We thank Sue Lindquist, Aaron Gitler, and Martin Duennwald for kindly sharing reagents. Our studies were supported by: an American Heart Association Post-Doctoral Fellowship (M.E.J); NIH Director's New Innovator Award DP2OD002177, NIH grants R21NS067354, R21HD074510, and R01GM099836, a Muscular Dystrophy Association Research Award (MDA277268), Packard Center for ALS Research at Johns Hopkins University, Target ALS, and an Ellison Medical Foundation New Scholar in Aging Award (J.S.).

1.ライブラリの生成<ol><li>ドメイン固有のエラープローンPCRを用いて、Hsp104ののライブラリーを構築するために、最初のエラーを起こしやすいDNAポリメラーゼ20で関心のあるドメインを増幅する。 </li><li>ゲル抽出によってPCR産物を精製する。 </li><li>標準的な部位特異的突然変異誘発プロトコルを使用して、メガプライマー伸長工程を実行する：PCR緩衝液中の50 ngの鋳型プラスミド、250ngののメガプライマー、200μMのdNTP類、および高忠実度DNAポリメラーゼを結合し、そしてPCRグレードの水20との50μlの総容量まで希釈する。標準的なPCRプログラムを実行します。 注：使用される特定のプライマーは、増幅される遺伝子の特定領域に基づいて異なります。 <ol><li> PCRに続いて、37℃で2時間の1μl のDpn I制限酵素で親テンプレートDNAを消化する。 </li></ol></li><li>ウルトラEにエレクトロポレーションまたは他の手段によってライブラリを変換する大腸菌は、ミニプレップによってそれを浄化し。 注：ライブラリ世代ほぼ所定の実験の目的に応じて変化する。 Hsp104のは、非常に大きなタンパク質であるので、私たちは、ドメイン固有のエラープローンPCRを利用した理由である全遺伝子を、ランダム化することは非現実的である。さらに、Hsp104のの構造は21に挑戦監督ライブラリの設計を行うこと、よくわかっていないままです。ライブラリは、唯一の制限は、テンプレートプラスミド骨格はデキストロース培地上で5-FOAの対抗選択を可能にするためにURA3遺伝子を含むべきであることであると共に、突然変異誘発またはランダムアプローチを用いて構築することができる。 </li></ol> Hsp104の図書館の2変容<ol><li>標準酢酸リチウム/ PEG形質転換プロトコル22を使用してW303aΔのHSP104の酵母に疾患関連基板を統合します。単一のコロニーを選択して、基板23を関連する疾患の高い毒性を持つ菌株を分離するために、毒性のためにそれらを選別。 注：統合されたひずみとイソラを使用してくださいすべてのセルに等しい発現を確実にするために単一のコロニーをteの。 pAG416GALプラスミド（ウラシルマーカー）24 ​​にウラシル以外の任意のマーカー（我々はヒスチジンを使用）と、Hsp104のライブラリ下にある遺伝子の組み込みを可能にするプラスミドに病気基板のクローンを作成。 5-FOAの対抗を可能にするために、ライブラリはウラシルマーカーとプラスミドから発現されていることを確認。他の酵母株を用いることもできる。我々は両方のWT及びΔHSP104背景 （MEJとJS、未発表の観察）にW303a及びBY4741酵母菌株を使用して同様の毒性抑制表現型を指摘している。 </li><li>同じ酢酸リチウム/ PEG形質転換プロトコル22を使用して、この株にHsp104のライブラリを変換します。ライブラリーの配列スペースを維持するために、予測ライブラリーサイズを維持するために適切に変換をスケールアップ。 </li><li>十分なプレートにを使用して非誘導、選択プレート（ 例えば 、SD-Hisを-URA）上でプレートを形質転換混合物を多数のコロニーの成長を確保する。必要なプレートの数を最小限にするために大規模なペトリ皿（150ミリメートル）を使用します。プレートを使用して形質転換体を回収すること形質転換効率を評価することができます。 </li><li>並行してHsp104のWT及びベクトル陰性対照を変換します。 </li></ol> Proteotoxicityの抑制3.スクリーニング<ol><li>ラフィノース補足ドロップアウト媒体（ 例えば SRaff-のHis-裏）を使用して、プレートからコロニーを洗ってください。プレートからコロニーを緩めるために血清用ピペット及び滅菌木製のアプリケーターを使用してください。を50mlコニカルチューブボルテックス液体の洗浄を転送することは完全に任意の細胞塊を分離する。わずかに濁っ文化、OD 600〜0.025に希釈します。 注：ここにラフィノースは、このようにガラクトース媒介誘導のために細胞をプライミング、誘導のグルコース媒介抑制の細胞を緩和するのに役立つ。 </li><li> 3で振盪しながらラフィノースドロップアウト培地で一晩希釈した培養を育てる0ºC。並行してHsp104のWT及びベクトル制御を育てる。 </li><li>翌朝、個々のガラクトース（ 例えば SGal-のHis-URA）プレート（400μlの総容量で1μlの2ミリリットルに濃縮した培養）の上に濃度の範囲をメッキ。広範囲の濃度をメッキすると、プレートは、単一のコロニーを持つことを保証します。実際に必要な濃度は、対象となる疾患基板の毒性に依存する。 </li><li>ライブラリのOD 600にベクトルとHsp104のWT培養を正常化し、コントロールとライブラリの相対的な成長を比較するために同じボリュームをプレート。 </li><li>選択ストリンジェンシーを評価するために、メディアを（抑制）グルコース上のライブラリをめっきする。コロニーが（ 図2）が表示されるまで、30ºCで2〜3日間プレートをインキュベートする。得られたコロニーを評価し、コントロールに比較してください。 注：多くの場合、両方の大小のコロニーが得られたが、コロニーの大きさと行動の間には相関関係ivityが観察される。配列決定に持ち越さ偽陽性を最小限にするために5-FOAカウンタ選択技術（工程4）を使用して、これらの潜在的なヒットをスクリーニングする。 </li></ol>偽陽性を排除するために4. 5-FOA対抗とスポッティング<ol><li>ダブルとシングルドロップアウトメディアに重複して、単一のコロニー（ 例えば 、SD-Hisを-浦とSD-Hisのプレート）、30ºCで一晩成長して連勝。ベクトルとHsp104のWTコントロールを繰り返します。上にプレーティングSD-Hisの前に5-FOAは、それらが5-FOA上に播種されたとき、細胞がHsp104のプラスミドを失った可能性を増加させることによって、5-FOA工程の効率を高める。 </li><li> 5-FOA画面を行いながら、乾燥からプレートを防止するために、4ºCでパラフィルムや店舗で、SD-Hisを-浦のプレートをラップ。これらのプレートは、最終的に配列決定のために使用される。 </li><li>ストリークからコロニーのSD-Hisを5-FOAプレート上の単一コロニーにプレート（YPD培地+ 1グラム/ Lの5-FOA）と、＆30で1〜2日間インキュベート＃186; Cシングルコロニーが現れるまで。ここでは、5-FOAは、URA3遺伝子を含む細胞中の毒性産物（5-フルオロ）に変換される。このように、まだHsp104のプラスミドをどの細胞が死んでしまう。 </li><li>ストリークSD-浦とSD-Hisをプレート上に重複した5-FOAプレートから単一コロニー。それぞれについて、試験3のコロニーを各ヒットするための少なくとも一つのコロニーHsp104のプラスミドを失った可能性を高めるために打つ。 30℃で1〜2日間インキュベートする。 Hsp104のプラスミドを失ったコロニーは、SD-Hisのプレートではなく、SD-浦プレート上で増殖します。 </li><li>アッセイをスポッティングするためのHsp104のプラスミドを失った菌株を栽培。振とうしながら30ºCで一晩96ディープウェル2ミリリットルプレート中で（ 例えば SRaff-His）をラフィノースドロップアウト培地で飽和するまでの株を栽培。 5-FOA処理対照株を含めるようにしてください。 </li><li>アッセイをスポッティングするためのプレートを準備します。マルチチャンネルピ ​​ペットを用いて、一定分量200μlのラフィノースドロップアウト媒体（ 例えば SRaff-His）のあたり96ウェルプレートのウェル、ニート培養の列1,7を予約する。アリコート列1およびプレートの7への16の飽和培養物の各々を250μl。 </li><li>シリアルにマルチチャンネルピペットで十分に混合し、プレートの各列に文化5倍に希釈する。各ウェルの最終容量は200μlであることを確認するために、列6および12から希釈した培養液50μlを削除します。これはさえスポッティング確保することが不可欠である。 </li><li> 96ボルトレプリケータツール（フロッガー）を使用して、SD-HisおよびSGal-彼のプレート上に重複しての文化を発見。 2-3日間30ºCでプレートをインキュベートする。 </li><li>コントロールと同様のSGal-Hisをプレート上に毒性を示す菌株の配列決定のために選択します。コントロールのように毒性がないなどの偽陽性株を捨てる。 5-FOAは、（自分自身でもしばしば有毒であるので、SD-Hisのプレートまたはその展示に単独の疾患基板よりも実質的に大きい毒性を成長欠損を示す菌株を破棄<stronG&gt;図3）。 </li></ol> 5.コロニーPCRによってHsp104のバリアントの配列決定<ol><li>のPCRストリップチューブ中の3μlの20 mMのNaOH中のSD-Hisの-浦プレートから掻き〜10μlの酵母および凍結融解によって細胞を溶解。 10分間のサーマルサイクラーで99ºCでインキュベート、その後、10分間、または液体窒素中で-80ºCの冷凍庫にストリップチューブを置きます。のPCRグレードの水で100μlに菌株を希釈します。 </li><li> PCR反応の準備：5μlのPCRの鋳型、0.5μlの100μMの各プライマー、0.5μlの10mMのdNTPを、PCRバッファー中0.25μlのDNAポリメラーゼを、そしてPCRグレードの水で25μlの総体積に占める。デザインは、プライマーのどちらかの端にランダム化された地域に加えて、約100bpのを増幅する。増幅の成功の可能性を高めるために増幅された領域のサイズを最小限に抑えます。標準的なPCRプログラムを実行します。 </li><li>適切なSIZのPCR産物の増幅を確認するためにアガロースゲル電気泳動によって試料を分析電子メール。何の製品が存在しない場合、PCRを繰り返します。 注：PCRの失敗は、典型的には、ステップ5.1で使用されすぎて、酵母に起因する。 </li><li> DNA配列決定のためのサンプルを準備します。そのようなエキソヌクレアーゼIおよびエビアルカリホスファターゼ酵素としてPCR精製またはPCR産物クリーンアップ試薬のいずれかを使用してPCRプライマーを除去する（ExoSAP-IT）は、一本鎖DNAを分解した。この試薬は、酵素的に、より高いスループットを可能にし、取り込まれていないプライマーおよびdNTPを分解する。 </li><li> PCR産物を配列決定する。徹底的に変異をシークエンスクロマトグラムを分析するようにしてください。多くの場合、複数のプラスミドを有する酵母として突然変異は、与えられた部位（ 図4）は 2つのヌクレオチドの混合物として観察され得る。 </li></ol>

