著者らはdysfusion cDNAを、Dys抗体とw 1118ハエのためにスティーブンクルーズを感謝したいと思います。

1。胚のコレクション<ol><li> 25℃ケージをセットアップ° 2から4日齢のワット1118 flies.Grapeジュースプレートを使用してCは、コレクションの前に1〜2日間にわたって卵のコレクションの同期をとるために日中時間ごとに変更されています</li><li> 25℃で1時間のために胚を集める° C </li><li>かみそりの刃を途中で軽くカットグレープジュース寒天の長方形の部分を、カット、寒天に線を残す</li><li>グレープジュース寒天のあなたの部分にブドウジュースのプレートから胚を転送するために金属製のプローブを使用して、寒天の真ん中の線に対して45度の角度で、胚の長軸と直線でそれらを並べる。約50胚の直線を取得します。ラインからポインティング後端と、すべての胚のポイント同じ方向を確認してください。 </li><li>ダブルスティックテープの長方形の部分をカットし、18x18mmのカバースリップに置きます。 </li><li>カバースリップ上でダブルスティックテープで胚を拾う</li><li>ガラスのスライドにカバースリップを置き、約10分間空気中で胚を乾燥させる。 </li><li> 700ハロカーボンのオイルの薄い層で胚をカバーする、今では注射の準備ができています。 </li></ol> 2。ニードルを引いてガラスキャピラリーチューブを引いて、マイクロインジェクションの針を作る。我々は、世界の精密機器（モデルPUL - 1）からニードルプラーを使用。針の引き手のプログラムの仕様は以下のとおりです。熱：1、ディレイ4。 3。針を充填エッペンドルフMicroloaderチップを搭載したエッペンドルフP20のピペットを使用して、バックロードの針は、通常、1.5〜2μLdsRNAをロードする 4。針を破る<ol><li>針を壊す前に、スライドにカバースリップを配置。 </li><li>スライドの真ん中にオイルのドロップでスライドを準備する</li><li>エア圧を調整するPicospritzer III picopumpをオンに</li><li>鋭いポイントを作成し、カバースリップの端に満たされた注射針を破る。 </li><li>先端が切断されると、Picospritzer III picopumpのフットペダルでのステップは、いくつかのdsRNAは、針の先端部から外れリークが発生します。 </li><li>カバーグラスとスライドを取り出してください。 </li><li>前準備、スライドの中央にある油滴の下に針を挿入します。 </li><li>あなたがフットペダルを踏む時に100 200pl dsRNAを得るためにPicospritzer III picopumpに設定を調整します。 dsRNAは、小さな液滴として見ることができます。 </li></ol> 5。注射<ol><li>同じ焦点面への最初の胚盤葉の胚の後方部分に針の先端を持参し、30〜50％、卵の長さの周りに中心から離れた位置に胚を注入する。 </li><li>あなたがPicospritzer III picopumpのフットペダルを踏むの後、dsRNAの少量の注射部位に小さなドットとして表示されます。 </li></ol> 6。表現型分析<ol><li>胚は、目的の胚の段階に発展するまで注入した後、18日にカバー湿室（プラスチックシャーレ）° Cにスライドを配置する。 </li><li>両面テープから胚を除去し、カバースリップの端にそれらをプッシュするプローブを使用してください。 ヘプタンを用いて下部のナイロンメッシュ付きコレクションバイアルにスライドから胚を洗浄、PBTで3回洗浄する。 Dechorionate 5分間50％の漂白剤の胚、とはPBTで3回洗浄する。 コレクションバイアルからナイロンメッシュの部分に胚を転送し、胚を解放するために固定溶液（5ミリリットルヘプタン、4.5ミリリットルPBS、0.5ミリリットルの37％ホルムアルデヒド）にナイロンメッシュを浸し。 30分のための固定液に胚を修正。 エッペンドルフチュー​​ブに胚を転送し、標準プロトコルを使用して胚を免疫染色、メタノールで胚をDevitellinize。 </li><li>胚はまた、適切な幼虫期に開発することができます。 </li><li>真ん中のハロカーボン700オイルのドロップでスライドを準備する</li><li>スライド一つ幼虫を転送する、上にカバースリップを入れて</li><li>明視野顕微鏡下で幼虫を観察。 </li></ol> 7。代表的な結果： dsRNAの例では、Fig1に示されているDrosophlia胚におけるdysfusion（dys）遺伝子をノックダウン。 GFP - dsRNAを注入胚ではネガティブコントロールです。 Dysは気管融合細胞で発現のbHLH PAS転写因子である。 GFP - dsRNAを注入された胚は、通常の気管の融合を示し、Dysタンパク質は、融合細胞（図1A）に存在しています。一方、悪dsRNAを注入された胚は、障害が発生した分岐の融合を示し、Dysタンパク質融合細胞（図1B）には存在しません。 dsRNAが胚は幼虫期に開発注入すると、悪dsRNAを注入された幼虫は、GFP - dsRNAを注入したネガティブコントロール（図1C）と比較して失敗した分岐の融合（Fig.1D）を示した。これらの結果は、 ショウジョウバエの胚にdsRNAを注入することにより内因性のdysの遺伝子の発現のノックダウン効果を示した<img src="/files/ftp_upload/2477/2477fig1.jpg" alt="図1" /> 図1。悪RNAiによるdys機能の除去は、気管融合dを明らかにefects。胚盤葉の胚（1118 W）はGFP - dsRNAが（ネガティブコントロール）または悪dsRNAをどちらかを注入し、気管の欠陥を測定した。 （A）ステージ16胚では、GFP - dsRNAを注射し、α- DysおよびMAb 2A12その汚れは、気管内腔で染色。目立つように融合している横方向のトランクは、示されている。矢印は横トランク悪正の融合細胞を指す。 （B）ステージ16胚では、悪dsRNAを注射し、α- DysおよびMAb 2A12で染色。胚は、横方向のトランクの融合（アスタリスクは対照胚における横幹の融合の正常な位置を示す）の欠如を示した。いいえ悪陽性細胞が悪RNAが効率的にDysのタンパク質を廃止したことを示す、観察されなかった。 GFP - dsRNA又は悪dsRNAをどちらかを含む第2齢幼虫（DのC）は気管の欠陥のための明視野顕微鏡で観察した。 （C）融合のサイトは、GFP - dsRNAを注入制御の幼虫のアスタリスクによって示されます。 （D）横方向のトランクが悪RNA注入幼虫の融合に失敗した、（*）は、横方向のトランクの融合の正常な位置を示している。

