このプロトコルは、現在我々の研究室で使用されており、博士デビッドシャープ、Mijungクォン、パトリツィアSommi、Dhanya Cheerambathurを含む多くの人々が長年にわたって洗練されています。我々はこのシステムでは我々の作業が開始されていた初期のショウジョウバエの胚の操作およびマイクロインジェクション（参考文献13）に優れたアドバイスを私たちに提供する博士ビルサリバン（UCSC）に感謝。我々はコーリーの研究室のすべてのメンバーに感謝。 ショウジョウバエの有糸分裂に関する私たちの仕事は、NIHの助成金GM55507によってサポートされています。

レシピ： グレープジュースプレート： <ul><li> 5.5グラムバクト寒天</li><li> 14.5グラムブドウ糖またはブドウ糖</li><li> 7.15グラムのショ糖</li><li> 45ミリリットルのグレープジュース濃縮物（100％ジュース）。 </li><li> 204.5ミリリットルH 2 0 </li><li> 625μlの10N NaOHを</li></ul>沸騰するまで、すべての材料と電子レンジを混ぜる。 2.8ミリリットルの酸ミックス（：20.9ミリリットルプロピオン酸、2.1 mlのリン酸、27ミリリットルH 2 0酸の混合）を追加します。 ミックスおよび35 mmシャーレに注ぐ。 1つまたは2つ日間室温で固化してみましょう。プレートは、すぐに使用されようとしてパラフィルムでシールし、4℃で（使用する前に、RTに平衡を許可）維持されていない場合。 酵母ペースト： 濃厚なペーストを形成するために水に酵母の茶さじ1杯（シグマYSC2、サッカロマイセスセレビシエII型から酵母）について溶解する。使用直前にそれぞれのグレープジュースのプレートでこの少量のを置きます。 G - PEMバッファー： <ul><li> 80mMのナトリウムPIPES、pHは6.9 </li><li> 1mMのMgCl 2 </li><li> 1mMのEGTA </li><li> 1mMのGTP </li></ul>インジェクションバッファー： <ul><li> 150mMのK -アスパラギン酸</li><li>の10mM K -リン酸</li><li> 20 mMイミダゾール、pH 7.2の</li></ul>ヘプタンの接着剤： 数日間50ミリリットル程度ヘプタン、ボトルを密封し、岩を追加し、100ミリリットル瓶でダブル粘着テープと場所を引き出します。接着剤が厚すぎる場合使用する場合は、ヘプタン追加。 脱水チャンバー： &quot;テーブル&quot;を作成し、DRIERITEの高さは、&quot;表&quot;とカバーより大きくしないようにその周りにDRIERITEを（無水硫酸カルシウム）を追加するために内部の35mm皿の一部を入れて100mmのペトリ皿を取る。胚とカバースリップは注射の前に脱水のためにこの&quot;テーブル&quot;に配置されます。それは、少なくともいくつかを示すDRIERITEを使用して、色を変更されたときにそれを変更することをお勧めします。 プロトコル： 例えば、どちらの観測や標的タンパク質の阻害剤またはその両方のための蛍光タンパク質：このプロトコルは、ショウジョウバエ多核体胚に実質的にあらゆる水溶性試薬の注入に使用することができます。 すべてが準備中： 1 .-実験の前に2〜3日、グレープジュースのプレートを作成し、設定するケージレイ：の反対側に二つの小さな穴を（2約1cm）カット、ペットボトル（6オンス、応用科学ショウジョウバエから丸底）に乗りボトルと綿の部分（これは空気が流れを許可する）で埋めるには、瓶にハエをタップし、グレープジュースのプレートでカバーしています。 25℃に保つ° C（または室温）、ハエは約10日間胚を置く必要があります。 実験は、注射用の針を引っ張る2 .-少なくとも1日の前に、我々はKopfはニードル/ピペットプラーモデル720と薄肉ガラスフィラメント（世界精密機器tw100f）を使用します。蛍光チューブリンの注入の場合は、℃の針で温度による重合を防ぐために4時に針を保つ。 チューブリンの準備： 注：チューブリンとのすべての仕事が4時に行われるべき° Cで重合を防止する。 <ol><li>冷凍庫のラベルが付いたチューブを取り出し、5〜15分間氷上に置きます。 </li><li> G - PEMのバッファ（必要な強度に応じて4〜10倍）で希釈する。 </li><li> 4で13K rpmで遠心10〜15分間。 </li><li>負荷1 - 針に2μlの。 </li></ol>抗体または化学的阻害剤： 適切な濃度（これはテストする必要があります）を使用して、チューブリンと同様に針を準備します。抗体は、注射のバッファ、G - PEMバッファーまたはPBSにすることができます。別のバッファが必要な場合は、このバッファは、このバッファには独自の上に損傷が生じないことを確認するコントロールとして注入する必要があります。 注：濃度で許可されている場合に阻害し、蛍光チューブリンを混合し、一緒に注入することができる。