私たちは、優れた技術支援のためのアンドレアKremsreiterに感謝したいと思います。マディソン - 私たちの研究室での作業は、NIH GM096088とウィスコンシン大学のラース·トラストによってサポートされています。

1。酵母ルミノメトリー方法 Saccharomyces cerevisiaeのひずみBYYTを使用してください。それはBY4741（MATA、ura3D0、his3D1、leu2D0、lys2D0、yvc1 :: HIS3、tok1 :: kanMX4）のyvc1-tok1-誘導体である。 Batiza ら 26とLoukin ら 24に記載されているように、 レイが選択可能なエクオリン発現プラスミドpEVP1/AeqとURA-選択可能なラットTRPV4発現プラスミドp416GPDV4で細胞を形質転換プラスミドの構築およびトランスフォーメーション27の使用標準的な方法。酵母株およびプラスミドのリクエストも承ります。 <ol><li>一晩1Mソルビトールを補足したLeu-URA-」DCD「ドロップアウト培地28（CMD-レイ-URA媒体29の硫酸塩が枯渇した変異体）、30℃のシェーカーにおけるポスト対数相への酵母細胞の培養2ミリリットル。 luciferi2μMの補充した新鮮な培地の1.8ミリリットルにこれらの培養液200μlを接種n個のセレンテラジン。振とうせずに、別の24時間室温で暗所に育つ。 </li><li>蒸気圧浸透圧計を使用して（下）（通常1,400ミリオスモルで）文化の浸透圧を測定し、他のソリューション。 </li><li>単管照度計のための12ミリメートルのルミノメーターチューブに一定分量新鮮な培養液20μlを。 </li><li>低張性ショックのために、必要なショックの程度に応じて25 mMのNaEGTA液（pH 7.2）または名前（pH7.2）にし、500から100のNaCl、（1,200-400ミリオスモルの合計オスモル濃度）を含む、溶液200μlを加える。連続して前に発光を監視し、少なくとも120秒の浸透downshockた後、唯一の簡単な希釈操作によって中断。相対発光単位（RUL）などのデスクトップコンピュータ上の照度計の出力を登録します。 </li></ol>しないトランスジェニックTRPV4の研究のためのけれども、我々は、酵母株の多数の応答を調べるために、自動化システムにおいて上記のプロトコルの変形を使用している。研究Oマイクロプレートルミノメーターを用いて低刺激30または酵母deletome（4906酵母単一遺伝子deletantsの集まり）の高浸透圧31刺激に対する応答Fと対応するソフトウェアで解析が成功している。 2。と3。卵母細胞電気生理学方法電気生理学は、基本的な方法32を使用しています。 PCRは、野生型または変異TRPV4は 、高忠実度PfuUtraポリメラーゼを使用しpGH19 33に統合のオープンリーディングフレームを増幅する。これは、 アフリカツメガエルの β-グロビン遺伝子およびT7 RNAポリメラーゼプロモーターの下流の5 &#39;および3&#39;UTRの間のORFの正確な挿入を伴う。 3 &#39;UTRの下流及びインビトロ T7 RNAポリメラーゼ反応においてテンプレートとして使用される構築物を線状化。標準の分子生物学的技術34を使用してください。 XからステージV〜VIの卵母細胞を使用ツメガエル 。畜産、部分的ovariecto私、defolliculation、そして卵黄エンベロープ除去は、標準的な手順を32,33,35に従ってください。ドラモンドNanoinject II自動マイクロインジェクターを使用して、卵母細胞当たりのcRNA液の2-40 NGを注入する。実験の各セットのために〜30卵母細胞を注入する。 TRPV4-cRNAを注入した後、ゲンタマイシン（100μg/ ml）をしてND96培地中で卵母細胞をインキュベートし、1μMのルテニウム14レッド 。 2。二枚の電極電圧クランプ<ol><li>ピペットプラーで個別に精度使い捨て100μlのマイクロピペットからホウケイ酸ガラス記録ピペットを引き出します。 </li><li>電圧測定の両方を引いて、現在の注入ピペット電極は、〜1μmの直径の先端の開口部を持つように引っ張られる。 </li><li> 2 M KClを埋め戻し電極は、0.1〜0.2mΩの抵抗を生じる。一緒VG-2Ax100仮想接地風呂クランプで、Digidata 1440デジタイザを通じてGeneClamp 500アンプインターフェイスに接続されているHS2Aのヘッドステージ、にマウント。取得する<html> pClamp10ソフトウェアを用いてデータを記憶する。 </li><li> 20Xの倍率で解剖顕微鏡のステージ上に取り付け作製したプラスチックチャンバー内の1ml浴中で試験される卵母細胞を配置する。浴溶液を3MのKCl寒天橋と塩素化銀線により、仮想接地されている卵母細胞の近くに配置し、VG-2A仮想接地段に接続。 </li><li>ソリューション入口と出口のようなプラスチックチューブで連続灌流用チャンバーを構築します。入口管は、6の50mlシリンジの殻、異なる溶液が充填された各々のマニホールドで製造された灌流システムに接続され、接続する。いかなる特定のソリューションの重力供給速度を制御していると〜2-3配管の「ケック」ランプクランプを使用してml /分。 </li><li>マイクロマニピュレータ上でのヘッドステージで両方の電極をマウントします。マイクロマニピュレーションによる卵母細胞の電極の貫通を実行します。 </li></ol> 3。直接機械的感受性のパッチクランプ試験&quot;&gt;ピペット製剤、GΩシールを形成し、オンセルの形成または切除されたモードを含むパッチクランプの基本的な方法は、ハミルらのものである。36及び32のConn。 <ol><li> 98のKCl、1mMのMgCl 2、及び10 mMのK +-HEPES、pH7.2で、先端に〜1μmの直径の開口（気泡数5-6）とホウケイ酸ガラスピペットを埋める。 </li><li>また、圧力計に、5ミリリットルの注射器にプラスチックチューブを通過して、T-ジョイントを介してパッチクランプピペットを取り付けます。電極と圧力計の出力の両方を処理するためにコンピュータを使用しています。 10kHzでデータを取得し、その後、分析のために1 kHzで8極ベッセルフィルタを介して再生する。 </li><li>異なるパッチが繰り返し同じ卵母細胞から切り出すことができる。この慣習は、TRPV4の分子活性の検査は、より効率的で信頼性の高いことができます。 </li></ol>

