国立科学財団（MF）で提供される資金。

はじめ OMTは、&quot;仮想プルームシミュレーター&quot;を組み込むために設計された[1]磁気テザーシステムの適応です。次のプロトコルは、どのように適切にテザーのハエ（パート1）を説明し、磁石（パート2）とマスフロー規制臭気デリバリーシステム（その3）を設定するための戦略を提供します。ここで説明する情報は、この特定のシステム用に最適化されており、他の技術仕様を持つコンポーネントによって異なる場合があります。 パート1：テザリングのハエここで説明するテザリングをするための手順は、多くの先行研究と類似している[2]しかし、OMT法での使用に適合[3]。適切なテザリングは、堅牢で再現性の実験的試行を確保することが重要です。慎重にテザリングの重要性は誇張することはできません。 <ol><li> 4-6日の後の羽化を飛ぶ収集し、4〜6時間のためにそれらを飢えさせる。キムワイプで乾燥を防ぐために、食べ物のないバイアルに水で湿らせた置きます。 </li><li>小さなバイアルに瓶からハエのバッチを転送し、摂氏約4度で設定されたペルチェ冷却ステージ上に真鍮のブロックに挿入します。適切な温度は約30秒以内にハエをanesthetizes。 </li><li>冷却ステージの冷たい表面上のハエをソートします。湿らせたキムワイプはキューティクルから一定の冷却と水分の上がりを確実にします。テザリングのための最大の女性を選択してください。 </li><li>細かいブラシを使用して、単一の野生型雌ミバエを収容する形特注の石棺の位置に単一のフライを押してください。 <img src="/files/ftp_upload/old/1063_2008_11_26_2/1063_Figure-01_thumb.png" alt="" /> 図1。 <a href="https://www.jove.com/files/1063_2008_10_28_1/1063_Figure-01.png">図の拡大バージョンをご覧いただくにはこちらをクリックしてください。</a> </li><li>我々は、マイクロマニピュレータに取り付けられた磁気ロッドとminutienピン（罰金科学のツール）を置きます。ピンは、平滑末端がちょうど2つの翼の間に頭の後ろに背胸部に触れるように配置されることです。目に向かって正面から見たときにピンが完全に垂直でなければなりません。フライとピンの間に&quot;ロール&quot;のであっても少量を許容することはできません。ピンと体の長軸の間にサイドビューの角度は水平線（図1）に対して相対的に30度の機首アップの角度になっているはず。注：磁石のテザーで最適な動作では、ピンはケーブル長を短くするためにトリミングする必要があります。これにより、サスペンションの磁石間の距離に完全に依存します。 </li><li> minutienのピンが整列されると、minutienピンの平滑末端にUV -活性化接着剤（ElectroLiteコーポレーション）の小滴を適用するために細い鋼線を使用してください。 </li><li>接着剤の液滴がちょうど頭の上にハエの胸部に触れるようにピンを操作します。適切なドロップサイズで、接着剤は、アニールすべきと胸部の上にピンから流れます。あまりにも多くの接着剤を使用しないように注意してください。 </li><li> UV光の2つ20秒のバーストで接着剤を治す。 </li><li>固定フライを脇に置き、繰り返し。キムワイプの小さな四角をスライスし、鉗子で飛行を抑制するために、各係留飛行するものを与える。 </li><li>回復の一時間後に、テザーハエは、実験の準備が整いました。 </li><li>不適切な接着には、磁石の分野で実行するハエの能力を妨げる可能性があります。すべてのハエは、ハエが適切に接着されるように、すべての実験の開始時に滑らかな回転のためにチェックする必要があります。これは、強力な微細運動の応答を引き出すために十分に大きな空間的な周期で縞模様を回転させることによって達成することができます[4]。ハエが間違っピッチ角に接着またはロール軸を基準にしてピンに傾いていると、ハエ5月は、（i）の動きの制限された範囲を持っている、または（iii）一貫性のないで回転（ii）全く回転しない速度。上記のケースではハエを破棄し、新しいものに置き換えられます。この問題が解決した場合、それはおそらく不適切な接着のケースだった。 （フライを交換しても問題が解決しない場合は、第2.4参照） </li></ol>パート2：磁石の位置を揃える磁石（希土類- magnets.com）を揃えることが適切動きのスムーズな360度の範囲を達成するためにフライが必要である。適切に磁石を整列させるには、次の手順に従います。 <ol><li>下のリング磁石は、プラスチックスリーブで水平に保持し、明確な真空チャンバーの上に直接配置されます（図1、パート3で詳しく説明）。上部の棒磁石を下側磁石の上に垂直に約3 / 4&quot;固定されています。 </li><li>ほぼ直接下部リング磁石の中心点の上に上部の棒磁石を置くことによって視覚的に上下の磁石の位置を合わせます。