トルク補償器の元の設計はカールゲッツに由来する。このプレゼンテーションで特定のセットアップは、ローンの大部分であり、もともとマーティンハイゼンベルクとラインハルトWolfによって開発されました。私は、引き続きご支援、励ましや専門知識のためのこの二人に特に感謝します。

培地を飛ばすフライ食品の組成は、（。Guoら、1996）学習のための非常に重要です。 <ul><li>水千ミリリットル</li><li>コー​​ンミール180グラム</li><li>大豆10グラム</li><li>酵母18.5グラム</li><li>寒天7.5グラム</li><li>糖蜜40グラム</li><li>シロップ（砂糖大根）40グラム</li><li> Nipagin 2.5グラム</li></ul>すべてのバイアルは、新鮮な、生きた酵母ペーストのDABとハエと蛹のための追加の表面を提供するために、フィルタ一枚の紙が付属しています。 繁殖とステージングを飛ぶ次の手順を正確に適切な密度で栽培した動物を段階的につながって、毎日実行されます。前日の最後の手順以降のすべての新しくecclosedハエは、繁殖や実験のために収集されます。残りの生きている蛹のない古いバイアルは廃棄されます。 4日目ハエが一晩卵の堆積のための新鮮なバイアルに追加されます。女性のハエの密度は約20各バイアル、バイアルのサイズを調整する必要がありますし、ひずみが使用。理想的な密度は、すべての幼虫が最初にハエのecclose前pupatedているように幼虫期の間にフライ媒体が液化するための十分に高いと十分に低いものです。前日から産卵ハエが削除され、破棄されます。 準備を飛ぶ 25℃で上記のようにハエは12時間light/12hr暗い政権との° C、60％の湿度標準のコーンミール/糖蜜培地に保持されます。簡単に冷麻酔によって24 - 48古いハエを固定化した後、ハ​​エが三角形の形をした銅製のフックに頭部と胸部に（瞬間接着剤UVガラス接着剤、505127A、ペーサーテクノロジー、クーカモンガ、カリフォルニア州。、米国）に接着されています（直径0.05ミリメートル実験の前に）一日。動物はその後、ショ糖のいくつかの粒子を含む小さな湿った室で一晩個別に保持されます。 装置セットアップのコアデバイスは、トルク補償（トルクメータ）（ゲッツ、1964）である。それは、意図された飛行の操縦に起因する垂直体軸周りのフライの角運動量を、測定します。前述のようにフックに接着ハエが、、均一に背後から照らされている円筒状のパノラマ（アリーナ、直径58ミリメートル）、の中心で静止飛行を達成するためにクランプを介してトルクメータに接続されています。光源は100W、12Vタングステン - ヨウ素電球です。アリーナの緑と青の照明の場合、光は単色広帯域コダックラッテンゼラチンフィルター（それぞれ＃47、＃99、）を介して渡されます。フィルタは、0.1秒以内の高速ソレノイドで交換することができます。また、アリーナは青緑色フィルター（ロスコ&quot;surfblue&quot;号5433）、またはまったくフィルターを通すことで、&quot;昼光&quot;で照らされている。本研究で使用したロスコ青緑色フィルタの透過スペクトルは、BG18フィルタ（Schott社、マインツ）と同等であり、コダックブルーとグリーンフィルター（Brembsとヘンペルデイバラ、2006年の間に中間体を構成し、Liuら、1999）。アリーナにはコンピュータ制御の電気モーターを使用してフライの周りに回転することができます。このような&quot;フライトシミュレータ&quot;状況では、アリーナの角速度に比例ですが、（係数K =- 11 ° /秒•10 - 10nmのカップリング）フライのヨートルクに対して向け。これは、パノラマを安定化させ、その角度方向を制御するためにハエが可能になります。この仮想の&quot;飛行の方向&quot;（すなわち、競技場の位置）は円形のポテンショメータ（Novotechnik、A4102a306）を経由して継続的に記録されます。