<ol>
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S., 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=Identification+and+characterization+of+a+novel+human+vanilloid+receptor-like+protein,+VRL-2.">Identification and characterization of a novel human vanilloid receptor-like protein, VRL-2.</a> Physiol. Genomics. 4, 165-174 (2001).</li><li>Rock, M. J., 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=Gain-of-function+mutations+in+TRPV4+cause+autosomal+dominant+brachyolmia.">Gain-of-function mutations in TRPV4 cause autosomal dominant brachyolmia.</a> Nat. 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Chem. 277, 33704-33710 (2002).</li><li>Watanabe, H., 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=Modulation+of+TRPV4+gating+by+intra-+and+extracellular+Ca2.">Modulation of TRPV4 gating by intra- and extracellular Ca2.</a> Cell Calcium. 33, 489-495 (2003).</li><li>Loukin, S., Zhou, X., Su, Z., Saimi, Y., Kung, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wild-type+and+brachyolmia-causing+mutant+TRPV4+channels+respond+directly+to+stretch+force.">Wild-type and brachyolmia-causing mutant TRPV4 channels respond directly to stretch force.</a> J. Biol. Chem. 285, 27176-27181 (2010).</li><li>Gao, X., Wu, L., O'Neil, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temperature-modulated+diversity+of+TRPV4+channel+gating:+activation+by+physical+stresses+and+phorbol+ester+derivatives+through+protein+kinase+C-dependent+and+-independent+pathways.">Temperature-modulated diversity of TRPV4 channel gating: activation by physical stresses and phorbol ester derivatives through protein kinase C-dependent and -independent pathways.</a> J. Biol. Chem. 278, 27129-27137 (2003).</li><li>Watanabe, H., 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=Anandamide+and+arachidonic+acid+use+epoxyeicosatrienoic+acids+to+activate+TRPV4+channels.">Anandamide and arachidonic acid use epoxyeicosatrienoic acids to activate TRPV4 channels.</a> Nature. 424, 434-438 (2003).</li><li>Vriens, J., 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=Cell+swelling,+heat,+and+chemical+agonists+use+distinct+pathways+for+the+activation+of+the+cation+channel+TRPV4.">Cell swelling, heat, and chemical agonists use distinct pathways for the activation of the cation channel TRPV4.</a> Proc. Natl. Acad. Sci. U.S.A. 101, 396-401 (2004).</li><li>Vincent, F., 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=Identification+and+characterization+of+novel+TRPV4+modulators.">Identification and characterization of novel TRPV4 modulators.</a> Biochem. Biophys. Res. Commun. 389, 490-494 (2009).</li><li>Willette, R. N., 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=Systemic+activation+of+the+transient+receptor+potential+vanilloid+subtype+4+channel+causes+endothelial+failure+and+circulatory+collapse:+Part+2.">Systemic activation of the transient receptor potential vanilloid subtype 4 channel causes endothelial failure and circulatory collapse: Part 2.</a> J. Pharmacol. Exp. Ther. 326, 443-452 (2008).</li><li>Thorneloe, K. S., 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=An+orally+active+TRPV4+channel+blocker+prevents+and+resolves+pulmonary+edema+induced+by+heart+failure.">An orally active TRPV4 channel blocker prevents and resolves pulmonary edema induced by heart failure.</a> Sci. Transl. Med. 4, (2012).</li><li>Kang, S. S., Shin, S. H., Auh, C. K., Chun, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+skeletal+dysplasia+caused+by+a+constitutive+activated+transient+receptor+potential+vanilloid+4+(TRPV4)+cation+channel+mutation.">Human skeletal dysplasia caused by a constitutive activated transient receptor potential vanilloid 4 (TRPV4) cation channel mutation.</a> Exp. Mol. Med. 44, 707-722 (2012).</li><li>Lamande, S. R., 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=Mutations+in+TRPV4+cause+an+inherited+arthropathy+of+hands+and+feet.">Mutations in TRPV4 cause an inherited arthropathy of hands and feet.</a> Nat. Genet. 43, 1142-1146 (2011).</li><li>Loukin, S., Su, Z., Kung, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+Basal+Activity+Is+a+Key+Determinant+in+the+Severity+of+Human+Skeletal+Dysplasia+Caused+by+TRPV4+Mutations.">Increased Basal Activity Is a Key Determinant in the Severity of Human Skeletal Dysplasia Caused by TRPV4 Mutations.</a> PLoS One. 6, (2011).</li><li>Loukin, S. H., Su, Z., Kung, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypotonic+shocks+activate+rat+TRPV4+in+yeast+in+the+absence+of+polyunsaturated+fatty+acids.">Hypotonic shocks activate rat TRPV4 in yeast in the absence of polyunsaturated fatty acids.</a> FEBS Lett. 583, 754-758 (2009).</li><li>Loukin, S., Su, Z., Zhou, X., Kung, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Forward+genetic+analysis+reveals+multiple+gating+mechanisms+of+TRPV4.">Forward genetic analysis reveals multiple gating mechanisms of TRPV4.</a> J. Biol. Chem. 285, 19884-19890 (2010).</li><li>Batiza, A. F., Schulz, T., Masson, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Yeast+respond+to+hypotonic+shock+with+a+calcium+pulse.">Yeast respond to hypotonic shock with a calcium pulse.</a> J. Biol. Chem. 271, 23357-23362 (1996).</li><li>Amberg, D. C., Burke, D., Strathern, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Methods in Yeast Genetics: a Cold Spring Harbor Laboratory Course Manual. , (2005).</li><li>Loukin, S., Zhou, X., Kung, C., Saimi, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+genome-wide+survey+suggests+an+osmoprotective+role+for+vacuolar+Ca2++release+in+cell+wall-compromised+yeast.">A genome-wide survey suggests an osmoprotective role for vacuolar Ca2+ release in cell wall-compromised yeast.</a> FASEB J. 22, 2405-2415 (2008).</li><li>Denis, V., Cyert, M. 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著者は、彼らが競合する経済的利益を持っていないことを宣言します。