それは、微細調整のためにマイクロマニピュレータ（シスキユー社）に、上部磁石を添付すると便利です。 </li><li>エポキシ回転摩擦を最小限にし、フライの位置を標準化するために、上部磁石の底面上にV -宝石軸受（スモールパーツ）。 </li><li>アリーナでフライを配置し、水平方向とvを調整するフライまで、上部磁石のertical位置は、円滑かつ着実にヨー軸に360度回転させることができます。すべてのハエは、磁石が適切に整列されており、個々のminutienピンは（パート1.11参照）損傷していないことを保証するためにすべての実験の開始時に滑らかな回転のためにチェックする必要があります。磁石が適切にフライを整列されていない場合5月は、（i）すべての（ⅱ）で回転しない動きの制限された範囲を（iii）の一貫性のない速度で回転する必要があります。問題は新しいものとフライを交換することによって解決されている場合、それは（パート1を参照）最も可能性の高い接着の問題によるものであった。これらの問題は、すべてのハエのために存在する場合、それは可能性が高い磁石とさらに調整が必要とされるのアラインメントの問題です。 </li><li>警告：楽しいことができる希土類磁石で遊んでいる間、彼らは非常に危険です。破損した機器及び/またはマイナー身体的危害の結果を悪用。これらの魅力的な、まだ簡単に過小評価して磁石では細心の注意を払ってください。 </li></ol>パート3：臭配信<ol><li>臭気の配信システムは、前述のいずれかに基づいている[5]および最適化するために過酷な部分（図1）である。それは忍耐と試行錯誤の多くを必要とします。安定した臭いの追跡を実現するためには、次の手順に従います。 </li><li>臭気のシステムの初期セットアップは、カスタム構築された水/臭バイアルへのガスマルチプレクサ（セーブルシステムズインターナショナル）から狭いゲージテフロンチューブ（スモールパーツ）を実行している必要があります。これらのバイアルの出口は、同じテフロンチューブを使用して皮下ポートのチューブに直接接続します。 </li><li>透明なアクリルの真空チャンバは、磁石をサポートするために、アリーナの下に配置されている。私たちは、ハエに近い開き真空を持参し、開口部の直径を減らすために、真空チャンバに接続されているフライの下にマウントさ4ミリメートルのガラス管を使用してください。 </li><li>鉗子を使用して舞台にテザーフライを置きます。 </li><li>魅力的な臭気（リンゴ酢がうまく機能）をオンにし、視覚的にハエが匂いを追跡することを確認してください。フライは、匂いに対する嗜好を持っていないように見える場合は、次の3つの手順に従ってください。これは、いくつかのハエ、そしていくつかの臨床試験が必要になる場合があります。 </li><li>フライは、匂いを追跡するために表示されるまで、臭気のポートの位置を調整します。我々は、マイクロマニピュレータ上に臭気のポートをマウント（シスキユー社）とハエの頭に4ミリメートル背側および3mm前方に配置されます。 </li><li>フライは、匂いを追跡するために表示されるまで、質量流量を調整します。私たちの設定が7ml/min（セーブルシステムズインターナショナルMFC - 4）です。 </li><li>フライは、匂いを追跡するために表示されるまで、真空の流量を調整します。我々は、フローレギュレータ（コールパーマー）を使用して13l/minで真空流量を設定する。注：建物の真空供給ラインは、プルームの構造の変動を引き起こして時間の経過とともに変動する可能性があります。それは一定の真空流量条件のフローレギュレータに外付けの真空ポンプを接続するために役に立つかもしれません。 </li><li>一度ポートの位置と流量が設定されているが、定期的にガスのマルチプレクサを使用して切替を使用し、視覚的にその場だけ臭いがオンになって臭いを追跡することを確認する水蒸気や臭気の蒸気を提供切り替える。時折、臭気のポート内部の残臭は臭いがオフになっている匂いの追跡を引き出すことができます。これは、エタノールで皮下注射管を充填し、キムワイプで配信終わりにそれを収集することにより適切な位置臭気のポートを削除せずに除去することができます。圧縮空気を使用して、残りのエタノールを吹き消すと実験を再開する。 </li></ol>代表的な結果： これらの手順が正しく実行されると二つのことが観察されるべきである。最初に、一定の回転視覚刺激下OMTに配置されたテザーフライは、スムーズにヨー軸に360度回転する必要があります。 &quot;ジャーキー&quot;ターンは、（サッカードと呼ばれる）を一定の視覚刺激の非存在下での自発的な転換動作の特徴です。ハエが明らかにスムーズに回転できない場合、不適切な接着（パート1を参照）とずれ磁石は（パート2を参照）せいにするかもしれない。第二に、臭気がオンになっているときに、ハエは臭いの方向に向かうはずです。匂いをオフにすると、フライは競技場内の任意の単一の位置のための希望なしに自発的にサッカードがあります。ハエが積極的に臭羽毛を追跡しない場合は、臭気のポートの位置、空気の流量および/または真空の流量は、（パート3を参照）調整が必要な場合があります。