そうでなければ、蛍光チューブリンを注入し、阻害剤を注入する前に、5〜10分待ちます。少ない胚が正常な状態に復元するので、二重注入を行うときは、、より多くの胚で始まります。 胚の収集： <ol><li>素人のケージに新しいブドウジュースのプレートを置く。 </li><li>一時間後にこのプレートを外し、この日の最初のコレクションであり、しばしば非常に良いではありません。それは破棄することができます。 </li><li>一時間ごとに収集する胚を保つためにプレートを毎時を変更します。 </li></ol>カバースリップの準備： <ol><li>顕微鏡のスライドの片側に50 × 22ミリメートルカバースリップを置く。スライドへのテープの四隅には移動しないように。 </li><li>綿を使用すると、アプリケーターをひっくり返した（接着剤は粘性すべきではないと数秒で乾燥させてください、そうでない場合よりヘプタン追加）カバースリップ上で1つの行にヘプタン接着剤の一つの層を置く。 </li><li>の部分を入れてカバースリップの側に向かってスライド上に粘着テープを倍増。 </li></ol>胚の準備： 注：胚の収集の開始後約2時間イメージ化されるはずなので、この時間枠内でそれらを完了するのに十分な時間を許可するには、次の手順を開始する。 <ol><li>湿らせたブラシで慎重にスライドをダブル粘着テープでグレープジュースのプレートと場所から胚を拾う。 </li><li>絨毛膜（外膜）が開いて解除するまでピンセットの外側の部分を使用して、ダブル粘着テープを介して胚をロールバックします。 </li><li>それはピンセットに付着し、カバースリップの長辺に胚の平行の長い側をカバースリップ上にヘプタンのりの上に置くように静かに絨毛膜の上に圧延して胚を拾う。つの行で10〜20の胚を設定します。 </li><li>カバーを取り外して、3〜8分（これは部屋の湿度に依存し、量を注入する）のための脱水槽に入れてください。 </li><li>真空グリースで、金属チャンバーの上に置きます。 </li><li>さらに脱水症状を避けるためにハロカーボンのオイル700の胚をカバーしています。胚はインジェクションのための準備が整いました。 </li></ol>胚の注入： <ol><li> 16倍の目標の下で胚を見つける。 </li><li>離れて胚を移動し、焦点面を移動せずに針を見つける。 </li><li>センター針と、xまたはyの方向に移動することなく、それを上に移動。 </li><li>ニードルが開いていない場合、ビューのフィールドのカバースリップの端を置くが、針がなるではない場所、および同じ焦点面に針を下ろします。それが針に当たると軽く開いて、それを解除するまで、非常に慎重にカバースリップを移動する。ニードルが開いている場合は、手順5に進みます。 （針を充填する前にフッ化水素酸で開くことができます）。 </li><li>ビュー（ただし、針がなる）に胚を入れ、油に針を下げるし、針から素敵な液滴を得ることを確認してください。 </li><li>慎重にしかし着実に、針に胚を移動し、胚にドロップを注入し、離れて胚を移動する。 （またはあなたの射出装置の指示に従ってください）​​。 </li><li>すべての胚が注入された後、彼らは共焦点顕微鏡で観察するための準備が整いました。 </li></ol>イメージング： 画像60または100倍を目的とした共焦点顕微鏡で胚を。 注意事項： <ol><li>胚の細胞分裂が非常に高速であるため、時間経過のシリーズでの時間間隔についても研究する正確なプロセスに応じて、最大で数秒にする必要があります。 </li><li> 3Dシリーズまたは単一平面は、関心のプロセスに応じて取得することができます。 </li><li>速効性阻害剤の場合は、注入の顕微鏡から、イメージング顕微鏡への転送が高速である必要があります。 </li></ol><img src="/files/ftp_upload/1382/1382fig1.jpg" alt="図1" /> 図1： ショウジョウバエ多核体胚の細胞分裂の研究。初期胚の核は、サイクル10 13を介して核の形の間に、細胞質分裂せずに皮質における単分子層を急速に分裂を受ける。。皮質での核と初期胚の概略図。胚では標識タンパク質GFPおよび/ ​​またはRFPを発現することができるし、他の標識タンパク質および/ ​​または阻害剤をマイクロインジェクションすることができます。サイクル11のGFP -チューブリンとポール-ポールの分離のRFP -ヒストン。C.プロットを表現する胚のB.のタイムラプスイメージング、野生型の胚で12と13。スピンドルの長さは非常に一貫性と再現性がアイソメトリック長さと伸びの期間の期間を、示す。スピンドル微小管のD.ダイナミクス。ローダミンのチューブリンの低濃度の注入は、微小管のダイナミクスを研究するために使用できるいくつかのチューブリンのサブユニットから形成された斑点につながります。