<ol>
	<li>St Johnston, D., Nusslein-Volhard, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+origin+of+pattern+and+polarity+in+the+Drosophila+embryo.">The origin of pattern and polarity in the Drosophila embryo.</a> Cell. 68, 201-219 (1992).</li><li>Venken, K. J., Bellen, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emerging+technologies+for+gene+manipulation+in+Drosophila+melanogaster.">Emerging technologies for gene manipulation in Drosophila melanogaster.</a> Nat. Rev. Genet. 6, 167-178 (2005).</li><li>Fire, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potent+and+specific+genetic+interference+by+double-stranded+RNA+in+Caenorhabditis+elegans.">Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans.</a> Nature. 391, 806-811 (1998).</li><li>Kennerdell, J. R., Carthew, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+dsRNA-mediated+genetic+interference+to+demonstrate+that+frizzled+and+frizzled+2+act+in+the+wingless+pathway.">Use of dsRNA-mediated genetic interference to demonstrate that frizzled and frizzled 2 act in the wingless pathway.</a> Cell. 95, 1017-1026 (1998).</li><li>Misquitta, L., Paterson, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+disruption+of+gene+function+in+Drosophila+by+RNA+interference+(RNA-i):+a+role+for+nautilus+in+embryonic+somatic+muscle+formation.">Targeted disruption of gene function in Drosophila by RNA interference (RNA-i): a role for nautilus in embryonic somatic muscle formation.</a> Proc. Natl. Acad. Sci. U. S. A. 96, 1451-1456 (1999).</li><li>Horowitz, A., Simons, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Branching+morphogenesis.">Branching morphogenesis.</a> Circ. Res. 103, 784-795 (2008).</li><li>Hogan, B. L., Kolodziej, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organogenesis:+molecular+mechanisms+of+tubulogenesis.">Organogenesis: molecular mechanisms of tubulogenesis.</a> Nat. Rev. Genet. 3, 513-523 (2002).</li><li>Ghabrial, A., Luschnig, S., Metzstein, M. M., Krasnow, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Branching+morphogenesis+of+the+Drosophila+tracheal+system.">Branching morphogenesis of the Drosophila tracheal system.</a> Annu. Rev. Cell Dev. Biol. 19, 623-647 (2003).</li><li>Jiang, L., Crews, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dysfusion+transcriptional+control+of+Drosophila+tracheal+migration,+adhesion,+and+fusion.">Dysfusion transcriptional control of Drosophila tracheal migration, adhesion, and fusion.</a> Mol. Cell. Biol. 26, 6547-6556 (2006).</li><li>Jiang, L., Crews, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Drosophila+dysfusion+basic+helix-loop-helix+(bHLH)-PAS+gene+controls+tracheal+fusion+and+levels+of+the+trachealess+bHLH-PAS+protein.">The Drosophila dysfusion basic helix-loop-helix (bHLH)-PAS gene controls tracheal fusion and levels of the trachealess bHLH-PAS protein.</a> Mol. Cell. Biol. 23, 5625-5637 (2003).</li></ol>