<ol>
	<li>Johnson, B. S., McCaffery, J. M., Lindquist, S., Gitler, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+yeast+TDP-43+proteinopathy+model:+Exploring+the+molecular+determinants+of+TDP-43+aggregation+and+cellular+toxicity.">A yeast TDP-43 proteinopathy model: Exploring the molecular determinants of TDP-43 aggregation and cellular toxicity.</a> Proc Natl Acad Sci USA. 105 (17), 6439-6444 (2008).</li><li>Sun, Z., 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=Molecular+Determinants+and+Genetic+Modifiers+of+Aggregation+and+Toxicity+for+the+ALS+Disease+Protein+FUS/TLS.">Molecular Determinants and Genetic Modifiers of Aggregation and Toxicity for the ALS Disease Protein FUS/TLS.</a> PLoS Biol. 9 (4), (2011).</li><li>Outeiro, T. F., Lindquist, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Yeast+Cells+Provide+Insight+into+Alpha-Synuclein+Biology+and+Pathobiology.">Yeast Cells Provide Insight into Alpha-Synuclein Biology and Pathobiology.</a> Science. 302 (5651), 1772-1775 (2003).</li><li>Forman, M. S., Trojanowski, J. Q., Lee, V. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurodegenerative+diseases:+a+decade+of+discoveries+paves+the+way+for+therapeutic+breakthroughs.">Neurodegenerative diseases: a decade of discoveries paves the way for therapeutic breakthroughs.</a> Nat Med. 10 (10), 1055-1063 (2004).</li><li>Morimoto, R. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stress,+aging,+and+neurodegenerative+disease.">Stress, aging, and neurodegenerative disease.</a> N Engl J Med. 355 (21), 2254-2255 (2006).</li><li>Elden, A. C., 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=Ataxin-2+intermediate-length+polyglutamine+expansions+are+associated+with+increased+risk+for+ALS.">Ataxin-2 intermediate-length polyglutamine expansions are associated with increased risk for ALS.</a> Nature. 466 (7310), 1069-1075 (2010).</li><li>Cooper, A. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alpha-synuclein+blocks+ER-Golgi+traffic+and+Rab1+rescues+neuron+loss+in+Parkinson's+models.">Alpha-synuclein blocks ER-Golgi traffic and Rab1 rescues neuron loss in Parkinson's models.</a> Science. 313 (5785), 324-328 (2006).</li><li>Gitler, A. D., 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=Alpha-synuclein+is+part+of+a+diverse+and+highly+conserved+interaction+network+that+includes+PARK9+and+manganese+toxicity.">Alpha-synuclein is part of a diverse and highly conserved interaction network that includes PARK9 and manganese toxicity.</a> Nature genetics. 41 (3), 308-315 (2009).</li><li>Su, L. 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=Compounds+from+an+unbiased+chemical+screen+reverse+both+ER-to-Golgi+trafficking+defects+and+mitochondrial+dysfunction+in+Parkinson's+disease+models.">Compounds from an unbiased chemical screen reverse both ER-to-Golgi trafficking defects and mitochondrial dysfunction in Parkinson's disease models.</a> Disease models &amp; mechanisms. (3-4), 194-208 (2010).</li><li>Tardiff, D. 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=Yeast+reveal+a+'druggable'+Rsp5/Nedd4+network+that+ameliorates+alpha-synuclein+toxicity+in+neurons.">Yeast reveal a 'druggable' Rsp5/Nedd4 network that ameliorates alpha-synuclein toxicity in neurons.</a> Science. 342 (6161), 979-983 (2013).</li><li>Tardiff, D. F., Tucci, M. L., Caldwell, K. A., Caldwell, G. A., Lindquist, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Different+8-hydroxyquinolines+protect+models+of+TDP-43+protein,+alpha-synuclein,+and+polyglutamine+proteotoxicity+through+distinct+mechanisms.">Different 8-hydroxyquinolines protect models of TDP-43 protein, alpha-synuclein, and polyglutamine proteotoxicity through distinct mechanisms.</a> The Journal of biological chemistry. 287 (6), 4107-4120 (2012).</li><li>Kim, H. 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=Therapeutic+modulation+of+eIF2alpha+phosphorylation+rescues+TDP-43+toxicity+in+amyotrophic+lateral+sclerosis+disease+models.">Therapeutic modulation of eIF2alpha phosphorylation rescues TDP-43 toxicity in amyotrophic lateral sclerosis disease models.</a> Nature. 46 (2), 152-160 (2014).</li><li>Ju, 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=A+yeast+model+of+FUS/TLS-dependent+cytotoxicity.">A yeast model of FUS/TLS-dependent cytotoxicity.</a> PLoS Biol. 9 (4), (2011).</li><li>Shorter, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hsp104:+a+weapon+to+combat+diverse+neurodegenerative+disorders.">Hsp104: a weapon to combat diverse neurodegenerative disorders.</a> Neurosignals. 16 (1), 63-74 (2008).</li><li>Vashist, S., Cushman, M., Shorter, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applying+Hsp104+to+protein-misfolding+disorders.">Applying Hsp104 to protein-misfolding disorders.</a> Biochem Cell Biol. 88 (1), 1-13 (2010).</li><li>DeSantis, M. E., 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=Operational+Plasticity+Enables+Hsp104+to+Disaggregate+Diverse+Amyloid+and+Nonamyloid+Clients.">Operational Plasticity Enables Hsp104 to Disaggregate Diverse Amyloid and Nonamyloid Clients.</a> Cell. 151 (4), 778-793 (2012).</li><li>Jackrel, M. E., 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=Potentiated+Hsp104+Variants+Antagonize+Diverse+Proteotoxic+Misfolding+Events.">Potentiated Hsp104 Variants Antagonize Diverse Proteotoxic Misfolding Events.</a> Cell. 156 (1-2), 170-182 (2014).</li><li>Wittrup, K. D., Verdine, G. L., Wittrup, K. D., Verdine, G. L. <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 Enzymology. 503, 13-14 (2012).</li><li>Boeke, J. D., Trueheart, J., Natsoulis, G., Fink, G. R. <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 Enzymology. 154, 164-175 (1987).</li><li>Miyazaki, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Creating+Random+Mutagenesis+Libraries+by+Megaprimer+PCR+of+Whole+Plasmid+(MEGAWHOP).+Directed+Evolution+Library+Creation:+Methods+in+Molecular+Biology.">Creating Random Mutagenesis Libraries by Megaprimer PCR of Whole Plasmid (MEGAWHOP). Directed Evolution Library Creation: Methods in Molecular Biology.</a> eds FrancesH Arnold &amp; George Georgiou. 231, 23-28 (2003).</li><li>Saibil, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chaperone+machines+for+protein+folding,+unfolding+and+disaggregation.">Chaperone machines for protein folding, unfolding and disaggregation.</a> Nat Rev Mol Cell Biol. 14 (10), 630-642 (2013).</li><li>Gietz, R. D., Schiestl, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-efficiency+yeast+transformation+using+the+LiAc/SS+carrier+DNA/PEG+method.">High-efficiency yeast transformation using the LiAc/SS carrier DNA/PEG method.</a> Nat. Protocols. 2 (1), 31-34 (2007).</li><li>Armakola, M., Hart, M. P., Gitler, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TDP-43+toxicity+in+yeast.">TDP-43 toxicity in yeast.</a> Methods. 53 (3), 238-245 (2011).</li><li>Alberti, S., Gitler, A. D., Lindquist, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+suite+of+Gateway&#174;+cloning+vectors+for+high-throughput+genetic+analysis+in+Saccharomyces+cerevisiae.">A suite of Gateway&#174; cloning vectors for high-throughput genetic analysis in Saccharomyces cerevisiae.</a> Yeast. 24 (10), 913-919 (2007).</li></ol>