<ol>
	<li>Brust-Mascher, I., Scholey, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitotic+spindle+dynamics+in+Drosophila.">Mitotic spindle dynamics in Drosophila.</a> Int Rev Cytol. 259, 139-172 (2007).</li><li>Duffy, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GAL4+system+in+Drosophila:+a+fly+geneticist's+Swiss+army+knife.">GAL4 system in Drosophila: a fly geneticist's Swiss army knife.</a> Genesis. 34, 1-15 (2002).</li><li>Morris, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microinjection+methods+for+analyzing+the+functions+of+kinesins+in+early+embryos.">Microinjection methods for analyzing the functions of kinesins in early embryos.</a> Methods Mol Biol. 164, 163-172 (2001).</li><li>Brust-Mascher, I., Sommi, P., Cheerambathur, D. K., Scholey, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinesin-5-dependent+poleward+flux+and+spindle+length+control+in+Drosophila+embryo+mitosis.">Kinesin-5-dependent poleward flux and spindle length control in Drosophila embryo mitosis.</a> Mol Biol Cell. 20, 1749-1762 (2009).</li><li>Brust-Mascher, I., Civelekoglu-Scholey, G., Kwon, M., Mogilner, A., Scholey, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Model+for+anaphase+B:+role+of+three+mitotic+motors+in+a+switch+from+poleward+flux+to+spindle+elongation.">Model for anaphase B: role of three mitotic motors in a switch from poleward flux to spindle elongation.</a> Proc Natl Acad Sci U S A. 101, 15938-15943 (2004).</li><li>Brust-Mascher, I., Scholey, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microtubule+flux+and+sliding+in+mitotic+spindles+of+Drosophila+embryos.">Microtubule flux and sliding in mitotic spindles of Drosophila embryos.</a> Mol Biol Cell. 13, 3967-3975 (2002).</li><li>Cheerambathur, D. K., Brust-Mascher, I., Civelekoglu-Scholey, G., Scholey, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+partitioning+of+mitotic+kinesin-5+crosslinkers+between+microtubule-bound+and+freely+diffusing+states.">Dynamic partitioning of mitotic kinesin-5 crosslinkers between microtubule-bound and freely diffusing states.</a> J Cell Biol. 182, 421-428 (2008).</li><li>Cheerambathur, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+analysis+of+an+anaphase+B+switch:+predicted+role+for+a+microtubule+catastrophe+gradient.">Quantitative analysis of an anaphase B switch: predicted role for a microtubule catastrophe gradient.</a> J Cell Biol. 177, 995-1004 (2007).</li><li>Kwon, 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+chromokinesin,+KLP3A,+dives+mitotic+spindle+pole+separation+during+prometaphase+and+anaphase+and+facilitates+chromatid+motility.">The chromokinesin, KLP3A, dives mitotic spindle pole separation during prometaphase and anaphase and facilitates chromatid motility.</a> Mol Biol Cell. 15, 219-233 (2004).</li><li>Sharp, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+coordination+of+three+mitotic+motors+in+Drosophila+embryos.">Functional coordination of three mitotic motors in Drosophila embryos.</a> Mol Biol Cell. 11, 241-253 (2000).</li><li>Sharp, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+bipolar+kinesin,+KLP61F,+cross-links+microtubules+within+interpolar+microtubule+bundles+of+Drosophila+embryonic+mitotic+spindles.">The bipolar kinesin, KLP61F, cross-links microtubules within interpolar microtubule bundles of Drosophila embryonic mitotic spindles.</a> J Cell Biol. 144, 125-138 (1999).</li><li>Sharp, D. J., Rogers, G. C., Scholey, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytoplasmic+dynein+is+required+for+poleward+chromosome+movement+during+mitosis+in+Drosophila+embryos.">Cytoplasmic dynein is required for poleward chromosome movement during mitosis in Drosophila embryos.</a> Nat Cell Biol. 2, 922-930 (2000).</li><li>Sharp, D. J., Yu, K. R., Sisson, J. C., Sullivan, W., Scholey, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antagonistic+microtubule-sliding+motors+position+mitotic+centrosomes+in+Drosophila+early+embryos.">Antagonistic microtubule-sliding motors position mitotic centrosomes in Drosophila early embryos.</a> Nat Cell Biol. 1, 51-54 (1999).</li><li>Waterman-Storer, C. M., Desai, A., Bulinski, J. C., Salmon, E. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+speckle+microscopy,+a+method+to+visualize+the+dynamics+of+protein+assemblies+in+living+cells.">Fluorescent speckle microscopy, a method to visualize the dynamics of protein assemblies in living cells.</a> Curr Biol. 8, 1227-1230 (1998).</li></ol>