デジタルコンバータカードにアナログ（PCL812、アドバンテック株式会社）は、後で分析するためのトレースを（周波数20Hzのサンプリング）を格納するコンピュータに競技場の位置とヨーのトルク信号を供給する。罰は、ハエの頭部と胸部に背後と上から指示さ調節可能な赤外線レーザー（825ナノメートル、150 mW）を、熱を適用することによって実現されます。レーザービームは、パルス（〜4Hzの時約200msのパルス幅）と、その強度はハエの生存を保証するために削減されます。 実験学習パターン伝統的なパターン学習の実験用（ディルとハイゼンベルク、1995;ディルら、1993、1995;。Liuら、2006;。Liuら、1998;。Liuら、1999;。ウルフとハイゼンベルク、1991） 、向きを交互に4つの黒、T字型のパターンは（すなわち、直立2つ、2つの逆方向）に均等に競技場の壁（パターン幅として見られるψ= 40 °、バーの高さはθ= 40 °、幅= 14 °、上離間しているフライの位置から）。コンピュータプログラムは、4つの仮想90 °の象限に競技場の360 °を分割する、のセンターは、パターンで示されます。ハエは、ヨートルク（フライトシミュレータの状況）とパターンの角度位置を制御します。訓練中に、熱の罰は、前頭葉視野内のパターンの方向の一つの出現と連続されています。各パターンの強化常にグループ内で均一化される。試験中に、熱は永久にオフになり、フライのパターンの優先順位は記録されます。 色の学習色の学習は、前に説明したように実行されます（Brembsとハイゼンベルク、2000; Brembsとヘンペルデイバラ、2006; Brembsとウィーン、2006;ウルフとハイゼンベルク、1997）。アリーナは、4つの仮想90 °の象限に分割されている、のセンターは4つの同一の縦縞（幅ψ= 14 °、高さθ= 40 °）で表されます。上記のT字型のパターンについて説明したようにフライはそのヨートルクを持つ4つの同一のストライプの角度位置を制御しています。仮想象限の境界線の一つは、ハエの目の前のポイントを通過するたびに、全体の競技場の照明の色が変更されます。訓練中に、熱の罰は、2色のいずれかに偶発されています。テスト中に、熱は、永久にオフになり、フライの色の好みが記録されます。もちろん、色は、化合物のコンディショニングのためのパターン（Brembsとハイゼンベルク、2001）と組み合わせることができます。 ヨートルクの学習ヨートルクの学習は、（;ハイゼンベルクやウルフ、1993 Brembsとハイゼンベルク、2000）前述のように実行されます。ハエの自発的なヨートルク範囲は、左または右巻きにおよそ対応する&quot;左&quot;と&quot;右&quot;のドメインに分割されている。アリーナの壁には模様はありません。訓練中に、熱がフライのヨートルクが1つのドメインにされるたびに適用し、トルクが他に通過するときにオフになっています。テストフェーズでは、熱は、永久にオフになり、ヨートルクドメインのフライの選択は記録されます。 複合学習 （Brembsとハイゼンベルク、2000）の前に説明したように総合学習では、ヨートルクの学習を拡張したものです。基本的には、ヨートルクの学習と色の学習は、同等のオペラント（ヨートルク）と予測（色）古典的な実験で結合されます。訓練中に、フライはフライのヨートルクは罰に関連付けられているドメインに通過するたびに加熱される。フライだけでなく、温度だけでなく、アリーナの着色（緑から青またはその逆に）変更され、ヨートルクドメインを切り替えたとき。このように、ヨートルクのドメインと色は、熱の等価な予測因子として機能する。テストフェーズでは、熱は、永久にオフになり、ヨートルクのドメイン/色の唯一のフライの選択が記録されます。 ディスカッションこの実験では、高度な遺伝的モデル生物での実験状況を見渡す見事なコントロールを兼ね備えています。このプレゼンテーションで説明する手順を使用して、行動特性の様々な分子と神経生物学的基盤は、を含め、調査に限定されないことができる、自発行動生成のメカニズムは、オペラントと古典的なコンディショニング、パターン認識、色覚やコースのコントロール。