利害の衝突は宣言されません。

dsRNAの注入法の存在はここにショウジョウバエ気管開発における遺伝子機能の非常に敏感かつ迅速な分析が可能になります。このメソッドは、潜在的に他の組織や臓器の開発のための遺伝子機能の解析に適用することができます。

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		<th>Comment</th>
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<td>Halocarbon oil 700</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>H8898</td>
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<td>Picospritzer III picopump</td>
<td>&nbsp;</td>
<td>Parker Precision Fluidics</td>
<td>051-0500-900</td>
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<td>Micro-pipettes</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>21170M</td>
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<td>Microloaders</td>
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<td>Eppendorf</td>
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rnai interference by dsrna injection into drosophila embryos

遺伝学的スクリーニングは、複雑な生物学的プロセス1への洞察を得るための最も強力な方法の一つである。年間で遺伝子操作のための多くの改良とツールは、 ショウジョウバエ 2で利用可能となっている。すぐに二本鎖RNAをノックダウンするために線虫（Caenorhabditis elegans）における個々の遺伝子の活動を使用することができることFrieとメロ3で最初の発見の後、RNA干渉（RNAi）は、 ショウジョウバエ器官開発における遺伝子機能を解析するための強力な逆遺伝学的なアプローチを提供することが示された4、5。 肺、腎臓、肝臓、および血管系を含む多くの臓器は、輸送の重要な液体や気体6、7、その分岐した管状のネットワークで構成されています。 ショウジョウバエ気管形成の分析は、他の管状の臓器8の形態形成を研究するための優れたモデルシステムを提供します。バークレーショウジョウバエゲノムプロジェクトでは、気管系で発現される遺伝子の数百を明らかにした。管形成の分子細胞メカニズムを調べるために、課題は、気管の発生におけるこれらの遺伝子の役割を理解することです。ここでは、ノックダウン、個々の遺伝子発現へのショウジョウバエの胚にdsRNAをインジェクションの詳細な方法を説明した。私たちは正常にdsRNAを注射により内因性dysfusion（dys）遺伝子の発現をノックダウン。 Dysは気管融合細胞で発現のbHLH - PASタンパク質であり、そしてそれが気管分岐融合9、10が必要です。悪RNAiは、完全にdysの発現を排除し、気管の融合の欠陥をもたらした。この比較的単純な方法は、 ショウジョウバエのtissureと臓器の開発のためのrequried遺伝子を同定するためのツールが用意されています。