このプロトコルは比較的簡単です、しかし、各ステップでは、胚が損傷していないことを確認する練習が必要です。慎重な対照実験は常に信頼性の高い結果が得られていることを保証するために行われる必要があります。この設定を開始する一つの良い方法は、有糸分裂が胚の表面（サイクル10から13）ですべてのサイクルを通じて正常に進行することを確認するためにGFP -チューブリンの発?...

microinjection techniques for studying mitosis in the drosophila melanogaster syncytial embryo

このプロトコルは、有糸分裂1を研究するためのキイロショウジョウバエ合胞体胚の使用について説明します。 ショウジョウバエは 、シーケンスされたゲノムを持つ有用な遺伝学があり、そしてそれは容易に維持して操作することができます。多くの有糸分裂変異体が存在し、（例えばGFP）蛍光機能を発現するトランスジェニックハエ - タグ分裂タンパク質がされていると生成されています。標的遺伝子の発現は、GAL4/UASシステム2を使って可能です。 ショウジョウバエ初期胚では、（細胞周期の期間、≈10分）非常に迅速に複数の分裂像を実施。サイクル10から13の間に、核がちょうど皮質下に単分子層で胚の表面に細胞質分裂を介在することなく、迅速にかつ同期的に分割するので、これはよく、画像有糸分裂に適しています。これらの急速に分裂する核は、おそらく他の細胞と同じ有糸分裂機構を使用しますが、それらは高速化のために最適化され、チェックポイントは、一般的に不在の強いチェックポイントを有する細胞の細胞周期停止を引き起こす細胞分裂タンパク質の研究を可能にする、厳格ではないと考えられている。またはGFP標識タンパク質を発現している胚は蛍光標識したタンパク質をマイクロインジェクションは、簡単にライブダイナミクス（図1）に従うために撮像することができます。さらに、胚は、その機能3の喪失または摂動の効果を研究するために特定のタンパク質の機能阻害抗体や阻害剤をマイクロインジェクションすることができます。これらの試薬は、その変異体の対立遺伝子のシリーズに匹敵する欠陥の勾配でターン結果の阻害剤の濃度の勾配を、生成するために多くのスピンドルに達し、胚全体に拡散することができます。理想的には、標的タンパク質を蛍光標識されている場合、阻害の勾配は、4を直接可視化することができる。それは正式に抗体を適用する必要があるまれなので、厳格なコントロールと慎重な解釈で支配的な影響を与えることがあるという可能性を排除するのは難しいですが、それは、最強の表現型がnull表現型と同程度であることを前提としています。遠くに注射部位から、タンパク質の機能は部分的にしか標的タンパク質の他の機能が明らかになることを可能にすることが失われている。

Watch this Scientific Journal Video about Microinjection Techniques for Studying Mitosis in the Drosophila melanogaster Syncytial Embryo at JoVE.com

Microinjection Techniques for Studying Mitosis in the Drosophila melanogaster Syncytial Embryo

このプロトコルは、マイクロインジェクションとで高解像度の画像の使用について説明<em&gt;キイロショウジョウバエ</em有糸分裂を勉強する&gt;シンシチウム胚。

に有糸分裂研究のためのマイクロインジェクション技術キイロショウジョウバエ合胞体胚

This protocol describes the use of the Drosophila melanogaster syncytial embryo  for studying mitosis1.  Drosophila has useful genetics with a sequenced ...

microinjection-techniques-for-studying-mitosis-drosophila

Research

JoVE Journal

Biology

Microinjection Techniques for Studying Mitosis in the Drosophila melanogaster Syncytial Embryo

14.0K ビュー.  University of California, Davis. このプロトコルは、マイクロインジェクションとで高解像度の画像の使用について説明キイロショウジョウバエシンシチウム胚。

ビデオ: Microinjection Techniques for Studying Mitosis in the Drosophila melanogaster Syncytial Embryo