<ol>
	<li>Bender, J. A., Dickinson, 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+stimulation+of+saccades+in+magnetically+tethered+Drosophila.">Visual stimulation of saccades in magnetically tethered Drosophila.</a> J. Exp. Biol. 209, 3170-3182 (2006).</li><li>Tammero, L. F., Dickinson, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collision-avoidance+and+landing+responses+are+mediated+by+separate+pathways+in+the+fruit+fly,+Drosophila+melanogaster.">Collision-avoidance and landing responses are mediated by separate pathways in the fruit fly, Drosophila melanogaster.</a> J. Exp. Biol. 205, 2785-2798 (2002).</li><li>Duistermars, B. J., Frye, 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=Crossmodal+visual+input+for+odor+tracking+during+fly+flight.">Crossmodal visual input for odor tracking during fly flight.</a> Curr. Biol. 18, 270-275 (2008).</li><li>Reiser, M. B., Dickinson, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+modular+display+system+for+insect+behavioral+neuroscience.">A modular display system for insect behavioral neuroscience.</a> J. Neurosci. Methods. 167, 127-139 (2008).</li><li>Frye, M. A., Dickinson, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Motor+output+reflects+the+linear+superposition+of+visual+and+olfactory+inputs+in+Drosophila.">Motor output reflects the linear superposition of visual and olfactory inputs in Drosophila.</a> J. Exp. Biol. 207, 123-131 (2004).</li></ol>

ハエをテザリングとこの分野で安定的な臭気の追跡を誘発することは比較的簡単です。それは練習、忍耐と試行錯誤が必要です。この分野の多くの部分は、さらに最適化や実験的な電位の変動をもたらすために代替できる場合もあります。例えば、真空チャンバ内の新しいバージョンは、はるかに小さいですし、舞台下に多くのスペースを許可する。また、希土類磁石の強さや種類が若干変化することがで?...