Watch this Scientific Journal Video about RNAi Interference by dsRNA Injection into Drosophila Embryos at JoVE.com

RNAi Interference by dsRNA Injection into Drosophila Embryos

RNA干渉は、遺伝子の機能を解析することは非常に効果的であることが証明されています<em&gt;ショウジョウバエ</em&gt;気管開発。ノックダウンの遺伝子発現にハエの胚にdsRNAを注入する江研究室で使用されている詳細なプロトコールが示されている。この手法はで組織や臓器の開発に必要なスクリーニングの遺伝子の可能性を秘めている<em&gt;ショウジョウバエ</em&gt;。

dsRNAをインジェクションによるRNAiの干渉にショウジョウバエ胚

Genetic screening is one of the most powerful methods available for gaining insights into complex biological process 1. Over the years many improvements ...

rnai-interference-by-dsrna-injection-into-drosophila-embryos

Research

JoVE Journal

Biology

RNAi Interference by dsRNA Injection into Drosophila Embryos

16.1K ビュー.  Oakland University. RNA干渉は、遺伝子の機能を解析することは非常に効果的であることが証明されていますショウジョウバエ気管開発。ノックダウンの遺伝子発現にハエの胚にdsRNAを注入する江研究室で使用されている詳細なプロトコールが示されている。この手法はで組織や臓器の開発に必要なスクリーニングの遺伝子の可能性を秘めているショウジョウ?...

ビデオ: RNAi Interference by dsRNA Injection into Drosophila Embryos

Injection of dsRNA into Female A. aegypti Mosquitos

Reverse genetic approaches have proven extremely useful for determining  which genes underly resistance to vector pathogens in mosquitoes.  This video protocol illustrates a method used by the James lab to inject dsRNA into female A. aegypti mosquitoes, which harbor the dengue virus.  The technique for calibrating injection needles, manipulating the injection setup, and injecting dsRNA into the thorax is illustrated.

Reverse genetic approaches have proven extremely useful for determining which genes underly resistance to vector pathogens in mosquitoes. This video protocol illustrates a method used by the James lab to inject dsRNA into female A. aegypti mosquitoes, which harbor the dengue virus. The technique for calibrating injection needles, manipulating the injection setup, and injecting dsRNA into the thorax is illustrated.

女性A.ネッタイシマカのモスキートへのdsRNAの注射

Reverse genetic approaches have proven extremely useful for determining  which genes underly resistance to vector pathogens in mosquitoes.  This video ...

生物学

Live Dissection of Drosophila Embryos: Streamlined Methods for Screening Mutant Collections by Antibody Staining

Drosophila embryos between stages 14 and 17 of embryonic development can be readily dissected to generate "fillet" preparations. In these preparations, the central nervous system runs down the middle, and is flanked by the body walls. Many different phenotypes have been examined using such preparations. In most cases, the fillets were generated by dissection of antibody-stained fixed whole-mount embryos. These "fixed dissections" have some disadvantages, however. They are time-consuming to execute, and it is difficult to sort mutant (GFP-negative) embryos from stocks in which mutations are maintained over GFP balancer chromosomes. Since 2002, our group has been conducting deficiency and ectopic expression screens to identify ligands for orphan receptors. In order to do this, we developed streamlined protocols for live embryo dissection and antibody staining of collections containing hundreds of balanced lines. We have concluded that it is considerably more efficient to examine phenotypes in large collections of stocks by live dissection than by fixed dissection. Using the protocol described here, a single trained individual can screen up to 10 lines per day for phenotypes, examining 4-7 mutant embryos from each line under a compound microscope. This allows the identification of mutations conferring subtle, low-penetrance phenotypes, since up to 70 hemisegments per line are scored at high magnification with a 40X water-immersion lens.