Dissection of Larval CNS in Drosophila Melanogaster

The central nervous system (CNS) of Drosophila larvae is complex and poorly understood.  One way to investigate the CNS is to use immunohistochemistry to examine the expression of various novel and marker proteins.  Staining of whole larvae is impractical because the tough cuticle prevents antibodies from penetrating inside the body cavity.  In order to stain these tissues it is necessary to dissect the animal prior to fixing and staining.  In this article we demonstrate how to dissect Drosophila larvae without damaging the CNS.  Begin by tearing the larva in half with a pair of fine forceps, and then turn the cuticle "inside-out" to expose the CNS.  If the dissection is performed carefully the CNS will remain attached to the cuticle.  We usually keep the CNS attached to the cuticle throughout the fixation and staining steps, and only completely remove the CNS from the cuticle just prior to mounting the samples on glass slides.  We also show some representative images of a larval CNS stained with Eve, a transcription factor expressed in a subset of neurons in the CNS.  The article concludes with a discussion of some of the practical uses of this technique and the potential difficulties that may arise.

In this article we demonstrate how to dissect the central nervous system from third instar Drosophila larvae.

キイロショウジョウバエの幼虫中枢神経系の解剖

The central nervous system (CNS) of Drosophila larvae is complex and poorly understood.  One way to investigate the CNS is to use immunohistochemistry to ...

生物学

High-Resolution Video Tracking of Locomotion in Adult Drosophila Melanogaster

Flies provide an important model for studying complex behavior due to the plethora of genetic tools available to researchers in this field. Studying locomotor behavior in Drosophila melanogaster relies on the ability to be able to quantify changes in motion during or in response to a given task. For this reason, a high-resolution video tracking system, such as the one we describe in this paper, is a valuable tool for measuring locomotion in real-time. Our protocol involves the use of an initial air pulse to break the flies momentum, followed by a thirty second filming period in a square chamber. A tracking program is then used to calculate the instantaneous speed of each fly within the chamber in 10 msec increments. Analysis software then compiles this data, and outputs a variety of parameters such as average speed, max speed, time spent in motion, acceleration, etc. This protocol will discuss proper feeding and management of flies for behavioral tasks, handling flies without anesthetization or immobilization, setting up a controlled environment, and running the assay from start to finish.

The study of complex locomotor behavior in Drosophila melanogaster is dependent upon the ability to quantify changes in a given fly's movement. This article demonstrates how to do this using a high-resolution tracking system.

アダルトキイロショウジョウバエのロコモーションの高解像度のビデオトラッキング

Flies provide an important model for studying complex behavior due to the plethora of genetic tools available to researchers in this field.  Studying ...

Electrophysiological Recording in the Drosophila Embryo

Drosophila is a premier genetic model for the study of both embryonic development and functional neuroscience. Traditionally, these fields are quite isolated from each other, with largely independent histories and scientific communities. However, the interface between these usually disparate fields is the developmental programs underlying acquisition of functional electrical signaling properties and differentiation of functional chemical synapses during the final phases of neural circuit formation. This interface is a critically important area for investigation. In Drosophila, these phases of functional development occur during a period of &lt;8 hours (at 25&deg;C) during the last third of embryogenesis. This late developmental period was long considered intractable to investigation owing to the deposition of a tough, impermeable epidermal cuticle. A breakthrough advance was the application of water-polymerizing surgical glue that can be locally applied to the cuticle to enable controlled dissection of late-stage embryos. With a dorsal longitudinal incision, the embryo can be laid flat, exposing the ventral nerve cord and body wall musculature to experimental investigation. Whole-cell patch-clamp techniques can then be employed to record from individually-identifiable neurons and somatic muscles. These recording configurations have been used to track the appearance and maturation of ionic currents and action potential propagation in both neurons and muscles. Genetic mutants affecting these electrical properties have been characterized to reveal the molecular composition of ion channels and associated signaling complexes, and to begin exploration of the molecular mechanisms of functional differentiation. A particular focus has been the assembly of synaptic connections, both in the central nervous system and periphery. The glutamatergic neuromuscular junction (NMJ) is most accessible to a combination of optical imaging and electrophysiological recording. A glass suction electrode is used to stimulate the peripheral nerve, with excitatory junction current (EJC) recordings made in the voltage-clamped muscle. This recording configuration has been used to chart the functional differentiation of the synapse, and track the appearance and maturation of presynaptic glutamate release properties. In addition, postsynaptic properties can be assayed independently via iontophoretic or pressure application of glutamate directly to the muscle surface, to measure the appearance and maturation of the glutamate receptor fields. Thus, both pre- and postsynaptic elements can be monitored separately or in combination during embryonic synaptogenesis. This system has been heavily used to isolate and characterize genetic mutants that impair embryonic synapse formation, and thus reveal the molecular mechanisms governing the specification and differentiation of synapse connections and functional synaptic signaling properties.