<ol>
	<li>Guo, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conditioned+visual+flight+orientation+in+Drosophila;+Dependence+on+age,+practice+and+diet.">Conditioned visual flight orientation in Drosophila; Dependence on age, practice and diet.</a> Learn. Mem.. 3, 49-59 (1996).</li><li>Goetz, K. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optomotorische+Untersuchung+des+visuellen+Systems+einiger+Augenmutanten+der+Fruchtfliege+Drosophila.">Optomotorische Untersuchung des visuellen Systems einiger Augenmutanten der Fruchtfliege Drosophila.</a> Kybernetik. 2, 77-92 (1964).</li><li>Liu, L., Wolf, R., Ernst, R., Heisenberg, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Context+generalization+in+Drosophila+visual+learning+requires+the+mushroom+bodies.">Context generalization in Drosophila visual learning requires the mushroom bodies.</a> Nature. 400, 753-756 (1999).</li><li>Brembs, B., Hempel de Ibarra, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Different+parameters+support+generalization+and+discrimination+learning+in+Drosophila+at+the+flight+simulator.">Different parameters support generalization and discrimination learning in Drosophila at the flight simulator.</a> Learn. Mem. 13, 629-637 (2006).</li><li>Liu, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+memory+traces+for+two+visual+features+in+the+Drosophila+brain.">Distinct memory traces for two visual features in the Drosophila brain.</a> Nature. 439, 551-556 (2006).</li><li>Wolf, R., Heisenberg, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basic+organization+of+operant+behavior+as+revealed+in+Drosophila+flight+orientation.">Basic organization of operant behavior as revealed in Drosophila flight orientation.</a> J. Comp. Physiol. A Neuroethol. Sens. Neural. Behav. Physiol. 169, 699-705 (1991).</li><li>Liu, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conditioned+visual+flight+orientation+in+Drosophila+melanogaster+abolished+by+benzaldehyde.">Conditioned visual flight orientation in Drosophila melanogaster abolished by benzaldehyde.</a> Pharmacol Biochem Behav. 61, 349-355 (1998).</li><li>Dill, M., Heisenberg, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visual+pattern+memory+without+shape+recognition.">Visual pattern memory without shape recognition.</a> Philos. Trans. R. Soc. Lond. B. Biol. Sci. 349, 143-152 (1995).</li><li>Dill, M., Wolf, R., Heisenberg, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visual+pattern+recognition+in+Drosophila+involves+retinotopic+matching.">Visual pattern recognition in Drosophila involves retinotopic matching.</a> Nature. 365, 751-753 (1993).</li><li>Dill, M., Wolf, R., Heisenberg, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavioral+analysis+of+Drosophila+landmark+learning+in+the+flight+simulator.">Behavioral analysis of Drosophila landmark learning in the flight simulator.</a> Learn. Mem. 2, 152-160 (1995).</li><li>Wolf, R., Heisenberg, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visual+Space+from+Visual+Motion:+Turn+Integration+in+Tethered+Flying+Drosophila.">Visual Space from Visual Motion: Turn Integration in Tethered Flying Drosophila.</a> Learn. Mem. 4, 318-327 (1997).</li><li>Brembs, B., Heisenberg, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+operant+and+the+classical+in+conditioned+orientation+in+Drosophila+melanogaster+at+the+flight+simulator.">The operant and the classical in conditioned orientation in Drosophila melanogaster at the flight simulator.</a> Learn. Mem. 7, 104-115 (2000).</li><li>Brembs, B., Wiener, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Context+generalization+and+occasion+setting+in+Drosophila+visual+learning.">Context generalization and occasion setting in Drosophila visual learning.</a> Learn. Mem. 13, 618-628 (2006).</li><li>Brembs, B., Heisenberg, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conditioning+with+compound+stimuli+in+Drosophila+melanogaster+in+the+flight+simulator.">Conditioning with compound stimuli in Drosophila melanogaster in the flight simulator.</a> J Exp Biol. 204, 2849-2859 (2001).</li><li>Heisenberg, M., Wolf, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+sensory-motor+link+in+motion-dependent+flight+control+of+flies.">The sensory-motor link in motion-dependent flight control of flies.</a> Rev. Oculomot. Res. 5, 265-283 (1993).</li></ol>