The authors have nothing to disclose.

ここでは、酵母proteinopathyモデルを用いた疾患関連基質の毒性を抑制増強Hsp104の変異体を単離するための我々のアプローチを提示します。このアプローチを用いて、変異体の大きなライブラリーは、唯一の制限は、5-FOA二次スクリーニングを渡すバリアントの数であると、高スループットでスクリーニングすることができる。 96ウェルフォーマットにこれらのステップを実行することにより、?...

酵母proteinopathyモデルは、筋萎縮性側索硬化症（ALS）およびパーキンソン病（PD）1-3を含む、タンパク質の誤った折りたたみに起因する疾患のために開発されている。 ALS患者において誤って折り畳まタンパク質TDP-43とFUSの発現は、酵母1,2-細胞質凝集体を形成するために有毒でmislocalizeある。同様に、PDに関与するαシヌクレイン（α-synの）の発現は、毒性であり、mislocalizes酵母3の細胞質の凝集体を形成する。これらの機能は、これらの疾患4,5を有する患者での表現型を再現。このように、酵母のモデルは防止又はこれらの表現型2,6-13を逆にタンパク質または小分子をスクリーニングするための有用なプラットフォームを提供します。私たちは、TDP-43、FUS、及びα-synのに凝集と毒性を逆転することができるタンパク質の開発に興味を持っています。我々はHsp104の、両方のFRタンパク質を脱凝集の一意可能な酵母からAAA +タンパク質に焦点を当てる酵母におけるアモルファス凝集体およびアミロイドOM、まだそれはないヒトホモログ14,15を持っていません。 Hsp104のは細かく、内因性酵母プリオンを分解するように調整し、それが通常16,17に遭遇したことがない人間の神経変性疾患に関与する基質を脱凝集する唯一の限られた能力を持っています。従って、我々は効果的にこれらのヒトの基質を分解することができますHsp104のの強化されたバージョンを設計することを目指しています。そうするために、我々はHsp104の大量のライブラリーを構築すると、エラープローンPCRを用いて、変異体;これらのライブラリーは、酵母proteinopathyモデル17を用いてスクリーニングすることができる。 Hsp104のは17非常に大きいように我々は、ライブラリーを構築およびスクリーニングへのドメインを標的とするアプローチを採用しています。同様のアプローチは他のドメインをスクリーニングするために用いることができるが、我々は最初に、Hsp104の17の中央ドメイン（MD）に焦点を当てた。そのような残りの表面ディスプレイなどの代替技術とは対照的に、これらのモデルは、直接disaggregase活性についてスクリーニングイネーブル18を結合監視するために使用するricted。 我々のプロトコルは、2スクリーニング工程（ 図1）に基づいています。まず、酵母における疾患基板の毒性を抑制Hsp104の変異体が選択されている。これを行うには、Hsp104の変異体および疾患関連基板は、ΔHSP104酵母に同時形質転換される。我々は、野生型（WT）Hsp104の17の非存在下ではHsp104のシーケンススペースを探検する、ΔHSP104酵母を採用している。重要なことは、Hsp104のの削除は、酵母中のα-synの、FUS、またはTDP-43の毒性には影響しません、とHsp104のWTの発現は最小限の救助1,13,17を提供しています。酵母は、両方のタンパク質の発現を誘導するためにメディアを誘導する上でメッキされる。コロニーの疾患関連基板トリシン成長の毒性を抑えるHsp104の変異体を保有する酵母。コロニーを毒性型を抑制しない変異体を維持しながら、これらの変異体は、さらなる分析のために選択される。しかし、偽陽性は、この画面の大幅な問題である。 TDP-43、FUS、及びα-synの発現は、発現されているHsp104の変種とは無関係の毒性の自発的な遺伝的抑制の出現のための強力な選択圧を作成する、非常に有毒である。従って、我々はまた、これらの非特異的毒性サプレッサ17を除去するために比較的高いスループットで二次スクリーニングを使用している。この二次スクリーニングでは、選択された酵母をHsp104のプラスミド19ためのセレクト対抗する5-Fluorootic酸（5-FOA）で処理する。株は、基板の毒性はHsp104のプラスミドの喪失後に復元されることを保証するためにアッセイをスポッティングを介して基板（TDP-43、FUS、またはα-SYN）毒性について評価される。したがって、毒性がこの二次スクリーニングで復元された酵母は、おそらく、もともとHsp104の変異体の存在に起因する毒性の抑制を示した。これらの酵母は、「ヒット」として指定され、Hsp104のプラスミドは、その後でなければならない回収しHsp104の遺伝子17（ 図1）における変異を同定するために配列決定。任意のヒットは、その後独立して、突然変異を構築する部位特異的変異誘発を使用して、その後、毒性抑制のため再試験により再確認する必要があります。このプロトコルの潜在的なアプリケーションが広範である。これらの方法を用いて、タンパク質の任意のタイプのライブラリーは、酵母における毒性である任意の基質蛋白質の毒性を抑制する変異体についてスクリーニングすることができる。