Electrophysiological Recording in the Drosophila Embryo

Electrophysiological recordings from Drosophila embryos allow analyses of developing muscle and neuron electrical properties, as well as characterization of functional synaptogenesis at the glutamatergic neuromuscular junction and central cholinergic and GABAergic synapses.

の電気生理学的記録ショウジョウバエ胚

Drosophila is a premier genetic model for the study of both embryonic development and functional neuroscience. Traditionally, these fields are quite ...

Monitoring Heart Function in Larval Drosophila melanogaster for Physiological Studies

We present various methods to record cardiac function in the larval Drosophila. The approaches allow heart rate to be measured in unrestrained and restrained whole larvae. For direct control of the environment around the heart another approach utilizes the dissected larvae and removal of the internal organs in order to bathe the heart in desired compounds. The exposed heart also allows membrane potentials to be monitored which can give insight of the ionic currents generated by the myocytes and for electrical conduction along the heart tube. These approaches have various advantages and disadvantages for future experiments that are discussed. The larval heart preparation provides an additional model besides the Drosophila skeletal NMJ to investigate the role of intracellular calcium regulation on cellular function. Learning more about the underlying ionic currents that shape the action potentials in myocytes in various species, one can hope to get a handle on the known ionic dysfunctions associated to specific genes responsible for various diseases in mammals.

Monitoring Heart Function in Larval Drosophila melanogaster for Physiological Studies

We present various ways to monitor heart function in the larva of Drosophila for assessing questions dealing with the function of gap junctions, ion channel mutations, modulation of pacemaker activity and pharmacological studies.

幼虫のモニタリングの心機能キイロショウジョウバエ生理学的研究のための

We present various methods to record cardiac function in the larval Drosophila. The approaches allow heart rate to be measured in unrestrained  and  ...

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may also be used to study the structure-function relationship for other ion channels and neurotransmitter receptors1. BK channels are widely expressed in different tissues and have been implicated in many physiological functions, including regulation of smooth muscle contraction, frequency tuning of inner hair cells and regulation of neurotransmitter release2-6. BK channels are activated by membrane depolarization and by intracellular Ca2+ and Mg2+ 6-9. Therefore, the protocol is designed to control both the membrane voltage and the intracellular solution. In this protocol, messenger RNA of BK channels is injected into Xenopus laevis oocytes (stage V-VI) followed by 2-5 days of incubation at 18&deg;C10-13. Membrane patches that contain single or multiple BK channels are excised with the inside-out configuration using patch clamp techniques10-13. The intracellular side of the patch is perfused with desired solutions during recording so that the channel activation under different conditions can be examined. To summarize, the mRNA of BK channels is injected into Xenopus laevis oocytes to express channel proteins on the oocyte membrane; patch clamp techniques are used to record currents flowing through the channels under controlled voltage and intracellular solutions.

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

Ionic current of BK channels is recorded using patch clamp techniques. BK channels are expressed in Xenopus oocytes by injecting messenger RNA. The intracellular solution during patch clamp recordings is controlled by a perfusion system.

イオンチャネル研究のためのパッチクランプおよび灌流のテクニックで表さアフリカツメガエル卵母細胞

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may ...

Studying Mitotic Checkpoint by Illustrating Dynamic Kinetochore Protein Behavior and Chromosome Motion in Living Drosophila Syncytial Embryos