The authors have nothing to disclose.

この実験では、高度な遺伝的モデル生物での実験状況を見渡す見事なコントロールを兼ね備えています。このプレゼンテーションで説明する手順を使用して、行動特性の様々な分子と神経生物学的基盤は、含めて、調査がこれらに限定されないが、自発的な行動生成のメカニズムは、オペラントと古典的条件付け、パターン認識、色覚やコースのコントロール。

operant learning of drosophila at the torque meter

トルクメータでの実験のために、ハエは25℃、標準フライ培地° Cおよび60％の12時間light/12hr暗い政権と湿度に保持されます。標準化された繁殖体制は適切な幼虫の密度と年齢をマッチさせたコホートを保証します。冷麻酔ハエは、三角形の形をしたフック実験前日に頭部と胸部で接着されています。クランプを介してトルクメータに接続され、フライの意図する飛行操縦は、その垂直の体軸周りの角運動量として測定されています。フライは、静止飛行を達成するために円筒状のパノラマの中央に配置されます。デジタルコンバータカードへのアナログは、後で解析するためにトレースを格納するコンピュータにヨートルク信号を供給する。コンピュータは、これらの刺激とヨートルクトレースの間にフィードバックループを閉じることにより、ハエの制御下に置くことができるさまざまな刺激を制御します。刑罰は、調整可能な赤外レーザーからの熱を適用することによって実現されます。

Watch this Scientific Journal Video about Operant Learning of Drosophila at the Torque Meter at JoVE.com

Operant Learning of Drosophila at the Torque Meter

トルクメータでテザーショウジョウバエのヨートルクを測定することは実験動物の刺激の状況の神経科学者絶妙なコントロールが可能になります。一緒にショウジョウバエで利用可能なユニークな遺伝的なツールで、このパラダイムは、神経生物学研究の幅広いさまざまな目的で使用されます。

トルクメーターでのショウジョウバエのオペラント学習

For experiments at the torque meter, flies are kept on standard fly medium at 25&deg;C and 60% humidity with a 12hr light/12hr dark regime. A standardized ...

operant-learning-of-drosophila-at-the-torque-meter

Research

JoVE Journal

Biology

13.4K ビュー.  Free University of Berlin. トルクメータでテザーショウジョウバエのヨートルクを測定することは実験動物の刺激の状況の神経科学者絶妙なコントロールが可能になります。一緒にショウジョウバエで利用可能なユニークな遺伝的なツールで、このパラダイムは、神経生物学研究の幅広いさまざまな目的で使用されます。

ビデオ: Operant Learning of Drosophila at the Torque Meter

Loading Drosophila Nerve Terminals with Calcium Indicators

Calcium plays many roles in the nervous system but none more impressive than as the trigger for neurotransmitter release, and none more profound than as the messenger essential for the synaptic plasticity that supports learning and memory. To further elucidate the molecular underpinnings of Ca2+-dependent synaptic mechanisms, a model system is required that is both genetically malleable and physiologically accessible. Drosophila melanogaster provides such a model. In this system, genetically-encoded fluorescent indicators are available to detect Ca2+ changes in nerve terminals. However, these indicators have limited sensitivity to Ca2+ and often show a non-linear response. Synthetic fluorescent indicators are better suited for measuring the rapid Ca2+ changes associated with nerve activity. Here we demonstrate a technique for loading dextran-conjugated synthetic Ca2+ indicators into live nerve terminals in Drosophila larvae. Particular emphasis is placed on those aspects of the protocol most critical to the technique's success, such as how to avoid static electricity discharges along the isolated nerves, maintaining the health of the preparation during extended loading periods, and ensuring axon survival by providing Ca2+ to promote sealing of severed axon endings. Low affinity dextran-conjugated Ca2+-indicators, such as fluo-4 and rhod, are available which show a high signal-to-noise ratio while minimally disrupting presynaptic Ca2+ dynamics. Dextran-conjugation helps prevent Ca2+ indicators being sequestered into organelles such as mitochondria. The loading technique can be applied equally to larvae, embryos and adults.

Calcium is a ubiquitous messenger in the nervous system, essential for triggering neurotransmitter release and changes in synaptic strength. Here we demonstrate a technique for loading Ca2+-indicators into Drosophila nerve terminals. We also demonstrate fabrication of the required apparatus and emphasize points critical for the technique's success.

カルシウムインジケータ付きのロードショウジョウバエの神経終末

Calcium plays many roles in the nervous system but none more impressive than as the trigger for neurotransmitter release, and none more profound than as ...