冒頭で述べたように、一般のTRPV4の機能を研究するために使用される方法は、時には矛盾し、論争となった。ここに記載された方法の二組は、いくつかの利点を提供し、既存の方法を補完することができる。我々はTRPV4でのみ研究を記載しているが、これらの方法は、他のイオンチャネルを研究するために拡張することができる。 1。酵母ルミノメトリー方法<p class="jove_...

のTRP（一過性受容体電位）が感覚機能を、1,2を果たす陽イオンチャネルの7のサブファミリーを構成する。哺乳類では、TRPV、のTRPのバニロイドサブファミリーは、6種類があります。 TRPV4（タイプ4） の3,4熱に反応する、特定の化学物質、浸透圧膨張、またはせん断応力。 TRPV4遺伝子を繰り返し、候補遺伝子および/ ​​または5-8発現クローニングによって単離した。後者の方法は、低刺激浸透圧モル濃度の遺伝子産物の応答を行った。 TRPV4は、開発、生理学、または多くの異なる細胞型の病理3,4のほぼ全ての器官および機能に発現される。 印象的には、末梢神経障害および/ ​​または骨格異形成（骨格の発達の異常）9月11日に発生し、&gt; 50人の常染色体優性のTRPV4変異である。セベ軽度brachyomiaタイプ3（タイプ3小人症）、spondylometaphyseal異形成コズロウスキータイプから骨格異形成範囲異形成RE、いくつかは、新生児または胚死を引き起こす。メカニズムのすべてのマナーが可能に見えるけど、どれも多様性、複雑性、多様性、およびこれらの疾患4の時々の重なりを説明していません。 他のTRPチャネル1と同様に、TRPV4は四量体である。ラットまたはヒトTRPV4では、各サブユニットは871残基から構成されている。その中心的な要素は、おそらく電位依存性K +チャネルと同様に配置された6つの膜貫通ヘリックス（S1-S6）である。そこでは、S1〜S4は、周辺領域を形成し、S5およびS6は、透過孔のドメインを形成する。 TRPV4のS5、S6の間、おそらく陽フィルタを形成するために収束そのうちの4つのシーケンスLDLFKLTIGMGDL、続く短いポアへリックスである。 470残基のN末端細胞質セグメントは、タンパク質または小リガンドを結合することが知られている6アンキリンリピートを含んでいます。 C末端152残基の細胞質セグメントは、OT結合する他の候補地の中でカルモジュリン結合配列を含む彼女の要素3。 TRPV4は、基本的に陰イオン1を除いた陽イオンチャネルである。その生理的機能は、Ca 2 +流入に刺激を伝達することですが、それは、P カルシウムの二価に有利、またアイゼンマンIV透過性配列と他の陽イオンを透過させる。P ナ 〜7 12。単一チャネルコンダクタンスは、〜90 psの外側へと〜40 psの内側6,13,14で整流する。異種発現（下）現在は低浸透腫れ、せん断応力、または暖かさ15によって活性化することができる。また、多価不飽和脂肪酸は16,17及び合成ホルボールエステルが18を 4αPDDによって活性化される。現時点では、どちらも高スループット、低分子スクリーニングによって発見された、10 -9〜10 -8 Mの20に効果的で、最も強力なアゴニストはGSK1016790A 19で、アンタゴニストはGSK2193874です。 TRPV4研究の二つの主要な分野は混乱のまま：TRPV4は、主にその機械的感受性のために研究されていても（1）、その分子基盤は、物議を醸している。 1つのモデルは、低刺激オスモル濃度が何らかの方法でエポキシゲナーゼによって酸（EET）をエポキシエイコサトリエンに変換される多価不飽和脂肪酸（PUFA）、アラキドン酸（AA）を生成するために、ホスホリパーゼA2（PLA2）を活性化し、EET ​​の結合は、TRPV4 16を活性化について説明17。しかし、TRPV4自体が直接簡単な説明を提供する、膜ストレッチ14（下）に応答することが示されている。 （2）TRPV4変異体の病状は途方に暮れるです。財団では、人は病気が消失、縮小、またはチャネル活動の増加によるものであるかどうかを知る必要があります。でも、ここで、文献は遠い透明からです。複数の骨格異形成の対立遺伝子は高い活性を有することが報告されたが、（機能獲得、GOF）4,21は 、いくつかは、活動（機能喪失、LOF）10,22が低下していると報告された。 14対立遺伝子の体系的な研究は、それらを発見すべてのGOFする変異（下）23。 TRPV4機能の完全な損失にもかかわらず、わずかな欠陥を持つ実行可能か、肥沃であるノックアウトマウス、、、 -いくつかのLOFSある主張は、TRPV4-/の表現型と矛盾するように思われる。 これらの論争の一部は方法論的な起源を持っています。研究所では、異なる方法、または1つの方法のバリアントを使用し、異なる判断基準を採用する。最も一般的には、TRPV4一過 （HEK、CHO、HeLa細胞）およびアゴニストまたは低張性刺激の際、内部の[Ca 2 +]の上昇をのCa 2 +感受性蛍光染料、フラ-2に登録されている培養哺乳動物細胞で発現される。この蛍光分析上の過度の依存は、1を批判されている。異なる集団における発現レベルは、その中に分布、ならびに効果的な色素濃度は、制御され、文書化される必要がある。より信頼性の高い、直接電気生理学的検査である。これさえ、commonlなどYは、練習問題もなくもありません。個々の培養細胞における発現レベルの制御が困難であるため、全細胞電流は大きな変動を有する。電流が小さいため、さらに、信頼できる統計は、多くの場合、実用的ではない大規模なサンプルサイズ、に依存する必要があります。パッチクランプ試験はほとんど行われていない。いくつかのそのような記録は、16,17に挑戦統計的評価を行い、活動のバーストのクラスタを示しています。 よりよい分子機械的感受性を理解するためには、TRPV4を調べるために2追加のシステムを開発しました。 （1）離れて通常の哺乳類のパートナーからTRPV4を単離するために、我々は、酵母24出芽にラットTRPV4を表明している。この進化的に遠い文脈におけるTRPV4の機能的発現は、それはまだ通常のパートナーなしで、浸透力に対応できることを示した。酵母は、AAまたはEET何らPUFAを行いませんし、そのゲノムは、PLA2またはエポキシゲの遺伝子を持っていない、このexpreので、ssionはまた、TRPV4力を感知するためにそれらが必要とされないことを示している。分子生物学的に最も影響を受けやすい真核生物では、TRPV4を持つことは、効率的な順方向または逆遺伝子操作25を可能にします。 （2）TRPV4の詳細な生物物理学的な分析のために、我々はアフリカツメガエル卵母細胞においてTRPV4を表明した。 NA（10 -10〜10 -9 A）の電流にサブ-NAをもたらす培養細胞とは異なり、卵母細胞はμAの（10 -6〜10 -4 A）の電流を表現しています。ノイズの上はるかに大きい信号がより良く定量化し、より多くの自信を持って比較することができます。 TRPV4は、そのように発現され、またパッチクランプを使用して、個々の分子として調べることができる。シングル卵母細胞は再び定量化は、より信頼性の高いことが繰り返さパッチのサンプリングを可能にします。このような研究は、TRPV4チャネル自体を直接、膜延伸力14によって活性化され得ることを示した。分析はまた、14の代表的な骨格異形成対立遺伝子がすべての機能獲得型突然変異であることが示された。さらに、DEGRこの構成のCa 2 +漏れのeeは、骨格疾患23の重大匹敵する。 ため、その新規性や有用性は、これらの2つの方法の詳細な手順は、TRPV4または類似のチャネルでの今後の研究で複製を可能にするためにここに組み立てられる。