<table border="0" cellpadding="0" cellspacing="0" width="766">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:320px;">Table of Specific Materials/Equipment</td>
			<td style="width:227px;"></td>
			<td style="width:132px;"></td>
			<td style="width:87px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">GeneMorphII EZClone Domain Mutagenesis Kit</td>
			<td>Agilent</td>
			<td>200552</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">150mm Petri dishes</td>
			<td>Falcon</td>
			<td>351058</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;">5-Fluorootic Acid</td>
			<td>Research Products International</td>
			<td>f10501-5.0</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">96-DeepWell 2mL Plates</td>
			<td>Eppendorf</td>
			<td>0030 502.302</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">96 bold replicator tool</td>
			<td>V&#38;P Scientific</td>
			<td>vp-404</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">ExoSAP-IT</td>
			<td>Affymetrix</td>
			<td>78200</td>
			<td></td>
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</table>

isolating potentiated hsp104 variants using yeast proteinopathy models

Many protein-misfolding disorders can be modeled in the budding yeast Saccharomyces cerevisiae. Proteins such as TDP-43 and FUS, implicated in amyotrophic lateral sclerosis, and &#945;-synuclein, implicated in Parkinson&#8217;s disease, are toxic and form cytoplasmic aggregates in yeast. These features recapitulate protein pathologies observed in patients with these disorders. Thus, yeast are an ideal platform for isolating toxicity suppressors from libraries of protein variants. We are interested in applying protein disaggregases to eliminate misfolded toxic protein conformers. Specifically, we are engineering Hsp104, a hexameric AAA+ protein from yeast that is uniquely capable of solubilizing both disordered aggregates and amyloid and returning the proteins to their native conformations. While Hsp104 is highly conserved in eukaryotes and eubacteria, it has no known metazoan homologue. Hsp104 has only limited ability to eliminate disordered aggregates and amyloid fibers implicated in human disease. Thus, we aim to engineer Hsp104 variants to reverse the protein misfolding implicated in neurodegenerative disorders. We have developed methods to screen large libraries of Hsp104 variants for suppression of proteotoxicity in yeast. As yeast are prone to spontaneous nonspecific suppression of toxicity, a two-step screening process has been developed to eliminate false positives. Using these methods, we have identified a series of potentiated Hsp104 variants that potently suppress the toxicity and aggregation of TDP-43, FUS, and &#945;-synuclein. Here, we describe this optimized protocol, which could be adapted to screen libraries constructed using any protein backbone for suppression of toxicity of any protein that is toxic in yeast.

私たちは、真ん中のドメインで無作為化Hsp104の変異体のライブラリーを構築し、TDP-43の毒性の抑制のためにそれをスクリーニングしている。ライブラリーは、形質転換され、画面のストリンジェンシーを評価するために、グルコースおよびガラクトースプレート（ 図2）上にプレーティングした。単一コロニーを選択した株は、カウンタHsp104のプラスミドを除去するために5-FOAを?...

Watch this Scientific Journal Video about Isolating Potentiated Hsp104 Variants Using Yeast Proteinopathy Models at JoVE.com

Isolating Potentiated Hsp104 Variants Using Yeast Proteinopathy Models

Yeast proteinopathy models are valuable tools to assess the toxicity and aggregation of proteins implicated in disease. Here, we present methods for screening Hsp104 variant libraries for toxicity suppressors. This protocol could be adapted to screen any protein library for toxicity suppressors of any protein that is toxic in yeast.

酵母Proteinopathyモデルを用いた増強Hsp104のバリアントを分離

Many protein-misfolding disorders can be modeled in the budding yeast Saccharomyces cerevisiae. Proteins such as TDP-43 and FUS, implicated in amyotrophic ...

isolating-potentiated-hsp104-variants-using-yeast-proteinopathy-models

Research

JoVE Journal

Biology

8.1K ビュー.  Perelman School of Medicine at the University of Pennsylvania. Yeast proteinopathy models are valuable tools to assess the toxicity and aggregation of proteins implicated in disease. Here, we present methods for screening Hsp104 variant libraries for toxicity suppressors. This protocol could be adapted to screen any protein library for toxicity suppressors of any protein that is toxic in yeast.

ビデオ: Isolating Potentiated Hsp104 Variants Using Yeast Proteinopathy Models

A Quantitative Assessment of The Yeast Lipidome using Electrospray Ionization Mass Spectrometry

Lipids are one of the major classes of biomolecules and play important roles membrane dynamics, energy storage, and signalling1-4. The budding yeast Saccharomyces cerevisiae, a genetically and biochemically manipulable unicellular eukaryote with annotated genome and very simple lipidome, is a valuable model for studying biological functions of various lipid species in multicellular eukaryotes2,3,5. S. cerevisiae has 10 major classes of lipids with chain lengths mainly of 16 or 18 carbon atoms and either zero or one degree of unsaturation6,7. Existing methods for lipid identification and quantification - such as high performance liquid chromatography, thin-layer chromatography, fluorescence microscopy, and gas chromatography followed by MS - are well established but have low sensitivity, insufficiently separate various molecular forms of lipids, require lipid derivitization prior to analysis, or can be quite time consuming. Here we present a detailed description of our experimental approach to solve these inherent limitations by using survey-scan ESI/MS for the identification and quantification of the entire complement of lipids in yeast cells. The described method does not require chromatographic separation of complex lipid mixtures recovered from yeast cells, thereby greatly accelerating the process of data acquisition. This method enables lipid identification and quantification at the concentrations as low as g/ml and has been successfully applied to assessing lipidomes of whole yeast cells and their purified organelles. Lipids extraction from whole yeast cells for using this method of lipid analysis takes two to three hours. It takes only five to ten minutes to run each sample of extracted and dried lipids on a Q-TOF mass spectrometer equipped with a nano-electrospray source.