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a magnetic tether system to investigate visual and olfactory mediated flight control in drosophila

それは虫が風の流れでその見出しを安定させるために視覚的な手がかりを使用することを長年にわたって明らかになっている。多くの動物は、風で運ばれた匂いを追跡。このように、風上の視覚的な安定は、臭気の追跡に直接補助追跡。しかし、嗅覚の信号は、直接風の手がかりから独立して視覚的な追跡の動作に影響を与えるのですか？また、ショウジョウバエの神経生理学と嗅覚の神経行動遺伝学に関する研究の最近の洪水では、これまで以上に技術的に洗練され、定量的な行動アッセイを動機としています。ここで、我々は元々狭い層流臭プルームでアリーナを搭載することにより、ビジョンの実験のために考案された磁気テザーシステムを変更。フライは小さなスチールピンに接着し、自由にヨーを可能にする磁場中に懸濁されている。小径食品の匂いのプルームは、空腹フライで安定したトラッキングを引き出す、ハエの頭の上に下向きにしている。ここでは、磁石を合わせ臭羽毛を考案し、安定的な臭気の追跡を確認、テザリングの重要なメカニズムに焦点を当てる。

Watch this Scientific Journal Video about A Magnetic Tether System to Investigate Visual and Olfactory Mediated Flight Control in Drosophila at JoVE.com

A Magnetic Tether System to Investigate Visual and Olfactory Mediated Flight Control in Drosophila

ここでは、どのようにテザー嗅覚磁気テザー（OMT）装置でフライをして説明します。我々は、希土類磁石と臭気のポート、およびどのように刺激の配信と最適な臭気の追跡を実現するために真空吸引の両方の質量流量を設定することを整列する方法について説明します。

ショウジョウバエの視覚と嗅覚を介したフライトコントロールを調査するために磁気テザーシステム

It has been clear for many years that insects use visual cues to stabilize their heading in a wind stream. Many animals track odors carried in the wind. ...

a-magnetic-tether-system-to-investigate-visual-olfactory-mediated

Research

JoVE Journal

Biology

11.2K ビュー.  University of California, Los Angeles. ここでは、どのようにテザー嗅覚磁気テザー（OMT）装置でフライをして説明します。我々は、希土類磁石と臭気のポート、およびどのように刺激の配信と最適な臭気の追跡を実現するために真空吸引の両方の質量流量を設定することを整列する方法について説明します。

ビデオ: A Magnetic Tether System to Investigate Visual and Olfactory Mediated Flight Control in Drosophila

Visually Mediated Odor Tracking During Flight in Drosophila

Flying insects use visual cues to stabilize their heading in a wind stream. Many animals additionally track odors carried in the wind. As such, visual stabilization of upwind tracking directly aids in odor tracking. But do olfactory signals directly influence visual tracking behavior independently from wind cues? Additionally, recent advances in olfactory molecular genetics and neurophysiology have motivated novel quantitative behavioral analyses to assess the behavioral influence of (e.g.) genetically inactivating specific olfactory activation circuits. We modified a magnetic tether system originally devised for vision experiments by equipping the arena with narrow laminar flow odor plumes. Here we focus on experiments that can be performed after a fly is tethered and is able to navigate in the magnetic arena. We show how to acquire video images optimized for measuring body angle, how to judge stable odor tracking, and we illustrate two experiments to examine the influence of visual cues on odor tracking.

Here we describe how to optimize the acquired video image for an olfactory magnetic-tether (OMT) apparatus. We also describe two sample experimental protocols for studying visuo-olfactory fusion.

ショウジョウバエのフライトの間に視覚的に仲介臭気の追跡

Flying insects use visual cues to stabilize their heading in a wind stream. Many animals additionally track odors carried in the wind. As such, visual ...

生物学

Single-cell Suction Recordings from Mouse Cone Photoreceptors

Rod and cone photoreceptors in the retina are responsible for light detection. In darkness, cyclic nucleotide-gated (CNG) channels in the outer segment are open and allow cations to flow steadily inwards across the membrane, depolarizing the cell. Light exposure triggers the closure of the CNG channels, blocks the inward cation current flow, and thus results in cell hyperpolarization. Based on the polarity of photoreceptors, a suction recording method was developed in 1970s that, unlike the classic patch-clamp technique, does not require penetrating the plasma membrane 1. Drawing the outer segment into a tightly-fitting glass pipette filled with extracellular solution allows recording the current changes in individual cells upon test-flash exposure. However, this well-established "outer-segment-in (OS-in)" suction recording is not suitable for mouse cone recordings, because of the low percentage of cones in the mouse retina (3%) and the difficulties in identifying the cone outer segments. Recently, an inner-segment-in (IS-in) recording configuration was developed to draw the inner segment/nuclear region of the photoreceptor into the recording pipette 2,3. In this video, we will show how to record from individual mouse cone photoresponses using single-cell suction electrode.