<table border="0" cellpadding="0" cellspacing="0" width="595">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Vapor Pressure Osmometer</td>
			<td style="width:93px;">Wescor</td>
			<td style="width:119px;">Wescor 5500</td>
			<td style="width:167px;">Logan, UT, USA</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Sirius Single Tube Luminometer </td>
			<td style="width:93px;">Titertek-Berhold</td>
			<td style="width:119px;">Sirius </td>
			<td>Huntsville, LA, USA</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Microplate luminometer</td>
			<td style="width:93px;">Titertek-Berthold </td>
			<td style="width:119px;">Mithras LB940</td>
			<td>Huntsville, LA, USA</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Luminometry software</td>
			<td style="width:93px;">Titertek-Berthold</td>
			<td style="width:119px;">MikroWin2000</td>
			<td>Huntsville, LA, USA</td>
		</tr>
		<tr height="210">
			<td height="210" style="height:210px;width:216px;">Patch clamp and software</td>
			<td style="width:93px;">Axon Instrument</td>
			<td style="width:119px;">HS2A headstage, VG-2Ax100 virtual grounded bath clamp, GeneClamp 500 amplifier, digidata 1440 digitizer, pClamp10 software </td>
			<td>Union City, CA, USA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">manometer</td>
			<td style="width:93px;">Bio-Tec</td>
			<td style="width:119px;"> </td>
			<td>Winooski, VT, USA</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Pipet puller</td>
			<td style="width:93px;">Sutter Instrument Co</td>
			<td style="width:119px;">Model P-97</td>
			<td>Novata, CA, USA</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Microinjector</td>
			<td style="width:93px;">Drummond Scientific</td>
			<td style="width:119px;">Nanoinject II</td>
			<td>Broomall, PA, USA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Material:</td>
			<td style="width:93px;"> </td>
			<td style="width:119px;"> </td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">luciferin coelenterazine</td>
			<td style="width:93px;">Invitrogen</td>
			<td style="width:119px;"> </td>
			<td>Carlsbad, CA, USA</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">T7 RNA polymerase reaction</td>
			<td style="width:93px;">Ambion</td>
			<td style="width:119px;">mMessage mMachine</td>
			<td>Austin, TX, USA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">gentamicin</td>
			<td style="width:93px;">Sigma</td>
			<td style="width:119px;"> </td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">ruthenium red </td>
			<td style="width:93px;">Sigma</td>
			<td style="width:119px;"> </td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">micropipets </td>
			<td style="width:93px;">Drummond</td>
			<td style="width:119px;">100 microliter micropipets</td>
			<td>Broomall, PA, USA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">high-fidelity PfuUtra polymerase </td>
			<td style="width:93px;">Stratagene</td>
			<td style="width:119px;"> </td>
			<td>La Jolla, CA, USA</td>
		</tr>
	</tbody>
</table>

yeast luminometric and xenopus oocyte electrophysiological examinations of the molecular mechanosensitivity of trpv4