生物学

Channelrhodopsin2 Mediated Stimulation of Synaptic Potentials at Drosophila Neuromuscular Junctions

The Drosophila larval neuromuscular preparation has proven to be a useful tool for studying synaptic physiology1,2,3. Currently, the only means available to evoke excitatory junctional potentials (EJPs) in this preparation involves the use of suction electrodes. In both research and teaching labs, students often have difficulty maneuvering and manipulating this type of stimulating electrode. In the present work, we show how to remotely stimulate synaptic potentials at the larval NMJ without the use of suction electrodes. By expressing channelrhodopsin2 (ChR2) 4,5,6 in Drosophila motor neurons using the GAL4-UAS system 7, and making minor changes to a basic electrophysiology rig, we were able to reliably evoke EJPs with pulses of blue light. This technique could be of particular use in neurophysiology teaching labs where student rig practice time and resources are limited.

Channelrhodopsin2 Mediated Stimulation of Synaptic Potentials at Drosophila Neuromuscular Junctions

This procedure uses a blue light-activated algal channel and cell-specific genetic expression tools to evoke synaptic potentials with light pulses at the neuromuscular junction (NMJ) in Drosophila larvae. This technique is an inexpensive and easy-to-use alternative to suction electrode stimulation for synaptic physiology studies in research and teaching laboratories.

シナプス電位のChannelrhodopsin2仲介刺激でショウジョウバエ神経筋接合部

The Drosophila larval neuromuscular preparation has proven to be a useful tool for studying synaptic physiology1,2,3. Currently, the only means available ...

Fluorescent Labeling of Drosophila Heart Structures

The Drosophila melanogaster dorsal vessel, or heart, is a tubular structure comprised of a single layer of contractile cardiomyocytes, pericardial cells that align along each side of the heart wall, supportive alary muscles and, in adults, a layer of ventral longitudinal muscle cells. The contractile fibers house conserved constituents of the muscle cytoarchitecture including densely packed bundles of myofibrils and cytoskeletal/submembranous protein complexes, which interact with homologous components of the extracellular matrix. Here we describe a protocol for the fixation and the fluorescent labeling of particular myocardial elements from the hearts of dissected larvae and semi-intact adult Drosophila. Specifically, we demonstrate the labeling of sarcomeric F-actin and of &alpha;-actinin in larval hearts. Additionally, we perform labeling of F-actin and &alpha;-actinin in myosin-GFP expressing adult flies and of &alpha;-actinin and pericardin, a type IV extracellular matrix collagen, in wild type adult hearts. Particular attention is given to a mounting strategy for semi-intact adult hearts that minimizes handling and optimizes the opportunity for maintaining the integrity of the cardiac tubes and the associated tissues. These preparations are suitable for imaging via fluorescent and confocal microscopy. Overall, this procedure allows for careful and detailed analysis of the structural characteristics of the heart from a powerful genetically tractable model system.

Fluorescent Labeling of Drosophila Heart Structures

Here we describe a basic protocol for fluorescent labeling of different elements of heart tubes from larva and adult Drosophila melanogaster. These specimens are well-suited for imaging via fluorescent or confocal microscopy. This technique permits detailed structural analysis of the features of the hearts from a powerful model organism.

の蛍光標識ショウジョウバエハートの構造

The Drosophila melanogaster dorsal vessel, or heart, is a tubular structure comprised of a single layer of contractile cardiomyocytes, pericardial cells ...

In vivo Imaging of Intact Drosophila Larvae at Sub-cellular Resolution

Recent improvements in optical imaging, genetically encoded fluorophores and genetic tools allowing efficient establishment of desired transgenic animal lines have enabled biological processes to be studied in the context of a living, and in some instances even behaving, organism. In this protocol we will describe how to anesthetize intact Drosophila larvae, using the volatile anesthetic desflurane, to follow the development and plasticity of synaptic populations at sub-cellular resolution1-3. While other useful methods to anesthetize Drosophila melanogaster larvae have been previously described4,5,6,7,8, the protocol presented herein demonstrates significant improvements due to the following combined key features: (1) A very high degree of anesthetization; even the heart beat is arrested allowing for lateral resolution of up to 150 nm1, (2) a high survival rate of &gt; 90% per anesthetization cycle, permitting the recording of more than five time-points over a period of hours to days2 and (3) a high sensitivity enabling us in 2 instances to study the dynamics of proteins expressed at physiological levels. In detail, we were able to visualize the postsynaptic glutamate receptor subunit GluR-IIA expressed via the endogenous promoter1 in stable transgenic lines and the exon trap line FasII-GFP1. (4) In contrast to other methods4,7 the larvae can be imaged not only alive, but also intact (i.e. non-dissected) allowing observation to occur over a number of days1. The accompanying video details the function of individual parts of the in vivo imaging chamber2,3, the correct mounting of the larvae, the anesthetization procedure, how to re-identify specific positions within a larva and the safe removal of the larvae from the imaging chamber.