We will show how to record flash responses from single mouse cones using a suction electrode.

マウスの錐体光受容体からの単一細胞サクション録音

Rod and cone photoreceptors in the retina are responsible for light detection. In darkness, cyclic nucleotide-gated (CNG) channels in the outer segment ...

A Practical Approach to Genetic Inducible Fate Mapping: A Visual Guide to Mark and Track Cells In Vivo

Fate maps are generated by marking and tracking cells in vivo to determine how progenitors contribute to specific structures and cell types in developing and adult tissue. An advance in this concept is Genetic Inducible Fate Mapping (GIFM), linking gene expression, cell fate, and cell behaviors in vivo, to create fate maps based on genetic lineage.
GIFM exploits X-CreER lines where X is a gene or set of gene regulatory elements that confers spatial expression of a modified bacteriophage protein, Cre recombinase (CreERT). CreERT contains a modified estrogen receptor ligand binding domain which renders CreERT sequestered in the cytoplasm in the absence of the drug tamoxifen. The binding of tamoxifen releases CreERT, which translocates to the nucleus and mediates recombination between DNA sequences flanked by loxP sites. In GIFM, recombination typically occurs between a loxP flanked Stop cassette preceding a reporter gene such as GFP.
Mice are bred to contain either a region- or cell type-specific CreER and a conditional reporter allele. Untreated mice will not have marking because the Stop cassette in the reporter prevents further transcription of the reporter gene. We administer tamoxifen by oral gavage to timed-pregnant females, which provides temporal control of CreERT release and subsequent translocation to the nucleus removing the Stop cassette from the reporter. Following recombination, the reporter allele is constitutively and heritably expressed. This series of events marks cells such that their genetic history is indelibly recorded. The recombined reporter thus serves as a high fidelity genetic lineage tracer that, once on, is uncoupled from the gene expression initially used to drive CreERT.
We apply GIFM in mouse to study normal development and ascertain the contribution of genetic lineages to adult cell types and tissues. We also use GIFM to follow cells on mutant genetic backgrounds to better understand complex phenotypes that mimic salient features of human genetic disorders.
This video article guides researchers through experimental methods to successfully apply GIFM. We demonstrate the method using our well characterized Wnt1-CreERT;mGFP mice by administering tamoxifen at embryonic day (E)8.5 via oral gavage followed by dissection at E12.5 and analysis by epifluorescence stereomicroscopy. We also demonstrate how to micro-dissect fate mapped domains for explant preparation or FACS analysis and dissect adult fate-mapped brains for whole mount fluorescent imaging. Collectively, these procedures allow researchers to address critical questions in developmental biology and disease models.

A Practical Approach to Genetic Inducible Fate Mapping: A Visual Guide to Mark and Track Cells In Vivo

Genetic Inducible Fate Mapping (GIFM) marks and tracks cells with fine spatial and temporal control in vivo and elucidates how cells from a specific genetic lineage contribute to developing and adult tissues. Demonstrated here are the techniques required to fate map E12.5 mouse embryos for epifluorescent and explant analysis.

遺伝子誘導の運命のマッピングへの実践的アプローチ：マークとトラックの細胞へのビジュアルガイドインビボでの</em

Fate maps are generated by marking and tracking cells in vivo to determine how progenitors contribute to specific structures and cell types in developing ...