Live Dissection of Drosophila Embryos: Streamlined Methods for Screening Mutant Collections by Antibody Staining

We describe a streamlined protocol for generating "fillet" preparations of Drosophila embryos of specific genotypes. This protocol allows efficient execution of a variety of genetic screens. It also allows excellent visualization of structures in the late embryo.

のライブ解剖ショウジョウバエ胚：抗体染色によってミュータントコレクションをスクリーニングするための合理化手法

Drosophila embryos between stages 14 and 17 of embryonic development can be readily dissected to generate "fillet" preparations.  In these preparations, ...

In-vivo Centrifugation of Drosophila Embryos

A major strategy for purifying and isolating different types of intracellular organelles is to separate them from each other based on differences in buoyant density. However, when cells are disrupted prior to centrifugation, proteins and organelles in this non-native environment often inappropriately stick to each other. Here we describe a method to separate organelles by density in intact, living Drosophila embryos. Early embryos before cellularization are harvested from population cages, and their outer egg shells are removed by treatment with 50% bleach. Embryos are then transferred to a small agar plate and inserted, posterior end first, into small vertical holes in the agar. The plates containing embedded embryos are centrifuged for 30 min at 3000g. The agar supports the embryos and keeps them in a defined orientation. Afterwards, the embryos are dug out of the agar with a blunt needle. Centrifugation separates major organelles into distinct layers, a stratification easily visible by bright-field microscopy. A number of fluorescent markers are available to confirm successful stratification in living embryos. Proteins associated with certain organelles will be enriched in a particular layer, demonstrating colocalization. Individual layers can be recovered for biochemical analysis or transplantation into donor eggs. This technique is applicable for organelle separation in other large cells, including the eggs and oocytes of diverse species.

In-vivo Centrifugation of Drosophila Embryos

We describe a method to separate organelles by density in living Drosophila embryos. Embryos are embedded in agar and centrifuged. This technique yields reproducible separation of major organelles along the anterior-posterior embryo axis. This method facilitates colocalization experiments and yields organelle fractions for biochemical analysis and transplantation experiments.

ショウジョウバエの胚の生体内の遠心

A major strategy for purifying and isolating different types of intracellular organelles is to separate them from each other based on differences in ...

Bacterial Delivery of RNAi Effectors: Transkingdom RNAi

RNA interference (RNAi) represents a high effective mechanism for specific inhibition of mRNA expression. Besides its potential as a powerful laboratory tool, the RNAi pathway appears to be promising for therapeutic utilization. For development of RNA interference (RNAi)-based therapies, delivery of RNAi-mediating agents to target cells is one of the major obstacles. A novel strategy to overcome this hurdle is transkingdom RNAi (tkRNAi). This technology uses non-pathogenic bacteria, e.g. Escherichia coli, to produce and deliver therapeutic short hairpin RNA (shRNA) into target cells to induce RNAi. A first-generation tkRNAi-mediating vector, TRIP, contains the bacteriophage T7 promoter for expression regulation of a therapeutic shRNA of interest. Furthermore, TRIP has the Inv locus from Yersinia pseudotuberculosis that encodes invasin, which permits natural noninvasive bacteria to enter &beta;1-integrin-positive mammalian cells and the HlyA gene from Listeria monocytogenes, which produces listeriolysin O. This enzyme allows the therapeutic shRNA to escape from entry vesicles within the cytoplasm of the target cell. TRIP constructs are introduced into a competent non-pathogenic Escherichia coli strain, which encodes T7 RNA polymerase necessary for the T7 promoter-driven synthesis of shRNAs. A well-characterized cancer-associated target molecule for different RNAi strategies is ABCB1 (MDR1/P-glycoprotein, MDR1/P-gp). This ABC-transporter acts as a drug extrusion pump and mediates the "classical" ABCB1-mediated multidrug resistance (MDR) phenotype of human cancer cells which is characterized by a specific cross resistance pattern. Different ABCB1-expressing MDR cancer cells were treated with anti-ABCB1 shRNA expression vector bearing E. coli. This procedure resulted in activation of the RNAi pathways within the cancer cells and a considerable down regulation of the ABCB1 encoding mRNA as well as the corresponding drug extrusion pump. Accordingly, drug accumulation was enhanced in the pristine drug-resistant cancer cells and the MDR phenotype was reversed. By means of this model the data provide the proof-of-concept that tkRNAi is suitable for modulation of cancer-associated factors, e.g. ABCB1, in human cancer cells.