In vivo Imaging of Intact Drosophila Larvae at Sub-cellular Resolution

This protocol describes a reliable method for anesthetization and imaging of intact Drosophila melanogaster larvae. We have utilized the volatile anesthetic desflurane to allow for repetitive imaging at sub-cellular resolution and re-identification of structures for up to a few days1.

サブセルラー解像度で手つかずのショウジョウバエの幼虫の in vivoイメージングで

Recent improvements in optical imaging, genetically encoded fluorophores and genetic tools allowing efficient establishment of desired transgenic animal ...

Quantitative Comparison of cis-Regulatory Element (CRE) Activities in Transgenic Drosophila melanogaster

Gene expression patterns are specified by cis-regulatory element (CRE) sequences, which are also called enhancers or cis-regulatory modules. A typical CRE possesses an arrangement of binding sites for several transcription factor proteins that confer a regulatory logic specifying when, where, and at what level the regulated gene(s) is expressed. The full set of CREs within an animal genome encodes the organism&prime;s program for development1, and empirical as well as theoretical studies indicate that mutations in CREs played a prominent role in morphological evolution2-4. Moreover, human genome wide association studies indicate that genetic variation in CREs contribute substantially to phenotypic variation5,6. Thus, understanding regulatory logic and how mutations affect such logic is a central goal of genetics. 
Reporter transgenes provide a powerful method to study the in vivo function of CREs. Here a known or suspected CRE sequence is coupled to heterologous promoter and coding sequences for a reporter gene encoding an easily observable protein product. When a reporter transgene is inserted into a host organism, the CRE&prime;s activity becomes visible in the form of the encoded reporter protein. P-element mediated transgenesis in the fruit fly species Drosophila (D.) melanogaster7 has been used for decades to introduce reporter transgenes into this model organism, though the genomic placement of transgenes is random. Hence, reporter gene activity is strongly influenced by the local chromatin and gene environment, limiting CRE comparisons to being qualitative. In recent years, the phiC31 based integration system was adapted for use in D. melanogaster to insert transgenes into specific genome landing sites8-10. This capability has made the quantitative measurement of gene and, relevant here, CRE activity11-13 feasible. The production of transgenic fruit flies can be outsourced, including phiC31-based integration, eliminating the need to purchase expensive equipment and/or have proficiency at specialized transgene injection protocols. 
Here, we present a general protocol to quantitatively evaluate a CRE&prime;s activity, and show how this approach can be used to measure the effects of an introduced mutation on a CRE&prime;s activity and to compare the activities of orthologous CREs. Although the examples given are for a CRE active during fruit fly metamorphosis, the approach can be applied to other developmental stages, fruit fly species, or model organisms. Ultimately, a more widespread use of this approach to study CREs should advance an understanding of regulatory logic and how logic can vary and evolve.

Quantitative Comparison of cis-Regulatory Element CRE Activities in Transgenic Drosophila melanogaster

Phenotypic variation for traits can result from mutations in cis-regulatory element (CRE) sequences that control gene expression patterns. Methods derived for use in Drosophila melanogaster can quantitatively compare the levels of spatial and temporal patterns of gene expression mediated by modified or naturally occurring CRE variants.

の定量的比較シス</emトランスジェニックで> -調節エレメント（CRE）の活動キイロショウジョウバエ</em

Gene expression patterns are specified by cis-regulatory element (CRE) sequences, which are also called enhancers or cis-regulatory modules. A typical CRE ...