TRPV4（一過性受容体電位バニロイドファミリー、タイプ4）が広く脊椎動物の組織において発現され、機械的な力によるものを含むいくつかの刺激によって活性化される。特定のTRPV4の変異は、人間の複雑な遺伝性骨や神経細胞の病状を引き起こす。野生型または変異TRPV4導入遺伝子は、一般的に培養された哺乳動物細胞で発現し、フラ-2蛍光法により、電極によって検査されています。機械的感受性のメカニズムと疾患の分子基盤の面では、現在の文献は混乱し、物議を醸している。既存のメソッドを補完するために、我々はTRPV4の分子特性を調べるために二つのメソッドを記述します。 （1）ラットTRPV4およびエクオリン導入遺伝子は、出芽酵母に形質転換する。形質転換体の人口の低刺激osmticショックはTRPV4チャネルを通じて放出によるCa 2 +を持つイクオリンの組み合わせに発光測定信号が得られます。ここでは、TRPV4は通常の哺乳類のパートナータンパク質から分離されているそして、独自の機械的感受性を明らかにする。 （2）TRPV4のcRNAをアフリカツメガエル卵母細胞に注入される。適当なインキュベーション期間の後、TRPV4巨視的な電流は、2電極電圧クランプを用いて試験される。卵母浴からの不活性浸透圧調節物質の除去の際に電流の上昇は機械的感受性の指標である。卵母細胞からのマイクロアンペア（10 -6 A -4〜10）の電流が明確に定量化し、より多くの自信を持って評価を得培養細胞からナノアンペアサブナノへ（10 -10 A -9〜10）の電流よりもはるかに大きい。個々のチャネルタンパク質の活性を反映微視的電流も上に直接細胞または切除されたモードでは、パッチクランプの下に登録することができる。同じ卵母細胞は、より良いデータ複製が可能、複数のパッチのサンプルが用意されています。パッチに適用される吸引は直接機械的感受性を評価するためにTRPV4を活性化することができる。これらの方法は、TRPチャネルの他のタイプの研究に有用であるはずである。

典型的な発光測定実験では、400-1,200ミリオスモルダウン低張性ショックの範囲は、1400ミリオスモルでの培養液20μlに異なる低浸透圧ショック溶液を200μLを添加することによって達成される。空p416GPDV4プラスミド又はイオンフィルタ（ 図1）24における変異を有するTRPV4遺伝子を含むもので形質転換された酵母細胞：実験のRLU二つの陰性対照と比較したときTRPV4活性が?...

Watch this Scientific Journal Video about Yeast Luminometric and Xenopus Oocyte Electrophysiological Examinations of the Molecular Mechanosensitivity of TRPV4 at JoVE.com

Yeast Luminometric and Xenopus Oocyte Electrophysiological Examinations of the Molecular Mechanosensitivity of TRPV4

TRPV4のを研究するためによく使用されるメソッドを補完するために、二つの方法が記載されている：その機械的感受性は、低浸透チャレンジの際に遺伝子組換え酵母中のCa 2 +-エクオリン発光測定により研究することができる。また、オンセルまたは切除されたモードでクランプ全細胞の2電極電圧クランプまたはパッチによりアフリカツメガエル卵母細胞に注入TRPV4-RNAで検査することができる。

酵母発光測定と<em&gt;アフリカツメガエル</emTRPV4の分子機械的感受性の&gt;卵母細胞電気生理学的検定試験

TRPV4 (Transient Receptor Potentials, vanilloid family, type 4) is widely expressed in vertebrate tissues and is activated by several stimuli, including ...

yeast-luminometric-xenopus-oocyte-electrophysiological-examinations

Research

JoVE Journal

Biology

Yeast Luminometric and Xenopus Oocyte Electrophysiological Examinations of the Molecular Mechanosensitivity of TRPV4

10.0K ビュー.  University of Wisconsin – Madison.  TRPV4のを研究するためによく使用されるメソッドを補完するために、二つの方法が記載されている：その機械的感受性は、低浸透チャレンジの際に遺伝子組換え酵母中のCa 2 +-エクオリン発光測定により研究することができる。また、オンセルまたは切除されたモードでクランプ全細胞の2電極電圧クランプまたはパッチによりアフリカツメガエ...

ビデオ: Yeast Luminometric and Xenopus Oocyte Electrophysiological Examinations of the Molecular Mechanosensitivity of TRPV4

A Rapid Technique for the Visualization of Live Immobilized Yeast Cells

We present here a simple, rapid, and extremely flexible technique for the immobilization and visualization of growing yeast cells by epifluorescence microscopy. The technique is equally suited for visualization of static yeast populations, or time courses experiments up to ten hours in length. My microscopy investigates epigenetic inheritance at the silent mating loci in S. cerevisiae. There are two silent mating loci, HML and HMR, which are normally not expressed as they are packaged in heterochromatin. In the sir1 mutant background silencing is weakened such that each locus can either be in the expressed or silenced epigenetic state, so in the population as a whole there is a mix of cells of different epigenetic states for both HML and HMR. My microscopy demonstrated that there is no relationship between the epigenetic state of HML and HMR in an individual cell. sir1 cells stochastically switch epigenetic states, establishing silencing at a previously expressed locus or expressing a previously silenced locus. My time course microscopy tracked individual sir1 cells and their offspring to score the frequency of each of the four possible epigenetic switches, and thus the stability of each of the epigenetic states in sir1 cells. See also  Xu et al., Mol. Cell 2006.

A rapid technique for the visualization of growing immobilized yeast cells, here applied to fluorescent reporters at the silent mating loci HML and HMR

ライブ固定化酵母細胞の可視化のための高速手法

We present here a simple, rapid, and extremely flexible technique for the immobilization and visualization of growing yeast cells by epifluorescence ...

