A. aegypti embrioni per ottenere zanzare transgeniche

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

Research

JoVE Journal

Biology

15.2K Views.  University of California, Irvine (UCI). In questo video, Nijole Jasinskiene dimostra la metodologia utilizzata per generare transgenici zanzare Aedes aegypti, che sono vettori per la febbre dengue. Le tecniche per la corretta preparazione di aghi microiniezione, disidratanti embrioni, e l'esecuzione di microiniezione sono dimostrati.

Video: 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.

Iniezione di An. Gli embrioni stephensi per generare la malaria resistenti zanzare

The introduction of exogenous genes into the genomes of mosquitoes requires microinjection techniques tailored to the specific species of interest. This ...

Ricerca

Biologia

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

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 the virus in the mosquito tissues. This protocol is routinely used for studying mosquito-virus interactions, especially for identification of novel host factors that are able to determine vector competence. The entire experiment must be conducted in a BSL2 laboratory. Similar to Plasmodium falciparum infections, proper attire including gloves and lab coat must be worn at all times. After the experiment, all the materials that came in contact with the virus need to be treated with 75% ethanol and bleached before proceeding with normal washing. All other materials need to be autoclaved before discarding them.

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.

Protocollo per la dengue in Zanzare (A. aegypti) e Fenotipo Determinazione infezione

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 ...

Dissection of Midgut and Salivary Glands from Ae. 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 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.

Dissezione delle ghiandole salivari e midgut da Ae. aegypti zanzare

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.

Costruire un Mosquito migliore: l&#39;identificazione dei geni Abilitazione malaria e febbre dengue Resistenza in A. gambiae e A. aegypti zanzare

In this interview, George Dimopoulos focuses on the physiological mechanisms used by mosquitoes to combat Plasmodium falciparum and dengue virus ...

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.

Generazione di Stabile transgenici C. elegans Utilizzando Microiniezione

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.

Microiniezione di embrioni di zebrafish per analizzare la funzione del gene

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.

Microiniezione di embrioni Medaka per essere utilizzato come organismo modello genetico

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. 
The protocol is setup in two parts: the first demonstration illustrates how to construct a simple mosquito injection device and how to prepare and inject dsRNA into the thorax of mosquitoes for RNAi-mediated gene knockdown. The second demonstration illustrates how to determine excretion rates in mosquitoes using an in vivo bioassay.

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.

RNAi-mediato Knockdown Gene e<em&gt; In Vivo</em&gt; Saggio La diuresi in femmina adulta<em&gt; Aedes aegypti</em&gt; Le zanzare

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.

Generazione di transgenici<em&gt; Hydra</em&gt; Da Embryo microiniezione

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

Maintaining Aedes aegypti Mosquitoes Infected with Wolbachia

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.

Mantenimento di Aedes aegypti zanzare infettate con Wolbachia

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

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Subtitles

Microiniezione di A. aegypti embrioni per ottenere zanzare transgeniche