Rapid Homogeneous Detection of Biological Assays Using Magnetic Modulation Biosensing System

A magnetic modulation biosensing system (MMB) [1,2] rapidly and homogeneously detected biological targets at low concentrations without any washing or separation step. When the IL-8 target was present, a 'sandwich'-based assay attached magnetic beads with IL-8 capture antibody to streptavidin coupled fluorescent protein via the IL-8 target and a biotinylated IL-8 antibody. The magnetic beads are maneuvered into oscillatory motion by applying an alternating magnetic field gradient through two electromagnetic poles. The fluorescent proteins, which are attached to the magnetic beads are condensed into the detection area and their movement in and out of an orthogonal laser beam produces a periodic fluorescent signal that is demodulated using synchronous detection. The magnetic modulation biosensing system was previously used to detect the coding sequences of the non-structural Ibaraki virus protein 3 (NS3) complementary DNA (cDNA) [2]. The techniques that are demonstrated in this work for external manipulation and condensation of particles may be used for other applications, e.g. delivery of magnetically-coupled drugs in-vivo or enhancing the contrast for in-vivo imaging applications.

Magnetic modulation biosensing system is utilized to rapidly, sensitively and simply detect biological assays, such as DNA molecules and proteins.

磁気変調バイオセンシングシステムを用いた生物学的アッセイの迅速な均質な検出

A magnetic modulation biosensing system (MMB) [1,2] rapidly and homogeneously detected biological targets at low concentrations without any washing or ...

Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting (LIMACS): a Novel Method to Analyze Protein-lipid Interaction

The analysis of lipid protein interaction is difficult because lipids are embedded in cell membranes and therefore, inaccessible to most purification procedures. As an alternative, lipids can be coated on flat surfaces as used for lipid ELISA and Plasmon resonance spectroscopy. However, surface coating lipids do not form microdomain structures, which may be important for the lipid binding properties. Further, these methods do not allow for the purification of larger amounts of proteins binding to their target lipids.
 To overcome these limitations of testing lipid protein interaction and to purify lipid binding proteins we developed a novel method termed lipid vesicle-mediated affinity chromatography using magnetic-activated cell sorting (LIMACS). In this method, lipid vesicles are prepared with the target lipid and phosphatidylserine as the anchor lipid for Annexin V MACS. Phosphatidylserine is a ubiquitous cell membrane phospholipid that shows high affinity to the protein Annexin V. Using magnetic beads conjugated to Annexin V the phosphatidylserine-containing lipid vesicles will bind to the magnetic beads. When the lipid vesicles are incubated with a cell lysate the protein binding to the target lipid will also be bound to the beads and can be co-purified using MACS. This method can also be used to test if recombinant proteins reconstitute a protein complex binding to the target lipid. 
 We have used this method to show the interaction of atypical PKC (aPKC) with the sphingolipid ceramide and to co-purify prostate apoptosis response 4 (PAR-4), a protein binding to ceramide-associated aPKC. We have also used this method for the reconstitution of a ceramide-associated complex of recombinant aPKC with the cell polarity-related proteins Par6 and Cdc42. Since lipid vesicles can be prepared with a variety of sphingo- or phospholipids, LIMACS offers a versatile test for lipid-protein interaction in a lipid environment that resembles closely that of the cell membrane. Additional lipid protein complexes can be identified using proteomics analysis of lipid binding protein co-purified with the lipid vesicles.

Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting LIMACS: a Novel Method to Analyze Protein-lipid Interaction

To test the interaction of a protein with its target lipid we used MACS and Annexin V-conjugated magnetic beads and lipid vesicles synthesized from the target lipid and Annexin V-binding phosphatidylserine. Proteins bound to the target lipid are co-purified and analyzed after elution from the beads.

磁気活性化細胞選別（LIMACS）を使用して、脂質小胞を介したアフィニティクロマトグラフィー：タンパク質脂質相互作用を解析する新手法

The analysis of lipid protein interaction is difficult because lipids are embedded in cell membranes and therefore, inaccessible to most purification ...

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated tools and resources for studies in this system are facilitating continued discovery of genes with more subtle phenotypes or roles. 
Here we present a generalized protocol we adapted for identifying C. elegans genes with postembryonic phenotypes of interest using RNAi2. This procedure is easily modified to assay the phenotype of choice, whether by light or fluorescence optics on a dissecting or compound microscope. This screening protocol capitalizes on the physical assets of the organism and molecular tools the C. elegans research community has produced. As an example, we demonstrate the use of an integrated transgene that expresses a fluorescent product in an RNAi screen to identify genes required for the normal localization of this product in late stage larvae and adults. First, we used a commercially available genomic RNAi library with full-length cDNA inserts. This library facilitates the rapid identification of multiple candidates by RNAi reduction of the candidate gene product. Second, we generated an integrated transgene that expresses our fluorecently tagged protein of interest in an RNAi-sensitive background. Third, by exposing hatched animals to RNAi, this screen permits identification of gene products that have a vital embryonic role that would otherwise mask a post-embryonic role in regulating the protein of interest. Lastly, this screen uses a compound microscope equipped for single cell resolution.