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

The proper elimination of unwanted or aberrant cells through apoptosis and subsequent phagocytosis (apoptotic cell clearance) is crucial for normal development in all metazoan organisms. Apoptotic cell clearance is a highly dynamic process intimately associated with cell death; unengulfed apoptotic cells are barely seen in vivo under normal conditions. In order to understand the different steps of apoptotic cell clearance and to compare 'professional' phagocytes - macrophages and dendritic cells to 'non-professional' - tissue-resident neighboring cells, in vivo live imaging of the process is extremely valuable. Here we describe a protocol for studying apoptotic cell clearance in live Drosophila embryos. To follow the dynamics of different steps in phagocytosis we use specific markers for apoptotic cells and phagocytes. In addition, we can monitor two phagocyte systems in parallel: 'professional' macrophages and 'semi-professional' glia in the developing central nervous system (CNS). The method described here employs the Drosophila embryo as an excellent model for real time studies of apoptotic cell clearance.

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

Here we describe an effective method for studying dynamics of apoptotic cell clearance in vivo. This method employs live Drosophila embryos as a powerful model for monitoring phagocytosis of apoptotic cells using specific labeling of apoptotic cells and phagocytes.

アポトーシス細胞のクリアランスのイメージング中に生きる<em&gt;ショウジョウバエ</em&gt;胚

The  proper elimination of unwanted or aberrant cells through apoptosis and  subsequent phagocytosis (apoptotic cell clearance) is crucial for normal  ...

The MultiBac Protein Complex Production Platform at the EMBL

Proteomics research revealed the impressive complexity of eukaryotic proteomes in unprecedented detail. It is now a commonly accepted notion that proteins in cells mostly exist not as isolated entities but exert their biological activity in association with many other proteins, in humans ten or more, forming assembly lines in the cell for most if not all vital functions.1,2 Knowledge of the function and architecture of these multiprotein assemblies requires their provision in superior quality and sufficient quantity for detailed analysis. The paucity of many protein complexes in cells, in particular in eukaryotes, prohibits their extraction from native sources, and necessitates recombinant production. The baculovirus expression vector system (BEVS) has proven to be particularly useful for producing eukaryotic proteins, the activity of which often relies on post-translational processing that other commonly used expression systems often cannot support.3 BEVS use a recombinant baculovirus into which the gene of interest was inserted to infect insect cell cultures which in turn produce the protein of choice. MultiBac is a BEVS that has been particularly tailored for the production of eukaryotic protein complexes that contain many subunits.4 A vital prerequisite for efficient production of proteins and their complexes are robust protocols for all steps involved in an expression experiment that ideally can be implemented as standard operating procedures (SOPs) and followed also by non-specialist users with comparative ease. The MultiBac platform at the European Molecular Biology Laboratory (EMBL) uses SOPs for all steps involved in a multiprotein complex expression experiment, starting from insertion of the genes into an engineered baculoviral genome optimized for heterologous protein production properties to small-scale analysis of the protein specimens produced.5-8 The platform is installed in an open-access mode at EMBL Grenoble and has supported many scientists from academia and industry to accelerate protein complex research projects.

Protein complexes catalyze key cellular functions. Detailed functional and structural characterization of many essential complexes requires recombinant production. MultiBac is a baculovirus/insect cell system particularly tailored for expressing eukaryotic proteins and their complexes. MultiBac was implemented as an open-access platform, and standard operating procedures developed to maximize its utility.

EMBLでMultiBacタンパク質複合体生産プラットフォーム

Proteomics  research revealed the impressive complexity of eukaryotic proteomes in  unprecedented detail. It is now a commonly accepted notion that ...

Recombineering Homologous Recombination Constructs in Drosophila

The continued development of techniques for fast, large-scale manipulation of endogenous gene loci will broaden the use of Drosophila melanogaster as a genetic model organism for human-disease related research. Recent years have seen technical advancements like homologous recombination and recombineering. However, generating unequivocal null mutations or tagging endogenous proteins remains a substantial effort for most genes. Here, we describe and demonstrate techniques for using recombineering-based cloning methods to generate vectors that can be used to target and manipulate endogenous loci in vivo. Specifically, we have established a combination of three technologies: (1) BAC transgenesis/recombineering, (2) ends-out homologous recombination and (3) Gateway technology to provide a robust, efficient and flexible method for manipulating endogenous genomic loci. In this protocol, we provide step-by-step details about how to (1) design individual vectors, (2) how to clone large fragments of genomic DNA into the homologous recombination vector using gap repair, and (3) how to replace or tag genes of interest within these vectors using a second round of recombineering. Finally, we will also provide a protocol for how to mobilize these cassettes in vivo to generate a knockout, or a tagged gene via knock-in. These methods can easily be adopted for multiple targets in parallel and provide a means for manipulating the Drosophila genome in a timely and efficient manner.