生物学

Patch Clamp Recording of Ion Channels Expressed in  Xenopus Oocytes

Since its development by Sakmann and Neher 1, 2, the patch clamp has become established as an extremely useful technique for electrophysiological measurement of single or multiple ion channels in cells. This technique can be applied to ion channels in both their native environment and expressed in heterologous cells, such as oocytes harvested from the African clawed frog, Xenopus laevis. Here, we describe the well-established technique of patch clamp recording from Xenopus oocytes. This technique is used to measure the properties of expressed ion channels either in populations (macropatch) or individually (single-channel recording). We focus on techniques to maximize the quality of oocyte preparation and seal generation. With all factors optimized, this technique gives a probability of successful seal generation over 90 percent. The process may be optimized differently by every researcher based on the factors he or she finds most important, and we present the approach that have lead to the greatest success in our hands.

This is intended as an introduction to patch clamp recording from Xenopus laevis oocytes. It covers vitelline membrane removal, formation of a gigaohm seal (gigaseal), and the optional conversion of the patch to the outside-out topology.

アフリカツメガエルの卵母細胞で発現イオンチャネルのパッチクランプ記録

Since its development by Sakmann and Neher 1, 2, the patch clamp has become established as an extremely useful technique for electrophysiological ...

Visualizing RNA Localization in Xenopus Oocytes

RNA localization is a conserved mechanism of establishing cell polarity. Vg1 mRNA localizes to the vegetal pole of Xenopus laevis oocytes and acts to spatially restrict gene expression of Vg1 protein. Tight control of Vg1 distribution in this manner is required for proper germ layer specification in the developing embryo. RNA sequence elements in the 3' UTR of the mRNA, the Vg1 localization element (VLE) are required and sufficient to direct transport. To study the recognition and transport of Vg1 mRNA in vivo, we have developed an imaging technique that allows extensive analysis of trans-factor directed transport mechanisms via a simple visual readout. 
To visualize RNA localization, we synthesize fluorescently labeled VLE RNA and microinject this transcript into individual oocytes. After oocyte culture to allow transport of the injected RNA, oocytes are fixed and dehydrated prior to imaging by confocal microscopy. Visualization of mRNA localization patterns provides a readout for monitoring the complete pathway of RNA transport and for identifying roles in directing RNA transport for cis-acting elements within the transcript and trans-acting factors that bind to the VLE (Lewis et al., 2008, Messitt et al., 2008). We have extended this technique through co-localization with additional RNAs and proteins (Gagnon and Mowry, 2009, Messitt et al., 2008), and in combination with disruption of motor proteins and the cytoskeleton (Messitt et al., 2008) to probe mechanisms underlying mRNA localization.

Visualizing RNA Localization in Xenopus Oocytes

Visualization of in vivo RNA transport is accomplished by microinjection of fluorescently labeled RNA transcripts into Xenopus oocytes, followed by confocal microscopy.

可視化RNAの局在にアフリカツメガエル卵母細胞

RNA localization is a conserved mechanism of establishing cell polarity.  Vg1 mRNA localizes to the vegetal pole of Xenopus laevis oocytes and acts to ...

A Fluorescence Microscopy Assay for Monitoring Mitophagy in the Yeast Saccharomyces cerevisiae

Autophagy is important for turnover of cellular components under a range of different conditions. It serves an essential homeostatic function as well as a quality control mechanism that can target and selectively degrade cellular material including organelles1-4. For example, damaged or redundant mitochondria (Fig. 1), not disposed of by autophagy, can represent a threat to cellular homeostasis and cell survival. In the yeast, Saccharomyces cerevisiae, nutrient deprivation (e.g., nitrogen starvation) or damage can promote selective turnover of mitochondria by autophagy in a process termed mitophagy 5-9.
We describe a simple fluorescence microscopy approach to assess autophagy. For clarity we restrict our description here to show how the approach can be used to monitor mitophagy in yeast cells. The assay makes use of a fluorescent reporter, Rosella, which is a dual-emission biosensor comprising a relatively pH-stable red fluorescent protein linked to a pH-sensitive green fluorescent protein. The operation of this reporter relies on differences in pH between the vacuole (pH ~ 5.0-5.5) and mitochondria (pH ~ 8.2) in living cells. Under growing conditions, wild type cells exhibit both red and green fluorescence distributed in a manner characteristic of the mitochondria. Fluorescence emission is not associated with the vacuole. When subjected to nitrogen starvation, a condition which induces mitophagy, in addition to red and green fluorescence labeling the mitochondria, cells exhibit the accumulation of red, but not green fluorescence, in the acidic vacuolar lumen representing the delivery of mitochondria to the vacuole. Scoring cells with red, but not green fluorescent vacuoles can be used as a measure of mitophagic activity in cells5,10-12.

A Fluorescence Microscopy Assay for Monitoring Mitophagy in the Yeast Saccharomyces cerevisiae

A robust approach to monitor the delivery of organelles to the acidic lumen of the yeast vacuole for degradation and recycling is described. The method relies on the specific labeling of target organelles with a genetically encoded dual-emission fluorescence pH-biosensor, and visualization of individual cells using fluorescence microscopy.

酵母でMitophagyを監視するための蛍光顕微鏡アッセイサッカロマイセスセレビシエ

Autophagy is important for turnover of cellular components under a range of different conditions. It serves an essential homeostatic function as well as a ...

