マイクロインジェクションの事前準備<ol><li>血液の供給蚊：月曜日から前の木曜日に、金曜日のフィード女性の注射用。 </li><li>プログラム3または2を使用して、ガラス針を準備する（資料を参照）。 </li><li>胚のために管を敷設するショウジョウバエの培養バイアルです。それを覆うフィルター紙のウェットディスクと底に湿った脱脂綿、。 </li><li>一方の端に両面テープを貼り付けることによってプラスチックのカバースリップを準備します。それはカバースリップの端で終わるようにスリップをカバーするためにテープをトリミング。 </li><li> （資料を参照してください）​​乾燥のためのオイルを準備</li><li>注入後の胚を保つためにろ紙のウェットディスク（80ミリメートルワットマン）で覆われた底に湿った綿のウール、と150ミリリットルのプラスチックビーカー。 </li></ol>強制産卵のセットアップ。 <ol><li>アスピレーターの使用で60から10の血液供給の女性を収集し、湿った綿やろ紙でショウジョウバエ培養バイアルに移す。 </li><li>暗闇の中でinsectory条件に蚊を戻し、および1hと15分間卵をすることができます。 </li><li>大人はケージに飛ぶと胚とろ紙ディスクを削除できます。 </li><li>卵を整列するために、解剖範囲で行ってください。微細な鉗子第5または罰金絵筆を使って、薄暗い灰色の胚に灰色ピックアップし、水に浸した3MMワットマン濾紙の正方形の上の行に配置します。注射は、後極にあるとする必要があるため、すべての胚は、同じ向きになっている必要があります。胚の前端は、後部より少し幅が広いです。 （ぬれたものがテープに付着しない）それに乾燥ろ紙を押すことによって胚と濾紙を乾燥させる。卵を転送するには、両面テープを含むスライドを反転し、静かに卵に対して押してください。卵の後端は、両面テープの端に非常に近いことがあります。 </li><li> 1分約胚を乾す。室温で。彼らは、乾くとわずかにディンプルを開始します。 </li><li>さらに乾燥を防ぐために、ハロカーボンのオイルで乾燥胚をカバーしています。 </li></ol>胚のマイクロインジェクション<ol><li>良い注射の最も重要な側面は、（資料を参照してください）​​針の品質です。 </li><li> microloaderを使用して注入されるようにDNA溶液を使用してニードルを埋めます。非常に少ない注射液が必要です：1〜2μlを。 </li><li>注入時間（マヌー）と圧力（600）を制御Transjector、、と背圧（250）に針を接続してください。マイクロインジェクションは、× 10の倍率とマイクロマニピュレータで移動ステージ（ライカ）と顕微鏡を使用して実行されます。必要に応じて、調達顕微鏡ステージは4-5の顕微鏡スライドを積層することにより準備されることがあります。発生段階に胚を運ぶカバースリップを置きます。 endochorionの引き裂き防止するために水平に胚を注入し、浸透が後極になっているはず。注射のために、私は針が静止して保持し、顕微鏡のステージに移動。 </li><li>胚の体積の約1から5パーセントに相当する解決策、の0.2から0.5 NLを注入する。射出圧力と時間を調整することで注入量を制御します。わずかに湾曲した、乾燥胚はその仰々しい外観は、次の注射を取り戻す必要があります。 </li><li>微細な鉗子や細かいブラシを使ってカバースリップから胚を選択し、ウェットコットンやウェットフィルター紙とプラスチック製のビーカーの中に置いてください。ティッシュペーパーでビーカーをカバー。リム周りゴムバンドとした場所に保管し、4日間昆虫館条件に戻ります。 </li></ol>ポスト噴射<ol><li>四日後には、慎重に食品の少量の大きなプラスチックボックスに水で、ダウン胚側、胚の用紙を置き。胚は非常に長い期間にわたってハッチ：少なくとも4-5週間後に注入するまでの文化を捨てていない。 </li></ol>ノート 注入用DNA 注射のためのプラスミドDNA溶液をEndoFreeプラスミドキットを用いて単離した。 izopropanolで沈殿、右側の濃度で一緒に構築し、ヘルパープラスミドミックス。ペレットは射出バッファーで再懸濁する前に70％エタノールで洗浄した。マイレクス- GVのカラムを使用して注入する前に、きれいなDNA。

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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

このビデオでは、Nijole Jasinskieneは、デング熱のためのベクトルであるトランスジェニックネッタイシマカ蚊を生成するために採用の方法論を、示しています。正しく、マイクロインジェクションの針を準備する胚をdessicating、およびマイクロインジェクションを行うための手法が実証されています。

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

トランスジェニック蚊を取得するA.ネッタイシマカの胚のマイクロインジェクション

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 ビュー.  University of California, Irvine (UCI). このビデオでは、Nijole Jasinskieneは、デング熱のためのベクトルであるトランスジェニックネッタイシマカ蚊を生成するために採用の方法論を、示しています。正しく、マイクロインジェクションの針を準備する胚をdessicating、およびマイクロインジェクションを行うための手法が実証されています。

ビデオ: 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.

の注入。ハマダラカの胚は、マラリア耐性の蚊を生成する

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

生物学

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.

デング熱蚊の感染症（A.ネッタイシマカ）と感染症の表現型決定のためのプロトコル

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.

AEから中腸と唾液腺の解剖。ネッタイシマカ蚊

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.

より良い蚊の構築：A. gambiaeとA.ネッタイシマカ蚊のマラリアとデング熱の抵抗を使用可能にする遺伝子を特定

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.

マイクロインジェクションを使用して安定したトランスジェニック線虫の世代

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.

遺伝子機能を解析するためにゼブラフィッシュ胚のマイクロインジェクション

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.

モデル遺伝生物として使用するためのメダカ胚のマイクロインジェクション

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による遺伝子ノックダウンと<emインビボで&gt;</em大人の女性の&gt;利尿アッセイ<em&gt;ネッタイシマカ</em&gt;蚊

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.

トランスジェニックの世代<em&gt;ハイドラ</em&gt;胚マイクロインジェクションによる

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.

ネッタイシマカ蚊ボルバキアに感染維持

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