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

We describe a sensitized method to identify postembryonic regulators of protein expression and localization in C. elegans using an RNAi-based genomic screen and an integrated transgene that expresses a functional, fluorescently tagged protein.

の後胚表現型を識別するためのRNAiスクリーニング C.エレガンス</em

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated ...

Quantitative Measurement of Invadopodia-mediated Extracellular Matrix Proteolysis in Single and Multicellular Contexts

Cellular invasion into local tissues is a process important in development and homeostasis. Malregulated invasion and subsequent cell movement is characteristic of multiple pathological processes, including inflammation, cardiovascular disease and tumor cell metastasis1. Focalized proteolytic degradation of extracellular matrix (ECM) components in the epithelial or endothelial basement membrane is a critical step in initiating cellular invasion. In tumor cells, extensive in vitro analysis has determined that ECM degradation is accomplished by ventral actin-rich membrane protrusive structures termed invadopodia2,3. Invadopodia form in close apposition to the ECM, where they moderate ECM breakdown through the action of matrix metalloproteinases (MMPs). The ability of tumor cells to form invadopodia directly correlates with the ability to invade into local stroma and associated vascular components3. 
Visualization of invadopodia-mediated ECM degradation of cells by fluorescent microscopy using dye-labeled matrix proteins coated onto glass coverslips has emerged as the most prevalent technique for evaluating the degree of matrix proteolysis and cellular invasive potential4,5. Here we describe a version of the standard method for generating fluorescently-labeled glass coverslips utilizing a commercially available Oregon Green-488 gelatin conjugate. This method is easily scaled to rapidly produce large numbers of coated coverslips. We show some of the common microscopic artifacts that are often encountered during this procedure and how these can be avoided. Finally, we describe standardized methods using readily available computer software to allow quantification of labeled gelatin matrix degradation mediated by individual cells and by entire cellular populations. The described procedures provide the ability to accurately and reproducibly monitor invadopodia activity, and can also serve as a platform for evaluating the efficacy of modulating protein expression or testing of anti-invasive compounds on extracellular matrix degradation in single and multicellular settings.

We describe the prototypical method for producing microscope coverslips coated with fluorescent gelatin for visualizing invadopodia-mediated matrix degradation. Computational techniques using available software are presented for quantifying the resultant levels of matrix proteolysis by single cells within a mixed population and for multicellular groups encompassing entire microscopic fields.

シングルおよび多細胞コンテキストでInvadopodia媒介細胞外マトリックスタンパク分解の定量測定

Cellular invasion into local tissues is a process important in development and homeostasis.  Malregulated invasion and subsequent cell movement is ...

Design and Analysis of Temperature Preference Behavior and its Circadian Rhythm in Drosophila

The circadian clock regulates many aspects of life, including sleep, locomotor activity, and body temperature (BTR) rhythms1,2. We recently identified a novel Drosophila circadian output, called the temperature preference rhythm (TPR), in which the preferred temperature in flies rises during the day and falls during the night 3. Surprisingly, the TPR and locomotor activity are controlled through distinct circadian neurons3. Drosophila locomotor activity is a well&#160;known circadian behavioral output and has provided strong contributions to the discovery of many conserved mammalian circadian clock genes and mechanisms4. Therefore, understanding TPR will lead to the identification of hitherto unknown molecular and cellular circadian mechanisms. Here, we describe how to perform and analyze the TPR assay. This technique not only allows for dissecting the molecular and neural mechanisms of TPR, but also provides new insights into the fundamental mechanisms of the brain functions that integrate different environmental signals and regulate animal behaviors. Furthermore, our recently published data suggest that the fly TPR shares features with the mammalian BTR3. Drosophila are ectotherms, in which the body temperature is typically behaviorally regulated. Therefore, TPR is a strategy used to generate a rhythmic body temperature in these flies5-8. We believe that further exploration of Drosophila TPR will facilitate the characterization of the mechanisms underlying body temperature control in animals.