The spindle assembly checkpoint (SAC) mechanism is an active signal, which monitors the interaction between chromosome kinetochores and spindle microtubules to prevent anaphase onset until the chromosomes are properly connected. Cells use this mechanism to prevent aneuploidy or genomic instability, and hence cancers and other human diseases like birth defects and Alzheimer's1. A number of the SAC components such as Mad1, Mad2, Bub1, BubR1, Bub3, Mps1, Zw10, Rod and Aurora B kinase have been identified and they are all kinetochore dynamic proteins2. Evidence suggests that the kinetochore is where the SAC signal is initiated. The SAC prime regulatory target is Cdc20. Cdc20 is one of the essential APC/C (Anaphase Promoting Complex or Cyclosome) activators3 and is also a kinetochore dynamic protein4-6. When activated, the SAC inhibits the activity of the APC/C to prevent the destruction of two key substrates, cyclin B and securin, thereby preventing the metaphase to anaphase transition7,8. Exactly how the SAC signal is initiated and assembled on the kinetochores and relayed onto the APC/C to inhibit its function still remains elusive.
Drosophila is an extremely tractable experimental system; a much simpler and better-understood organism compared to the human but one that shares fundamental processes in common. It is, perhaps, one of the best organisms to use for bio-imaging studies in living cells, especially for visualization of the mitotic events in space and time, as the early embryo goes through 13 rapid nuclear division cycles synchronously (8-10 minutes for each cycle at 25 &deg;C) and gradually organizes the nuclei in a single monolayer just underneath the cortex9.
Here, I present a bio-imaging method using transgenic Drosophila expressing GFP (Green Fluorescent Protein) or its variant-targeted proteins of interest and a Leica TCS SP2 confocal laser scanning microscope system to study the SAC function in flies, by showing images of GFP fusion proteins of some of the SAC components, Cdc20 and Mad2, as the example.

Studying Mitotic Checkpoint by Illustrating Dynamic Kinetochore Protein Behavior and Chromosome Motion in Living Drosophila Syncytial Embryos

The kinetochore is where the SAC initiates its signal monitoring the mitotic segregation of the sister chromatids. A method is described to visualize the recruitment and turnover of one of the kinetochore proteins and its coordination with the chromosome motion in Drosophila embryos using a Leica laser scanning confocal system.

リビングでの動的動原体タンパク質の挙動と染色体の動きを図解して有糸分裂チェックポイントを勉強<em&gt;ショウジョウバエ</em&gt;合胞体胚

The spindle assembly checkpoint (SAC) mechanism is an active signal, which monitors the interaction between chromosome kinetochores and spindle ...

Measurement of Lifespan in Drosophila melanogaster

Aging is a phenomenon that results in steady physiological deterioration in nearly all organisms in which it has been examined, leading to reduced physical performance and increased risk of disease. Individual aging is manifest at the population level as an increase in age-dependent mortality, which is often measured in the laboratory by observing lifespan in large cohorts of age-matched individuals. Experiments that seek to quantify the extent to which genetic or environmental manipulations impact lifespan in simple model organisms have been remarkably successful for understanding the aspects of aging that are conserved across taxa and for inspiring new strategies for extending lifespan and preventing age-associated disease in mammals.
The vinegar fly, Drosophila melanogaster, is an attractive model organism for studying the mechanisms of aging due to its relatively short lifespan, convenient husbandry, and facile genetics. However, demographic measures of aging, including age-specific survival and mortality, are extraordinarily susceptible to even minor variations in experimental design and environment, and the maintenance of strict laboratory practices for the duration of aging experiments is required. These considerations, together with the need to practice careful control of genetic background, are essential for generating robust measurements. Indeed, there are many notable controversies surrounding inference from longevity experiments in yeast, worms, flies and mice that have been traced to environmental or genetic artifacts1-4. In this protocol, we describe a set of procedures that have been optimized over many years of measuring longevity in Drosophila using laboratory vials. We also describe the use of the dLife software, which was developed by our laboratory and is available for download (<a href="http://sitemaker.umich.edu/pletcherlab/software">http://sitemaker.umich.edu/pletcherlab/software</a>). dLife accelerates throughput and promotes good practices by incorporating optimal experimental design, simplifying fly handling and data collection, and standardizing data analysis. We will also discuss the many potential pitfalls in the design, collection, and interpretation of lifespan data, and we provide steps to avoid these dangers.

Measurement of Lifespan in Drosophila melanogaster

Drosophila melanogaster is a powerful model organism for exploring the molecular basis of longevity regulation. This protocol will discuss the steps involved in generating a reproducible, population-based measurement of longevity as well as potential pitfalls and how to avoid them.

における寿命の測定<em&gt;キイロショウジョウバエ</em

Aging is a phenomenon that results in steady physiological deterioration in nearly all organisms in which it has been examined, leading to reduced ...