For development of RNA interference (RNAi)-based therapies, a novel strategy was developed, transkingdom RNAi (tkRNAi). This technology uses non-pathogenic bacteria to produce and deliver therapeutic short hairpin RNA (shRNA) into target cells. Here, tkRNAi was successfully applied for reversal of classical ABCB1-mediated multidrug resistance (MDR) of cancer cells.

RNAiのエフェクターの細菌配達：Transkingdom RNAiの

RNA interference (RNAi) represents a high effective mechanism for specific inhibition of mRNA expression. Besides its potential as a powerful laboratory ...

Intraperitoneal Injection into Adult Zebrafish

A convenient method for chemically treating zebrafish is to introduce the reagent into the tank water, where it will be taken up by the fish. However, this method makes it difficult to know how much reagent is absorbed or taken up per fish. Some experimental questions, particularly those related to metabolic studies, may be better addressed by delivering a defined quantity to each fish, based on weight. Here we present a method for intraperitoneal (IP) injection into adult zebrafish. Injection is into the abdominal cavity, posterior to the pelvic girdle. This procedure is adapted from veterinary methods used for larger fish. It is safe, as we have observed zero mortality. Additionally, we have seen bleeding at the injection site in only 5 out of 127 injections, and in each of those cases the bleeding was brief, lasting several seconds, and the quantity of blood lost was small. Success with this procedure requires gentle handling of the fish through several steps including fasting, weighing, anesthetizing, injection, and recovery. Precautions are required to minimize stress throughout the procedure. Our precautions include using a small injection volume and a 35G needle. We use Cortland salt solution as the vehicle, which is osmotically balanced for freshwater fish. Aeration of the gills is maintained during the injection procedure by first bringing the fish into a surgical plane of anesthesia, which allows slow operculum movements, and second, by holding the fish in a trough within a water-saturated sponge during the injection itself. We demonstrate the utility of IP injection by injecting glucose and monitoring the rise in blood glucose level and its subsequent return to normal. As stress is known to increase blood glucose in teleost fish, we compare blood glucose levels in vehicle-injected and non-injected adults and show that the procedure does not cause a significant rise in blood glucose.

We demonstrate intraperitoneal injection into adult zebrafish. We use a 10 &mu;l NanoFil microsyringe controlled by a Micro4 controller and UltraMicroPump III. This demonstration includes the use of cold water as an anesthetic.

アダルトゼブラフィッシュへの腹腔内注射

A convenient method for chemically treating zebrafish is to introduce the reagent into the tank water, where it will be taken up by the fish. However, ...

Upright Imaging of Drosophila Embryos

Several well-known morphogenetic gradients and cellular movements occur along the dorsal/ventral axis of the Drosophila embryo. However, the current techniques used to view such processes are somewhat limited. The following protocol describes a new technique for mounting fixed and labeled Drosophila embryos for coronal viewing with confocal imaging. This method consists of embedding embryos between two layers of glycerin jelly mounting media, and imaging jelly strips positioned upright. The first step for sandwiching the embryos is to make a thin bedding of glycerin jelly on a slide. Next, embryos are carefully aligned on this surface and covered with a second layer of jelly. After the second layer is solidified, strips of jelly are cut and flipped upright for imaging. Alternatives are described for visualizing the embryos depending upon the type of microscope stand to be used. Since all cells along the dorsal-ventral axis are imaged within a single confocal Z-plane, our method allows precise measurement and comparison of fluorescent signals without photobleaching or light scattering common to 3D reconstructions of longitudinally mounted embryos.

