Preparación antes de la microinyección <ol><li> Los mosquitos la sangre de alimentación: para la inyección de lunes a viernes en hembras se alimentan el jueves anterior. </li><li> Prepare las agujas de vidrio mediante el programa de 3 o 2 (ver Materiales). </li><li> Por la que se tubo para los embriones de Drosophila vial cultura. Algodón húmedo en la parte inferior, con un disco de papel de filtro húmedo que lo cubre. </li><li> Prepare cubreobjetos de plástico adhesiva de doble cara de la cinta a un extremo. Cinta de ajuste para cubrir deslizamiento de forma que termine en el borde del cubreobjetos. </li><li> Preparar el aceite para la desecación (ver Materiales) </li><li> 150 ml vaso de plástico con un algodón húmedo en la parte inferior, cubierto con un disco de papel de filtro húmedo (80 mm Whatman) para mantener los embriones después de la inyección. </li></ol> Puesta en marcha de por la que se obligó <ol><li> Recoger 60-10 hembras alimentadas con sangre con el uso de un aspirador y transferirlos a la cultura vial Drosophila con algodón húmedo y papel de filtro. </li><li> Puesto que los mosquitos de nuevo en condiciones insectory en la oscuridad, y permitir a poner huevos durante 1 hora y 15 minutos. </li><li> Permiten a los adultos vuelan en la jaula y retire el disco de papel filtro con embriones. </li><li> Para alinear los huevos, lo hacen bajo microscopio de disección. Con unas pinzas finas N º 5 o un pincel fino, de color gris a recoger oscuro-gris embriones y los arreglos en línea en la plaza de papel Whatman 3MM empapado de agua. Todos los embriones debe estar en la misma orientación, como la inyección tiene que estar en el polo posterior. El extremo anterior del embrión es ligeramente más ancha que la posterior. Seque el papel de filtro con embriones pulsando un papel de filtro seco para (ropa mojada no se pegue a la cinta). Para transferir los huevos, invertir la diapositiva que contiene la cinta de doble cara y presione suavemente contra los huevos. El extremo posterior de los huevos tienen que estar muy cerca de la orilla de la cinta de doble cara. </li><li> Desecar los embriones de 1 min. a temperatura ambiente. Empiezan a hoyuelo poco cuando se secan. </li><li> Cubrir los embriones desecados con aceite para evitar la desecación de halocarbonos más. </li></ol> Microinyección de los embriones <ol><li> El aspecto más importante de la inyección es la calidad de la aguja (ver Materiales). </li><li> Llenará una aguja con la solución de ADN que se inyecta mediante una microloader. Muy solución inyectable que se necesita poco: 1 a 2 l. </li><li> Conecte la aguja a la Transjector, que controla el tiempo de inyección (Manu) y presión (600), y la contrapresión (250). Microinyección se realizó con un microscopio con una etapa de movimiento (Leica) en x 10 aumentos y un micromanipulador. Si es necesario, una platina del microscopio puede ser levantado por el apilamiento preparar diapositivas 5.4 microscopio. Coloque la hoja de la cubierta lleva los embriones en etapa levantada. Inyectar embriones horizontal para evitar la ruptura de la endochorion, la penetración debe ser en el polo posterior. Para la inyección, que mantener la aguja fija y mover la platina del microscopio. </li><li> Inyectan 0.2-0.5 nl de solución, que corresponde a aproximadamente 1.5% del volumen del embrión. Controlar el volumen de inyección mediante el ajuste de la presión de inyección y el tiempo. El se inclinó ligeramente, los embriones desecados debe recuperar su turgencia después de la aparición de la inyección. </li><li> Recoger los embriones de la hoja de la cubierta con unas pinzas finas o un pincel fino, y colocarlos en un recipiente de plástico con algodón húmedo y papel de filtro húmedo. Cubrir vaso con papel de seda. Mantenga en su lugar con una banda elástica alrededor de la llanta y el retorno a las condiciones de insectario para 4 días. </li></ol> Después de la inyección <ol><li> Cuatro días después, se coloca cuidadosamente el papel del embrión, el embrión hacia abajo, sobre el agua en la caja grande de plástico con una pequeña cantidad de comida. Embriones eclosionan en un periodo muy largo: no se deshaga de la cultura por lo menos hasta 4-5 semanas después de la inyección. </li></ol> Notas ADN para la inyección Solución de ADN plásmido para la inyección fue aislado utilizando el kit plásmido EndoFree. Construir y mezclar plásmido ayudante juntos en la concentración de la derecha, se precipitó con izopropanol. El sedimento se lavó con etanol al 70% antes de resuspender en tampón de inyección. Antes de la inyección, el ADN limpia utilizando Millex-GV columna.

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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

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.

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

Microinyección de A. aegypti embriones para obtener los mosquitos transgénicos

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

15.1K 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

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.

La inyección de una. Los embriones stephensi para generar la malaria resistente a los mosquitos

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

Investigación

Biología

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.

Protocolo para las infecciones de dengue en mosquitos (Aedes aegypti) y determinación de fenotipo infección

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.

La disección de las glándulas salivales y del intestino medio de Ae. aegypti

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.

La construcción de un mosquito mejor: La identificación de los genes que permiten resistencia a la malaria y el dengue en A. gambiae y A. aegypti

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.

Generación de transgénicos estable C. elegans con microinyección

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.

Microinyección de embriones de pez cebra para analizar la función génica

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.

Microinyección de embriones de medaka para su uso como un organismo modelo genético

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 mediada por Gene y Derribo<em&gt; En Vivo</em&gt; Ensayo de diuresis en Mujeres Adultas<em&gt; Aedes aegypti</em&gt; Los mosquitos

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.

Generación de transgénicos<em&gt; Hydra</em&gt; Por microinyección de embriones

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.

Mantener los mosquitos Aedes aegypti infectados con Wolbachia

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