We describe a new quantitative lipidomics method for identifying numerous lipid species in yeast using survey-scan electrospray ionization mass spectrometry (ESI/MS). This method exceeds currently available methods for lipid identification and quantification in the ability to resolve various molecular forms of lipids, sensitivity, and speed.

エレクトロスプレーイオン化質量分析法を用いて酵母Lipidomeの定量評価

Lipids are one of the major classes of biomolecules and play important roles membrane dynamics, energy storage, and signalling1-4. The budding yeast ...

生物学

Genetic Studies of Human DNA Repair Proteins Using Yeast as a Model System 

Understanding the roles of human DNA repair proteins in genetic pathways is a formidable challenge to many researchers. Genetic studies in mammalian systems have been limited due to the lack of readily available tools including defined mutant genetic cell lines, regulatory expression systems, and appropriate selectable markers. To circumvent these difficulties, model genetic systems in lower eukaryotes have become an attractive choice for the study of functionally conserved DNA repair proteins and pathways. We have developed a model yeast system to study the poorly defined genetic functions of the Werner syndrome helicase-nuclease (WRN) in nucleic acid metabolism. Cellular phenotypes associated with defined genetic mutant backgrounds can be investigated to clarify the cellular and molecular functions of WRN through its catalytic activities and protein interactions. The human WRN gene and associated variants, cloned into DNA plasmids for expression in yeast, can be placed under the control of a regulatory plasmid element. The expression construct can then be transformed into the appropriate yeast mutant background, and genetic function assayed by a variety of methodologies. Using this approach, we determined that WRN, like its related RecQ family members BLM and Sgs1, operates in a Top3-dependent pathway that is likely to be important for genomic stability. This is described in our recent publication [1] at <a href="http://www.impactaging.com">www.impactaging.com</a>. Detailed methods of specific assays for genetic complementation studies in yeast are provided in this paper.

Genetic Studies of Human DNA Repair Proteins Using Yeast as a Model System

Genetic studies in yeast can be employed to investigate the molecular and cellular functions of human genes in cellular DNA metabolism. Methods are described for the genetic characterization of the human WRN gene product defective in the premature aging disorder Werner syndrome in functionally conserved pathways using yeast as a tractable model system.

人間のDNA修復タンパク質の遺伝的研究は、モデルシステムとして酵母を使用

Understanding the roles of human DNA repair proteins in genetic pathways is a formidable challenge to many researchers.  Genetic studies in mammalian ...

Yeast Colony Embedding Method

Patterning of different cell types in embryos is a key mechanism in metazoan development. Communities of microorganisms, such as colonies and biofilms also display patterns of cell types. For example, in the yeast S. cerevisiae, sporulated cells and pseudohyphal cells are not uniformly distributed in colonies. The functional importance of patterning and the molecular mechanisms that underlie these patterns are still poorly understood.
One challenge with respect to investigating patterns of cell types in fungal colonies is that unlike metazoan tissue, cells in colonies are relatively weakly attached to one another. In particular, fungal colonies do not contain the same extensive level of extracellular matrix found in most tissues . Here we report on a method for embedding and sectioning yeast colonies that reveals the interior patterns of cell types in these colonies. The method can be used to prepare thick sections (0.5 &mu;) useful for light microscopy and thin sections (0.1 &mu;) suitable for transmission electron microscopy. Asci and pseudohyphal cells can easily be distinguished from ovoid yeast cells by light microscopy , while the interior structure of these cells can be visualized by EM.
The method is based on surrounding colonies with agar, infiltrating them with Spurr's medium, and then sectioning. Colonies with a diameter in the range of 1-2 mm are suitable for this protocol. In addition to visualizing the interior of colonies, the method allows visualization of the region of the colony that invades the underlying agar.

A method for embedding yeast colonies allowing sectioning for light and electron microscopy. This protocol allows determination of the distribution of sporulated cells and pseudohyphal cells within colonies providing a new tool toward understanding the organization of cell types within a fungal community.

酵母のコロニーの埋め込み法

Patterning of  different cell types in embryos is a key mechanism in metazoan development. Communities of microorganisms, such as colonies and biofilms ...

Principles of Rodent Surgery for the New Surgeon

For both scientific and animal welfare reasons, training in basic surgical concepts and techniques should be undertaken before ever seeking to perform surgery on a rodent. Students, post-doctoral scholars, and others interested in performing surgery on rodents as part of a research protocol may not have had formal surgical training as part of their required coursework. Surgery itself is a technical skill, and one that will improve with practice. The principles of aseptic technique, however, often remain unexplained or untaught. For most new surgeons, this vital information is presented in piecemeal fashion or learned on the job, neither of which is ideal. It may also make learning how to perform a particular surgery difficult, as the new surgeon is learning both a surgical technique and the principles of asepsis at the same time. This article summarizes and makes recommendations for basic surgical skills and techniques necessary for successful rodent surgery. This article is designed to supplement hands-on training by the user's institution.

Before attempting surgery, a new surgeon should have training in basic surgical techniques and concepts. This article will present basic surgical considerations with an emphasis on rodents.

新外科医のための齧歯類の外科の原則

For both scientific and animal welfare reasons, training in basic surgical concepts and techniques should be undertaken before ever seeking to perform ...

Manual Restraint and Common Compound Administration Routes in Mice and Rats

Being able to safely and effectively restrain mice and rats is an important part of conducting research. Working confidently and humanely with mice and rats requires a basic competency in handling and restraint methods. This article will present the basic principles required to safely handle animals. One-handed, two-handed, and restraint with specially designed restraint objects will be illustrated. Often, another part of the research or testing use of animals is the effective administration of compounds to mice and rats. Although there are a large number of possible administration routes (limited only by the size and organs of the animal), most are not used regularly in research. This video will illustrate several of the more common routes, including intravenous, intramuscular, subcutaneous, and oral gavage. The goal of this article is to expose a viewer unfamiliar with these techniques to basic restraint and substance administration routes. This video does not replace required hands-on training at your facility, but is meant to augment and supplement that training.