Mouse Oocyte Microinjection, Maturation and Ploidy Assessment

Mistakes in chromosome segregation lead to aneuploid cells. In somatic cells, aneuploidy is associated with cancer but in gametes, aneuploidy leads to infertility, miscarriages or developmental disorders like Down syndrome. Haploid gametes form through species-specific developmental programs that are coupled to meiosis. The first meiotic division (MI) is unique to meiosis because sister chromatids remain attached while homologous chromosomes are segregated. For reasons not fully understood, this reductional division is prone to errors and is more commonly the source of aneuploidy than errors in meiosis II (MII) or than errors in male meiosis 1,2. 
In mammals, oocytes arrest at prophase of MI with a large, intact germinal vesicle (GV; nucleus) and only resume meiosis when they receive ovulatory cues. Once meiosis resumes, oocytes complete MI and undergo an asymmetric cell division, arresting again at metaphase of MII. Eggs will not complete MII until they are fertilized by sperm. Oocytes also can undergo meiotic maturation using established in vitro culture conditions 3. Because generation of transgenic and gene-targeted mouse mutants is costly and can take long periods of time, manipulation of female gametes in vitro is a more economical and time-saving strategy. 
Here, we describe methods to isolate prophase-arrested oocytes from mice and for microinjection. Any material of choice may be introduced into the oocyte, but because meiotically-competent oocytes are transcriptionally silent 4,5 cRNA, and not DNA, must be injected for ectopic expression studies. To assess ploidy, we describe our conditions for in vitro maturation of oocytes to MII eggs. Historically, chromosome-spreading techniques are used for counting chromosome number 6. This method is technically challenging and is limited to only identifying hyperploidies. Here, we describe a method to determine hypo-and hyperploidies using intact eggs 7-8. This method uses monastrol, a kinesin-5 inhibitor, that collapses the bipolar spindle into a monopolar spindle 9 thus separating chromosomes such that individual kinetochores can readily be detected and counted by using an anti-CREST autoimmune serum. Because this method is performed in intact eggs, chromosomes are not lost due to operator error.

Oocytes are prone to aneuploidy due to errors in chromosome segregation during meiotic maturation. Aneuploid eggs can cause infertility, miscarriages or developmental disorders like Down syndrome. Here, we describe methods to introduce materials of choice into oocytes and methods to study meiotic maturation and assess ploidy.

マウス卵母細胞のマイクロインジェクション、成熟および倍数性評価

Mistakes in chromosome segregation lead to aneuploid cells.  In somatic cells, aneuploidy is associated with cancer but in gametes, aneuploidy leads to ...

Competitive Genomic Screens of Barcoded Yeast Libraries

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily. The tempo of these advances is accelerating, promising greater depth and breadth. In light of these extraordinary advances, the need for fast, parallel methods to define gene function becomes ever more important. Collections of genome-wide deletion mutants in yeasts and E. coli have served as workhorses for functional characterization of gene function, but this approach is not scalable, current gene-deletion approaches require each of the thousands of genes that comprise a genome to be deleted and verified. Only after this work is complete can we pursue high-throughput phenotyping. Over the past decade, our laboratory has refined a portfolio of competitive, miniaturized, high-throughput genome-wide assays that can be performed in parallel. This parallelization is possible because of the inclusion of DNA 'tags', or 'barcodes,' into each mutant, with the barcode serving as a proxy for the mutation and one can measure the barcode abundance to assess mutant fitness. In this study, we seek to fill the gap between DNA sequence and barcoded mutant collections. To accomplish this we introduce a combined transposon disruption-barcoding approach that opens up parallel barcode assays to newly sequenced, but poorly characterized microbes. To illustrate this approach we present a new Candida albicans barcoded disruption collection and describe how both microarray-based and next generation sequencing-based platforms can be used to collect 10,000 - 1,000,000 gene-gene and drug-gene interactions in a single experiment.

We have developed comprehensive, unbiased genome-wide screens to understand gene-drug and gene-environment interactions. Methods for screening these mutant collections are presented.

バーコード酵母ライブラリーの競争力のあるゲノム画面

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily.  The tempo of these advances is ...

Chromatographic Purification of Highly Active Yeast Ribosomes

Eukaryotic ribosomes are much more labile as compared to their eubacterial and archael counterparts, thus posing a significant challenge to researchers. Particularly troublesome is the fact that lysis of cells releases a large number of proteases and nucleases which can degrade ribosomes. Thus, it is important to separate ribosomes from these enzymes as quickly as possible. Unfortunately, conventional differential ultracentrifugation methods leaves ribosomes exposed to these enzymes for unacceptably long periods of time, impacting their structural integrity and functionality. To address this problem, we utilize a chromatographic method using a cysteine charged Sulfolink resin. This simple and rapid application significantly reduces co-purifying proteolytic and nucleolytic activities, producing high yields of intact, highly biochemically active yeast ribosomes. We suggest that this method should also be applicable to mammalian ribosomes. The simplicity of the method, and the enhanced purity and activity of chromatographically purified ribosome represents a significant technical advancement for the study of eukaryotic ribosomes.

Contamination of preparations of eukaryotic ribosomes purified by traditional methods by co-purifying nucleases and proteases negatively impacts on downstream biochemical and structural analyses. A rapid and simple chromatographic purification method is used to solve this problem using yeast ribosomes as a model system.

高活性酵母のリボソームのクロマトグラフィー精製

Eukaryotic ribosomes are much more labile as compared to their eubacterial and archael counterparts, thus posing a significant challenge to researchers.  ...