Imaging Through the Pupal Case of Drosophila melanogaster

The longstanding use of Drosophila as a model for cell and developmental biology has yielded an array of tools. Together, these techniques have enabled analysis of cell and developmental biology from a variety of methodological angles. Live imaging is an emerging method for observing dynamic cell processes, such as cell division or cell motility. Having isolated mutations in uncharacterized putative cell cycle proteins it became essential to observe mitosis in situ using live imaging. Most live imaging studies in Drosophila have focused on the embryonic stages that are accessible to manipulation and observation because of their small size and optical clarity. However, in these stages the cell cycle is unusual in that it lacks one or both of the gap phases. By contrast, cells of the pupal wing of Drosophila have a typical cell cycle and undergo a period of rapid mitosis spanning about 20 hr of pupal development. It is easy to identify and isolate pupae of the appropriate stage to catch mitosis in situ. Mounting intact pupae provided the best combination of tractability and durability during imaging, allowing experiments to run for several hours with minimal impact on cell and animal viability. The method allows observation of features as small as, or smaller than, fly chromosomes. Adjustment of microscope settings and the details of mounting, allowed extension of the preparation to visualize membrane dynamics of adjacent cells and fluorescently labeled proteins such as tubulin. This method works for all tested fluorescent proteins and can capture submicron scale features over a variety of time scales. While limited to the outer 20 &#181;m of the pupa with a conventional confocal microscope, this approach to observing protein and cellular dynamics in pupal tissues in vivo may be generally useful in the study of cell and developmental biology in these tissues.

Imaging Through the Pupal Case of Drosophila melanogaster

This paper demonstrates the use of a fast scanning confocal microscope to image cell behavior directly through the puparium. By leaving the pupal case intact, this method allows observation and measurement of dynamic cell processes at a stage of Drosophila development that is difficult to study directly.

の蛹ケースを画像化<em&gt;キイロショウジョウバエ</em

The longstanding use of Drosophila as a model for cell and developmental biology has yielded an array of tools. Together, these techniques have enabled ...

Enhanced Northern Blot Detection of Small RNA Species in Drosophila Melanogaster

The last decades have witnessed the explosion of scientific interest around gene expression control mechanisms at the RNA level. This branch of molecular biology has been greatly fueled by the discovery of noncoding RNAs as major players in post-transcriptional regulation. Such a revolutionary perspective has been accompanied and triggered by the development of powerful technologies for profiling short RNAs expression, both at the high-throughput level (genome-wide identification) or as single-candidate analysis (steady state accumulation of specific species). Although several state-of-art strategies are currently available for dosing or visualizing such fleeing molecules, Northern Blot assay remains the eligible approach in molecular biology for immediate and accurate evaluation of RNA expression. It represents a first step toward the application of more sophisticated, costly technologies and, in many cases, remains a preferential method to easily gain insights into RNA biology. Here we overview an efficient protocol (Enhanced Northern Blot) for detecting weakly expressed microRNAs (or other small regulatory RNA species) from Drosophila melanogaster whole embryos, manually dissected larval/adult tissues or in vitro cultured cells. A very limited amount of RNA is required and the use of material from flow cytometry-isolated cells can be also envisaged.

Enhanced Northern Blot Detection of Small RNA Species in Drosophila Melanogaster

The aim of this publication is to visualize and discuss the operative steps of an Enhanced Northern Blot protocol on RNA extracted from Drosophila melanogaster embryos, cells, and tissues. This protocol is particularly useful for the efficient detection of small RNA species.

低分子RNA種のノーザンブロット検出における強化<em&gt;キイロショウジョウバエ</em

The last decades have witnessed the explosion of scientific interest around gene expression control mechanisms at the RNA level. This branch of molecular ...

Generation of Transgenic Hydra by Embryo Microinjection

As a member of the phylum Cnidaria, the sister group to all bilaterians, Hydra can shed light on fundamental biological processes shared among multicellular animals. Hydra is used as a model for the study of regeneration, pattern formation, and stem cells. However, research efforts have been hampered by lack of a reliable method for gene perturbations to study molecular function. The development of transgenic methods has revitalized the study of Hydra biology1. Transgenic Hydra allow for the tracking of live cells, sorting to yield pure cell populations for biochemical analysis, manipulation of gene function by knockdown and over-expression, and analysis of promoter function. Plasmid DNA injected into early stage embryos randomly integrates into the genome early in development. This results in hatchlings that express transgenes in patches of tissue in one or more of the three lineages (ectodermal epithelial, endodermal epithelial, or interstitial). The success rate of obtaining a hatchling with transgenic tissue is between 10% and 20%. Asexual propagation of the transgenic hatchling is used to establish a uniformly transgenic line in a particular lineage. Generating transgenic Hydra is surprisingly simple and robust, and here we describe a protocol that can be easily implemented at low cost.

Generation of Transgenic Hydra by Embryo Microinjection

Stably transgenic Hydra are made by microinjection of plasmid DNA into embryos followed by random genomic integration and asexual propagation to establish a uniform line. Transgenic Hydra are used to track cell movements, overexpress genes, study promoter function, or knock down gene expression using RNAi.