Recombineering Homologous Recombination Constructs in Drosophila

Homologous recombination techniques greatly advance Drosophila genetics by enabling the creation of molecularly precise mutations. The recent adoption of recombineering allows one to manipulate large pieces of DNA and transform them into Drosophila6. The methods presented here combine these techniques to rapidly generate large homologous recombination vectors.

における相同組換えコンストラクトを遺伝子改変<em&gt;ショウジョウバエ</em

The continued development of techniques for fast, large-scale manipulation of endogenous gene loci will broaden the use of Drosophila melanogaster as a ...

Culturing of Human Nasal Epithelial Cells at the Air Liquid Interface

In vitro models using human primary epithelial cells are essential in understanding key functions of the respiratory epithelium in the context of microbial infections or inhaled agents. Direct comparisons of cells obtained from diseased populations allow us to characterize different phenotypes and dissect the underlying mechanisms mediating changes in epithelial cell function. Culturing epithelial cells from the human tracheobronchial region has been well documented, but is limited by the availability of human lung tissue or invasiveness associated with obtaining the bronchial brushes biopsies. Nasal epithelial cells are obtained through much less invasive superficial nasal scrape biopsies and subjects can be biopsied multiple times with no significant side effects. Additionally, the nose is the entry point to the respiratory system and therefore one of the first sites to be exposed to any kind of air-borne stressor, such as microbial agents, pollutants, or allergens. 
Briefly, nasal epithelial cells obtained from human volunteers are expanded on coated tissue culture plates, and then transferred onto cell culture inserts. Upon reaching confluency, cells continue to be cultured at the air-liquid interface (ALI), for several weeks, which creates more physiologically relevant conditions. The ALI culture condition uses defined media leading to a differentiated epithelium that exhibits morphological and functional characteristics similar to the human nasal epithelium, with both ciliated and mucus producing cells. Tissue culture inserts with differentiated nasal epithelial cells can be manipulated in a variety of ways depending on the research questions (treatment with pharmacological agents, transduction with lentiviral vectors, exposure to gases, or infection with microbial agents) and analyzed for numerous different endpoints ranging from cellular and molecular pathways, functional changes, morphology, etc. 
In vitro models of differentiated human nasal epithelial cells will enable investigators to address novel and important research questions by using organotypic experimental models that largely mimic the nasal epithelium in vivo.

Nasal epithelial cells, obtained through superficial scrape biopsy of human volunteers, are expanded and transferred onto tissue culture inserts. Upon reaching confluency, cells are grown at air liquid interface, yielding cultures of ciliated and non-ciliated cells. Differentiated nasal epithelial cell cultures provide viable experimental models for studying the respiratory mucosa.

エア·液界面での人間の鼻上皮細胞の培養

In vitro models using human primary  epithelial cells are essential in understanding key functions of the respiratory  epithelium in the context of ...

Dissection and Mounting of Drosophila Pupal Eye Discs

The Drosophila melanogaster eye disc is a powerful system that can be used to study many different biological processes. It contains approximately 800 separate eye units, termed ommatidia1. Each ommatidium contains eight neuronal photoreceptors that develop from undifferentiated cells following the passage of the morphogenetic furrow in the third larval instar2. Following the sequential differentiation of the photoreceptors, non-neuronal cells develop, including cone and pigment cells, along with mechanosensory bristle cells3. Final differentiation processes, including the structured arrangement of all the ommatidial cell types, programmed cell death of undifferentiated cell types and rhodopsin expression, occurs through the pupal phase4-7. This technique focuses on manipulating the pupal eye disc, providing insight and instruction on how to dissect the eye disc during the pupal phase, which is inherently more difficult to perform than the commonly dissected third instar eye disc. This technique also provides details on immunostaining to allow the visualization of various proteins and other cell components.

Dissection and Mounting of Drosophila Pupal Eye Discs

The goal of this technique is to enable researchers to perform dissection, immunostaining and mounting of pupal eye discs from Drosophila melanogaster of any age.

解剖と実装<em&gt;ショウジョウバエ</em&gt;蛹アイディスク

The Drosophila melanogaster eye disc is a powerful system that can be used to study many different biological processes.  It contains approximately 800 ...