Working safely and humanely with research rodents requires a core competency in handling and restraint methods. This article will present the basic principles required to safely handle and effectively administer compounds to mice and rats.

マウスとラットの拘束マニュアルと共通の複合投与経路

Being able to safely and effectively restrain mice and rats is an important part of conducting research.  Working confidently and humanely with mice and ...

High-throughput Yeast Plasmid Overexpression Screen

The budding yeast, Saccharomyces cerevisiae, is a powerful model system for defining fundamental mechanisms of many important cellular processes, including those with direct relevance to human disease. Because of its short generation time and well-characterized genome, a major experimental advantage of the yeast model system is the ability to perform genetic screens to identify genes and pathways that are involved in a given process. Over the last thirty years such genetic screens have been used to elucidate the cell cycle, secretory pathway, and many more highly conserved aspects of eukaryotic cell biology 1-5. In the last few years, several genomewide libraries of yeast strains and plasmids have been generated 6-10. These collections now allow for the systematic interrogation of gene function using gain- and loss-of-function approaches 11-16. Here we provide a detailed protocol for the use of a high-throughput yeast transformation protocol with a liquid handling robot to perform a plasmid overexpression screen, using an arrayed library of 5,500 yeast plasmids. We have been using these screens to identify genetic modifiers of toxicity associated with the accumulation of aggregation-prone human neurodegenerative disease proteins. The methods presented here are readily adaptable to the study of other cellular phenotypes of interest.

Here we describe a plasmid overexpression screen in Saccharomyces cerevisiae, using an arrayed plasmid library and a high-throughput yeast transformation protocol with a liquid handling robot.

ハイスループット酵母プラスミドの過剰発現の画面

The budding yeast, Saccharomyces cerevisiae, is a powerful model system for defining fundamental mechanisms of many important cellular processes, ...

Purification of Hsp104, a Protein Disaggregase

Hsp104 is a hexameric AAA+ protein1 from yeast, which couples ATP hydrolysis to protein disaggregation2-10 (Fig. 1). This activity imparts two key selective advantages. First, renaturation of disordered aggregates by Hsp104 empowers yeast survival after various protein-misfolding stresses, including heat shock3,5,11,12. Second, remodeling of cross-beta amyloid fibrils by Hsp104 enables yeast to exploit myriad prions (infectious amyloids) as a reservoir of beneficial and heritable phenotypic variation13-22. Remarkably, Hsp104 directly remodels preamyloid oligomers and amyloid fibrils, including those comprised of the yeast prion proteins Sup35 and Ure223-30. This amyloid-remodeling functionality is a specialized facet of yeast Hsp104. The E. coli orthologue, ClpB, fails to remodel preamyloid oligomers or amyloid fibrils26,31,32.
Hsp104 orthologues are found in all kingdoms of life except, perplexingly, animals. Indeed, whether animal cells possess any enzymatic system that couples protein disaggregation to renaturation (rather than degradation) remains unknown33-35. Thus, we and others have proposed that Hsp104 might be developed as a therapeutic agent for various neurodegenerative diseases connected with the misfolding of specific proteins into toxic preamyloid oligomers and amyloid fibrils4,7,23,36-38. There are no treatments that directly target the aggregated species associated with these diseases. Yet, Hsp104 dissolves toxic oligomers and amyloid fibrils composed of alpha-synuclein, which are connected with Parkinson's Disease23 as well as amyloid forms of PrP39. Importantly, Hsp104 reduces protein aggregation and ameliorates neurodegeneration in rodent models of Parkinson's Disease23 and Huntington's disease38. Ideally, to optimize therapy and minimize side effects, Hsp104 would be engineered and potentiated to selectively remodel specific aggregates central to the disease in question4,7. However, the limited structural and mechanistic understanding of how Hsp104 disaggregates such a diverse repertoire of aggregated structures and unrelated proteins frustrates these endeavors30,40-42.
To understand the structure and mechanism of Hsp104, it is essential to study the pure protein and reconstitute its disaggregase activity with minimal components. Hsp104 is a 102kDa protein with a pI of ~5.3, which hexamerizes in the presence of ADP or ATP, or at high protein concentrations in the absence of nucleotide43-46. Here, we describe an optimized protocol for the purification of highly active, stable Hsp104 from E. coli. The use of E. coli allows simplified large-scale production and our method can be performed quickly and reliably for numerous Hsp104 variants. Our protocol increases Hsp104 purity and simplifies His6-tag removal compared to a previous purification method from E. coli47. Moreover, our protocol is more facile and convenient than two more recent protocols26,48.

Here, we describe a protocol for the purification of highly active Hsp104, a hexameric AAA+ protein from yeast, which couples ATP hydrolysis to protein disaggregation. This scheme exploits a His6-tagged construct for affinity purification from E. coli followed by anion-exchange chromatography, His6-tag removal with TEV protease, and size-exclusion chromatography.

Hsp104の精製、タンパク質Disaggregase

Hsp104 is a hexameric AAA+ protein1 from yeast, which couples ATP hydrolysis to protein disaggregation2-10 (Fig. 1). This activity imparts two key ...

Mouse Sperm Cryopreservation and Recovery using the I&middot;Cryo Kit

Thousands of new genetically modified (GM) strains of mice have been created since the advent of transgenesis and knockout technologies. Many of these valuable animals exist only as live animals, with no backup plan in case of emergency. Cryopreservation of embryos can provide this backup, but is costly, can be a lengthy procedure, and generally requires a large number of animals for success. Since the discovery that mouse sperm can be successfully cryopreserved with a basic cryoprotective agent (CPA) consisting of 18&#37; raffinose and 3&#37; skim milk, sperm cryopreservation has become an acceptable and cost-effective procedure for archiving, distributing and recovery of these valuable strains.
 Here we demonstrate a newly developed I&bull;Cryo kit for mouse sperm cryopreservation. Sperm from five commonly-used strains of inbred mice were frozen using this kit and then recovered. Higher protection ratios of sperm motility (&gt; 60&#37;) and rapid progressive motility (&gt; 45&#37;) compared to the control (basic CPA) were seen for sperm frozen with this kit in 5 inbred mouse strains. Two cell stage embryo development after IVF with the recovered sperm was improved consistently in all 5 mouse strains examined. Over a 1.5 year period, 49 GM mouse lines were archived by sperm cryopreservation with the I&bull;Cryo kit and later recovered by IVF.