Upright Imaging of Drosophila Embryos

Here we present a mounting protocol for stained Drosophila embryos in an upright position that allows imaging of cross-sections using Confocal microscopy.

の直立イメージングショウジョウバエ胚

Several well-known morphogenetic gradients and cellular movements occur along the dorsal/ventral axis of the Drosophila embryo. However, the current ...

Primary Cell Cultures from Drosophila Gastrula Embryos

Here we describe a method for preparing and culturing primary cells dissociated from Drosophila gastrula embryos. In brief, a large amount of staged embryos from young and healthy flies are collected, sterilized, and then physically dissociated into a single cell suspension using a glass homogenizer. After being plated on culture plates or chamber slides at an appropriate density in culture medium, these cells can further differentiate into several morphologically-distinct cell types, which can be identified by their specific cell markers. Furthermore, we present conditions for treating these cells with double stranded (ds) RNAs to elicit gene knockdown. Efficient RNAi in Drosophila primary cells is accomplished by simply bathing the cells in dsRNA-containing culture medium. The ability to carry out effective RNAi perturbation, together with other molecular, biochemical, cell imaging analyses, will allow a variety of questions to be answered in Drosophila primary cells, especially those related to differentiated muscle and neuronal cells.

Primary Cell Cultures from Drosophila Gastrula Embryos

We provide a detailed protocol for preparing primary cells dissociated from Drosophila embryos. The ability to carry out the effective RNAi perturbation, together with other molecular, biochemical and cell imaging methods will allow a variety of questions to be addressed in Drosophila primary cells.

から初代細胞培養ショウジョウバエ原腸胚

Here we describe a method for preparing and culturing primary cells dissociated from Drosophila gastrula embryos.   In brief, a large amount of staged ...

Gene Transfer into Older Chicken Embryos by ex ovo Electroporation

The chicken embryo provides an excellent model system for studying gene function and regulation during embryonic development. In ovo electroporation is a powerful method to over-express exogenous genes or down-regulate endogenous genes in vivo in chicken embryos1. Different structures such as DNA plasmids encoding genes2-4, small interfering RNA (siRNA) plasmids5, small synthetic RNA oligos6, and morpholino antisense oligonucleotides7 can be easily transfected into chicken embryos by electroporation. However, the application of in ovo electroporation is limited to embryos at early incubation stages (younger than stage HH20 - according to Hamburg and Hamilton)8 and there are some disadvantages for its application in embryos at later stages (older than stage HH22 - approximately 3.5 days of development). For example, the vitelline membrane at later stages is usually stuck to the shall membrane and opening a window in the shell causes rupture of the vessels, resulting in death of the embryos; older embryos are covered by vitelline and allantoic vessels, where it is difficult to access and manipulate the embryos; older embryos move vigorously and is difficult to control the orientation through a relatively small window in the shell.
In this protocol we demonstrate an ex ovo electroporation method for gene transfer into chicken embryos at late stages (older than stage HH22). For ex ovo electroporation, embryos are cultured in Petri dishes9 and the vitelline and allantoic vessels are widely spread. Under these conditions, the older chicken embryos are easily accessed and manipulated. Therefore, this method overcomes the disadvantages of in ovo electroporation applied to the older chicken embryos. Using this method, plasmids can be easily transfected into different parts of the older chicken embryos10-12.

Gene Transfer into Older Chicken Embryos by ex ovo Electroporation

A method of gene transfer into chicken embryos at later incubation stages (older than Hamburger and Hamilton stage (HH) 22) is described. This method overcomes disadvantages of in ovo electroporation applied to older chicken embryos and is a useful technique to study gene function and regulation at older developmental stages.