The Green Monster Process for the Generation of Yeast Strains Carrying Multiple Gene Deletions

Phenotypes for a gene deletion are often revealed only when the mutation is tested in a particular genetic background or environmental condition1,2. There are examples where many genes need to be deleted to unmask hidden gene functions3,4. Despite the potential for important discoveries, genetic interactions involving three or more genes are largely unexplored. Exhaustive searches of multi-mutant interactions would be impractical due to the sheer number of possible combinations of deletions. However, studies of selected sets of genes, such as sets of paralogs with a greater a priori chance of sharing a common function, would be informative.
In the yeast Saccharomyces cerevisiae, gene knockout is accomplished by replacing a gene with a selectable marker via homologous recombination. Because the number of markers is limited, methods have been developed for removing and reusing the same marker5,6,7,8,9,10. However, sequentially engineering multiple mutations using these methods is time-consuming because the time required scales linearly with the number of deletions to be generated.
Here we describe the Green Monster method for routinely engineering multiple deletions in yeast11. In this method, a green fluorescent protein (GFP) reporter integrated into deletions is used to quantitatively label strains according to the number of deletions contained in each strain (Figure 1). Repeated rounds of assortment of GFP-marked deletions via yeast mating and meiosis coupled with flow-cytometric enrichment of strains carrying more of these deletions lead to the accumulation of deletions in strains (Figure 2). Performing multiple processes in parallel, with each process incorporating one or more deletions per round, reduces the time required for strain construction.
The first step is to prepare haploid single-mutants termed 'ProMonsters,' each of which carries a GFP reporter in a deleted locus and one of the 'toolkit' loci&#x2014;either Green Monster GMToolkit-a or GMToolkit-&alpha; at the can1&Delta; locus (Figure 3). Using strains from the yeast deletion collection12, GFP-marked deletions can be conveniently generated by replacing the common KanMX4 cassette existing in these strains with a universal GFP-URA3 fragment. Each GMToolkit contains: either the a- or &alpha;-mating-type-specific haploid selection marker1 and exactly one of the two markers that, when both GMToolkits are present, collectively allow for selection of diploids.
The second step is to carry out the sexual cycling through which deletion loci can be combined within a single cell by the random assortment and/or meiotic recombination that accompanies each cycle of mating and sporulation.

The Green Monster method enables the rapid assembly of multiple deletions marked with a reporter gene encoding green fluorescent protein. This method is based on driving yeast strains through repeated cycles of sexual assortment of deletions and fluorescence-based enrichment of cells carrying more deletions.

複数の遺伝子欠失を持つ酵母菌株の世代のためのグリーンモンスタープロセス

Phenotypes  for a gene deletion are often revealed only when the mutation is tested in a  particular genetic background or environmental condition1,2. ...

The Xenopus Oocyte Cut-open Vaseline Gap Voltage-clamp Technique With Fluorometry

The cut-open oocyte Vaseline gap (COVG) voltage clamp technique allows for analysis of electrophysiological and kinetic properties of heterologous ion channels in oocytes. Recordings from the cut-open setup are particularly useful for resolving low magnitude gating currents, rapid ionic current activation, and deactivation. The main benefits over the two-electrode voltage clamp (TEVC) technique include increased clamp speed, improved signal-to-noise ratio, and the ability to modulate the intracellular and extracellular milieu.
Here, we employ the human cardiac sodium channel (hNaV1.5), expressed in Xenopus oocytes, to demonstrate the cut-open setup and protocol as well as modifications that are required to add voltage clamp fluorometry capability.
The properties of fast activating ion channels, such as hNaV1.5, cannot be fully resolved near room temperature using TEVC, in which the entirety of the oocyte membrane is clamped, making voltage control difficult. However, in the cut-open technique, isolation of only a small portion of the cell membrane allows for the rapid clamping required to accurately record fast kinetics while preventing channel run-down associated with patch clamp techniques.
In conjunction with the COVG technique, ion channel kinetics and electrophysiological properties can be further assayed by using voltage clamp fluorometry, where protein motion is tracked via cysteine conjugation of extracellularly applied fluorophores, insertion of genetically encoded fluorescent proteins, or the incorporation of unnatural amino acids into the region of interest1. This additional data yields kinetic information about voltage-dependent conformational rearrangements of the protein via changes in the microenvironment surrounding the fluorescent molecule.

The Xenopus Oocyte Cut-open Vaseline Gap Voltage-clamp Technique With Fluorometry

The cut-open Vaseline gap approach is used to obtain low noise recordings of ionic and gating currents from voltage-dependent ion channels expressed in Xenopus oocytes with high resolution of fast channel kinetics. With minor modification, voltage clamp fluorometry can be coupled to the cut-open oocyte protocol.

ザ·<em&gt;アフリカツメガエル</em蛍光測定で&gt;卵母細胞のカットオープンワセリンギャップ電圧クランプ法

The cut-open oocyte Vaseline gap (COVG) voltage clamp technique allows for analysis of electrophysiological and kinetic properties of heterologous ion ...

Surface Spreading and Immunostaining of Yeast Chromosomes

The small size of nuclei of the budding yeast Saccharomyces cerevisiae limits the utility of light microscopy for analysis of the subnuclear distribution of chromatin-bound proteins. Surface spreading of yeast nuclei results in expansion of chromatin without loss of bound proteins. A method for surface spreading balances fixation of DNA bound proteins with detergent treatment. The method demonstrated is slightly modified from that described by Josef Loidl and Franz Klein1,2. The method has been used to characterize the localization of many chromatin-bound proteins at various stages of the mitotic cell cycle, but is especially useful for the study of meiotic chromosome structures such as meiotic recombinosomes and the synaptonemal complex. We also describe a modification that does not require use of Lipsol, a proprietary detergent, which was called for in the original procedure, but no longer commercially available. An immunostaining protocol that is compatible with the chromosome spreading method is also described.

A method for surface-spreading chromosomes from budding yeast is presented. This method is derived from a method previously described by Loidl and Klein. In addition, we demonstrate a procedure for immunostaining of spread chromosomes.

表面拡散と酵母染色体の免疫染色

The small size of nuclei of the budding yeast Saccharomyces cerevisiae limits the utility of light microscopy for analysis of the subnuclear distribution ...