Design and Analysis of Temperature Preference Behavior and its Circadian Rhythm in Drosophila

We recently identified a novel Drosophila circadian output, temperature preference rhythm (TPR), in which the preferred temperature in flies rises during the day and falls during the night. TPR is regulated independently from another circadian output, locomotor activity. Here&#160;we describe the design and analysis of TPR in Drosophila.

ショウジョウバエにおける温度嗜好挙動とその概日リズムの設計と解析

The circadian clock regulates many aspects of life, including sleep, locomotor activity, and body temperature (BTR) rhythms1,2. We recently identified a ...

An Improved Method for Accurate and Rapid Measurement of Flight Performance in Drosophila

Drosophila has proven to be a useful model system for analysis of behavior, including flight. The initial flight tester involved dropping flies into an oil-coated graduated cylinder; landing height provided a measure of flight performance by assessing how far flies will fall before producing enough thrust to make contact with the wall of the cylinder. Here we describe an updated version of the flight tester with four major improvements. First, we added a &#34;drop tube&#34; to ensure that all flies enter the flight cylinder at a similar velocity between trials, eliminating variability between users. Second, we replaced the oil coating with removable plastic sheets coated in Tangle-Trap, an adhesive designed to capture live insects. Third, we use a longer cylinder to enable more accurate discrimination of flight ability. Fourth we use a digital camera and imaging software to automate the scoring of flight performance. These improvements allow for the rapid, quantitative assessment of flight behavior, useful for large datasets and large-scale genetic screens.

An Improved Method for Accurate and Rapid Measurement of Flight Performance in Drosophila

Here we describe a method for rapid and accurate measurement of flight performance in Drosophila, enabling high-throughput screening.

飛行性能の正確かつ迅速な測定のための改善された方法での<em&gt;ショウジョウバエ</em

Drosophila has proven to be a useful model system for analysis of behavior, including flight. The initial flight tester involved dropping flies into an ...

Validation of a Mouse Model to Disrupt LINC Complexes in a Cell-specific Manner

Nuclear migration and anchorage within developing and adult tissues relies heavily upon large macromolecular protein assemblies called LInkers of the Nucleoskeleton and Cytoskeleton (LINC complexes). These protein scaffolds span the nuclear envelope and connect the interior of the nucleus to components of the surrounding cytoplasmic cytoskeleton. LINC complexes consist of two evolutionary-conserved protein families, Sun proteins and Nesprins that harbor C-terminal molecular signature motifs called the SUN and KASH domains, respectively. Sun proteins are transmembrane proteins of the inner nuclear membrane whose N-terminal nucleoplasmic domain interacts with the nuclear lamina while their C-terminal SUN domains protrudes into the perinuclear space and interacts with the KASH domain of Nesprins. Canonical Nesprin isoforms have a variable sized N-terminus that projects into the cytoplasm and interacts with components of the cytoskeleton. This protocol describes the validation of a dominant-negative transgenic mouse strategy that disrupts endogenous SUN/KASH interactions in a cell-type specific manner. Our approach is based on the Cre/Lox system that bypasses many drawbacks such as perinatal lethality and cell nonautonomous phenotypes that are associated with germline models of LINC complex inactivation. For this reason, this model provides a useful tool to understand the role of LINC complexes during development and homeostasis in a wide array of tissues.

Nuclear envelope proteins play a central role in many basic biological processes and have been implicated in a variety of human diseases. This protocol describes a new Cre/Lox-based mouse model that allows for the spatiotemporal control of LINC complexes disruption.

細胞特異的にLINC複合体を破壊するマウスモデルの検証

Nuclear migration and anchorage within developing and adult tissues relies heavily upon large macromolecular protein assemblies called LInkers of the ...