して、古いニワトリ胚への遺伝子導入<em&gt; EX OVO</em&gt;エレクトロポレーション

The chicken embryo provides an  excellent model system for studying gene function and regulation during  embryonic development. In ovo electroporation is ...

RNAi Trigger Delivery into Anopheles gambiae Pupae

RNA interference (RNAi), a naturally occurring phenomenon in eukaryotic organisms, is an extremely valuable tool that can be utilized in the laboratory for functional genomic studies. The ability to knockdown individual genes selectively via this reverse genetic technique has allowed many researchers to rapidly uncover the biological roles of numerous genes within many organisms, by evaluation of loss-of-function phenotypes. In the major human malaria vector Anopheles gambiae, the predominant method used to reduce the function of targeted genes involves injection of double-stranded (dsRNA) into the hemocoel of the adult mosquito. While this method has been successful, gene knockdown in adults excludes the functional assessment of genes that are expressed and potentially play roles during pre-adult stages, as well as genes that are expressed in limited numbers of cells in adult mosquitoes. We describe a method for the injection of Serine Protease Inhibitor 2 (SRPN2) dsRNA during the early pupal stage and validate SRPN2 protein knockdown by observing decreased target protein levels and the formation of melanotic pseudo-tumors in SRPN2 knockdown adult mosquitoes. This evident phenotype has been described previously for adult stage knockdown of SRPN2 function, and we have recapitulated this adult phenotype by SRPN2 knockdown initiated during pupal development. When used in conjunction with a dye-labeled dsRNA solution, this technique enables easy visualization by simple light microscopy of injection quality and distribution of dsRNA in the hemocoel.

RNAi Trigger Delivery into Anopheles gambiae Pupae

RNA interference (RNAi) is an extremely valuable tool for uncovering gene function. However, the ability to target genes using RNAi during pre-adult stages is limited in the major human malaria vector Anopheles gambiae. We describe an RNAi protocol to reduce gene function via direct injection during pupal development.

RNAiのトリガ配信へ<em&gt;ハマダラカ</em&gt;蛹

RNA interference (RNAi), a naturally occurring phenomenon in eukaryotic organisms, is an extremely valuable tool that can be utilized in the laboratory ...

Effective Oral RNA Interference (RNAi) Administration to Adult Anopheles gambiae Mosquitoes

RNA interference has been a heavily utilized tool for reverse genetic analysis for two decades. In adult mosquitoes, double-stranded RNA (dsRNA) administration has been accomplished primarily via injection, which requires significant time and is not suitable for field applications. To overcome these limitations, here we present a more efficient method for robust activation of RNAi by oral delivery of dsRNA to adult Anopheles gambiae. Long dsRNAs were produced in Escherichia coli strain HT115 (DE3), and a concentrated suspension of heat-killed dsRNA-containing bacteria in 10% sucrose was offered on cotton balls ad-libitum to adult mosquitoes. Cotton balls were replaced every 2 days for the duration of the treatment. Use of this method to target doublesex (a gene involved in sex differentiation) or fork head (which encodes a salivary gland transcription factor) resulted in reduced target gene expression and/or protein immunofluorescence signal, as measured by quantitative Real-Time PCR (qRT-PCR) or fluorescence confocal microscopy, respectively. Defects in salivary gland morphology were also observed. This highly flexible, user-friendly, low-cost, time-efficient method of dsRNA delivery could be broadly applicable to target genes important for insect vector physiology and beyond.

Effective Oral RNA Interference RNAi Administration to Adult Anopheles gambiae Mosquitoes

The oral administration of dsRNA produced by bacteria, a delivery method for RNA interference (RNAi) that is routinely used in Caenorhabditis elegans, was successfully applied here to adult mosquitoes. Our method allows for robust reverse genetics studies and transmission-blocking vector studies without the use of injection.