Here we demonstrate the newly developed I&#x2022;Cryo kit for mouse sperm cryopreservation. Two-cell stage embryo development with frozen-thawed sperm was improved consistently in 5 mouse strains with the use of this kit. Over a 1.5 year period, 49 genetically modified mouse lines were archived by sperm cryopreservation with the I&#x2022;Cryo kit and later successfully recovered by IVF.

I ·クライオキットを使用してマウスの精子の凍結保存と回復

Thousands of new genetically modified (GM) strains of mice have been created since the advent of transgenesis and knockout technologies.  Many of these ...

Detection of Rare Genomic Variants from Pooled Sequencing Using SPLINTER

As DNA sequencing technology has markedly advanced in recent years2, it has become increasingly evident that the amount of genetic variation between any two individuals is greater than previously thought3. In contrast, array-based genotyping has failed to identify a significant contribution of common sequence variants to the phenotypic variability of common disease4,5. Taken together, these observations have led to the evolution of the Common Disease / Rare Variant hypothesis suggesting that the majority of the "missing heritability" in common and complex phenotypes is instead due to an individual's personal profile of rare or private DNA variants6-8. However, characterizing how rare variation impacts complex phenotypes requires the analysis of many affected individuals at many genomic loci, and is ideally compared to a similar survey in an unaffected cohort. Despite the sequencing power offered by today's platforms, a population-based survey of many genomic loci and the subsequent computational analysis required remains prohibitive for many investigators.
To address this need, we have developed a pooled sequencing approach1,9 and a novel software package1 for highly accurate rare variant detection from the resulting data. The ability to pool genomes from entire populations of affected individuals and survey the degree of genetic variation at multiple targeted regions in a single sequencing library provides excellent cost and time savings to traditional single-sample sequencing methodology. With a mean sequencing coverage per allele of 25-fold, our custom algorithm, SPLINTER, uses an internal variant calling control strategy to call insertions, deletions and substitutions up to four base pairs in length with high sensitivity and specificity from pools of up to 1 mutant allele in 500 individuals. Here we describe the method for preparing the pooled sequencing library followed by step-by-step instructions on how to use the SPLINTER package for pooled sequencing analysis (<a href="http://www.ibridgenetwork.org/wustl/splinter" target="_blank">http://www.ibridgenetwork.org/wustl/splinter</a>). We show a comparison between pooled sequencing of 947 individuals, all of whom also underwent genome-wide array, at over 20kb of sequencing per person. Concordance between genotyping of tagged and novel variants called in the pooled sample were excellent. This method can be easily scaled up to any number of genomic loci and any number of individuals. By incorporating the internal positive and negative amplicon controls at ratios that mimic the population under study, the algorithm can be calibrated for optimal performance. This strategy can also be modified for use with hybridization capture or individual-specific barcodes and can be applied to the sequencing of naturally heterogeneous samples, such as tumor DNA.

Pooled DNA sequencing is a fast and cost-effective strategy to detect rare variants associated with complex phenotypes in large cohorts. Here we describe the computational analysis of pooled, next-generation sequencing of 32 cancer-related genes using the SPLINTER software package. This method is scalable, and applicable to any phenotype of interest.

SPLINTERを使用してプールされたシーケンスからレアゲノム変異の検出

As DNA sequencing technology has markedly advanced in recent years2, it has become increasingly evident that the amount of genetic variation between any ...

Screening for Functional Non-coding Genetic Variants Using Electrophoretic Mobility Shift Assay (EMSA) and DNA-affinity Precipitation Assay (DAPA)

Population and family-based genetic studies typically result in the identification of genetic variants that are statistically associated with a clinical disease or phenotype. For many diseases and traits, most variants are non-coding, and are thus likely to act by impacting subtle, comparatively hard to predict mechanisms controlling gene expression. Here, we describe a general strategic approach to prioritize non-coding variants, and screen them for their function. This approach involves computational prioritization using functional genomic databases followed by experimental analysis of differential binding of transcription factors (TFs) to risk and non-risk alleles. For both electrophoretic mobility shift assay (EMSA) and DNA affinity precipitation assay (DAPA) analysis of genetic variants, a synthetic DNA oligonucleotide (oligo) is used to identify factors in the nuclear lysate of disease or phenotype-relevant cells. For EMSA, the oligonucleotides with or without bound nuclear factors (often TFs) are analyzed by non-denaturing electrophoresis on a tris-borate-EDTA (TBE) polyacrylamide gel. For DAPA, the oligonucleotides are bound to a magnetic column and the nuclear factors that specifically bind the DNA sequence are eluted and analyzed through mass spectrometry or with a reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by Western blot analysis. This general approach can be widely used to study the function of non-coding genetic variants associated with any disease, trait, or phenotype.

Screening for Functional Non-coding Genetic Variants Using Electrophoretic Mobility Shift Assay EMSA and DNA-affinity Precipitation Assay DAPA

We present a strategic plan and protocol for identifying non-coding genetic variants affecting transcription factor (TF) DNA binding. A detailed experimental protocol is provided for electrophoretic mobility shift assay (EMSA) and DNA affinity precipitation assay (DAPA) analysis of genotype-dependent TF DNA binding.

機能電気泳動移動度シフトアッセイ（EMSA）を用いた非コーディング遺伝的変異体およびDNA親和性沈殿アッセイ（DAPA）のスクリーニング

Population and family-based genetic studies typically result in the identification of genetic variants that are statistically associated with a clinical ...