この作品は、とShanghaiTech大学、上海、中国（DP1CA174426カルロスF.バルバスへ）（JL）の国立衛生研究所によってサポートされていました。分子グラフィックスはPyMOLをを使用して生成された。

1.クローニング<ol><li>をpET-28の発現ベクター系にサブクローン化し、要求（PET-2F-ZIF）承りますされてきたアラニン置換二本指ZIFドメインを取得します。34 </li><li> PCRはプライマーを用いて、プラスミドエメラルド-のpBADからEmGFPを増幅5 &#39;のXmaI-EmGFP（5&#39;-GGAAATTG CCCGGG ATGGTGAGCAAGGGCGAGGAGCTGTTCAC-3&#39;;太字でXmaIで I部位）および3 &#39;のSacI-EmGFP（5&#39;-CGGATCT GAGCTC TTACTTGTACAGCTCGTCCATGCCGAG-3&#39; ;太字のSacI部位）。 <ol><li>蒸留/脱イオン水で構成された残りの容量は100μlの溶液中で鋳型DNA 5ngの、10×ポリメラーゼバッファー、Taq DNAポリメラーゼの1単位（U）、0.2mMの10μlの各dNTP、各プライマー、0.2μMを使用。サイクル条件を使用してください：95℃、5分間。 1分30秒、72℃30秒間、55℃、95℃の30サイクル; 10分間72℃である。ゲルextractiによってPCR産物を精製し上とABS 260×50 / mlの測定分光光度計を用いてDNA濃度を決定する。 </li></ol></li><li>ダイジェストをpET-2F-ZIFおよび1μgのDNAあたりの酵素10 Uを使用して、37℃で3時間のための推奨バッファ内の制限酵素XmaIで IとSacIでEmGFPをコードするインサート。臭化エチジウム等の蛍光インターカレート色素を用いたアガロースゲル電気泳動によりDNAを可視化する。 </li><li>ゲル抽出キットによって消化されたDNAを精製し、腹筋260×50 / mlのを測定する分光光度計でDNA濃度を決定する。 </li><li> RTで少なくとも1時間のT4 DNAリガーゼの1 Uを用いたPET-2F-ZIFの50-100 NGに精製されたEmGFPをコードするDNAを連結。 1のインサート·ベクターのモル比：最高の結果を得るには、6を使用してライゲーション反応を行う。 </li><li>氷の上の任意の化学的にコンピXL-1ブルー大腸菌細胞50μlを解凍し、連結し、pET-2F-ZIF-EmGFPの10-20 NGで軽く混ぜる。 </li><li> 3氷上で保管してください0分。 90秒間、42℃でヒートショックの混合物を、振とうしながら37℃で1時間、異化代謝産物抑制（SOC）とスーパー最適ブロスの2ミリリットルで細胞を回収する。 </li><li> 50μg/ mlのカナマイシンを含むLB培地（LB）寒天プレート上に回復培養物100μlを広げ、37℃でO / Nインキュベートする。 </li><li>翌日、スーパーブロス（SB）または50μg/ mlのカナマイシン、37℃でLB寒天プレート培養O / Nから1つのコロニーとを含むLB培地6mlの接種。 注：コロニーPCRプライマー5 &#39;のXmaI-EmGFPおよび3&#39;を用いてのSacI-EmGFPをミニプレップする前に、陽性クローンを同定するために使用することができる。 </li><li>ミニプレップによりたpET-2F-ZIF-EmGFPを精製し、T7プロモーターを用いてDNA配列決定によりプラスミドを確認し（5&#39;-TAATACGACTCACTATAGGG-3 &#39;）。 </li></ol> 2.発現および精製<ol><li>化学的にコンピテントBL21 E.50μlの解凍氷の上で大腸菌の細胞およびPLをpET-2F-ZIF-EmGFP 100ngので軽く混ぜるasmid。ステップ1.7から1.8に記載されているように変換します。 </li><li>翌日、単一コロニーを50μg/ mlのカナマイシンを含むLB培地10mlに接種し、振盪しながら37℃でO / Nを成長させる。 </li><li>翌日、50μg/ mlのカナマイシン、0.2％グルコースおよび100μMのZnCl 2を補充したLB培地1L中にO / N培養物を10mlに希釈。 0.8の600nmで（OD 600）での光学密度に振とうしながら37℃で培養物を成長させ、2 mMのイソプロピルβ-D-1-チオガラクトピラノシド（IPTG）を用いてタンパク質発現を誘導する。 37℃での増殖の6時間後に、4℃で10分間、5000×gでの遠心分離により収穫細胞。 注意：誘導条件は非常に可変であり、発現しているタンパク質の安定性に依存します。 0.8のOD 600に達するまでOD 600を 30分ごとに監視します。 </li><li>溶解緩衝液5ml（50mMトリス-HCl、500mMのNaCl中で細胞ペレットを再懸濁し、100μMのZnCl 2を 、1メートルMジチオスレイトール（DTT）、1mMのMgCl 2、1mMのフェニルメチルスルホニルフルオリド（PMSF）、および10 mMイミダゾール、pHは8.0）。溶解以下の設定で超音波処理することにより氷上で細胞：/ 10秒オフの間隔で5秒で50％の電力出力、4分の処理時間。ソリューションの過熱を防ぐ。 </li><li> 4℃で30分間25000×gで細胞溶解物を遠心分離し、新鮮な収集チューブに上清を移す。最良の結果を得るには、4℃でのすべての次の手順を実行します。 </li><li>平衡化されたNi-NTAスラリーの1ミリリットルで予め充填したカラムに上清を実行します。洗浄緩衝液を20ml（50mMトリス-HCl、500mMのNaCl、100μMのZnCl 2、1mMのDTT、1mMのMgCl 2、30 mMイミダゾール、pH8.0）で樹脂を洗浄する。 </li><li>溶出緩衝液5ml（50mMトリス-HCl、500mMのNaCl、100μMのZnCl 2、1mMのDTT、1mMのMgCl 2、300 mMイミダゾール、pH8.0）でタンパク質を溶出する。 </li><li>バッファは、ストレージ·バッファーで溶離したタンパク質（50mMの交換をトリス-HCl、500mMのNaCl、100μMのZnCl 2、1mMのDTT、1mMのMgCl 2、10％グリセロール、pH8.0）で、製造業者の説明書に従って遠心分離によってスピン濃縮器を用いて、少なくとも40μM、タンパク質を濃縮する。 </li><li> BCAやBradfordアッセイによりタンパク質濃度を決定します。 、2μlの2×SDS-PAGEローディング色素で精製タンパク質を2μlを混合し、10分間95℃で沸騰した後、タンパク質純度（ 図2）を評価するために、4％-20％トリス-グリシンSDS-PAGE上で解決する。 </li></ol> 3.タンパク質貯蔵<ol><li>濃縮タンパク質、-80℃で液体窒素とストア内のフラッシュ凍結アリコート。 EmGFP融合タンパク質については、光からタンパク質を保護するためにアルミホイルでチューブをカバーする。 </li><li>タンパク質溶液の凍結融解の繰り返しは避けてください。注意：ZIF融合タンパク質は、少なくとも1ヶ月間、これらの条件下で安定である。不適切または&gt; 4月のストレージがつながる可能性タンパク質沈殿またはEmGFPの光退色に。 </li></ol> 4.タンパク質導入<ol><li> 10％（v / v）のウシ胎児血清（FBS）および1％抗生物質-抗真菌37℃、5％CO 2に完全に加湿雰囲気中を含むダルベッコ改変イーグル培地（DMEM）中でHeLa細胞を維持する。 注：細胞は30倍以上のタンパク質導入のために推奨されていません継代した。 </li><li> 25℃で30から60分間ポリリジンを50μg/ mlの500μlのプレコート24ウェルプレート。ウェルあたり2×10 5細胞の密度で24ウェルプレート上にシード細胞。播種後24時間で、または細胞に一度80パーセント-90％コンフルエントの間にある、各ウェルから培地を除去し、予め温めておいた無血清DMEM（SFM）を500μlで洗浄する。 </li><li>各ウェルに、ZIF-EmGFPタンパク質及び100μMのZnCl 2の2μMを含むSFMの250μlを添加する。 1時間37℃で細胞をインキュベート。注意：ZIFドメインが細胞に入るPRimarilyエネルギー依存性機構であるマクロピノサイトーシス、34を介して。したがって、細胞は、効率的なタンパク質の内在化のために37℃でインキュベートされなければならない。 </li><li>細胞から培地を除去および0.5mg / mlのヘパリンを補足したカルシウムおよびマグネシウムを含まないダルベッコのリン酸緩衝食塩水（DPBS）500μlで3回洗浄する。注：ヘパリン下流分析を複雑にする可能性があり、表面に結合したタンパク質を除去する必要がある。 </li><li>強化された配信のための3回まで（オプション）を繰り返しトリートメント。 </li><li>すぐに2分間37℃で、0.05％トリプシン-EDTAを用いて消化することにより洗浄した後、ヘパリン処理した細胞を単離する。 1％FBSを補充したDPBS 0.5mlに剥離した細胞を再懸濁する。 </li><li>フルオレセインイソチオシアネート（FITC）チャンネル（ 図3）を用いてフローサイトメトリーにより35を 、各サンプルの蛍光強度を測定する。の人口を配置する前方散乱（FSC）および側方散乱（SSC）を調整異なる特性を持つ集団が相互に解決されることを保証する規模の利息、。 </li><li> SFMのみで処置した細胞にZIF-EmGFPで処理したHeLa細胞から36ノーマライズ蛍光を、各サンプル10,000ライブイベントを収集し、フローサイトメトリーデータ分析ソフトウェアを用いてデータを分析する。 </li></ol>

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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=A+class+of+human+proteins+that+deliver+functional+proteins+into+mammalian+cells+in+vitro+and+in+vivo.">A class of human proteins that deliver functional proteins into mammalian cells in vitro and in vivo.</a> Chem. Biol. 18, 833-838 (2011).</li><li>Gaj, T., Liu, J., Anderson, K. E., Sirk, S. J., Barbas, C. F., 3rd, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+delivery+using+Cys2-His2+zinc-finger+domains.">Protein delivery using Cys2-His2 zinc-finger domains.</a> ACS Chem. Biol. 9, 1662-1667 (2014).</li><li>Radcliff, G., Jaroszeski, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basics+of+flow+cytometry.">Basics of flow cytometry.</a> Methods Mol. Biol. 91, 1-24 (1998).</li><li>Mercer, A. C., Gaj, T., Sirk, S. J., Lamb, B. M., Barbas, C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+endogenous+human+gene+expression+by+ligand-inducible+TALE+transcription+factors.">Regulation of endogenous human gene expression by ligand-inducible TALE transcription factors.</a> ACS Synth. Biol. 3, 723-730 (2014).</li><li>Segal, D. J., Crotty, J. W., Bhakta, M. S., Barbas, C. F., Horton, N. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+of+Aart,+a+designed+six-finger+zinc+finger+peptide,+bound+to+DNA.">Structure of Aart, a designed six-finger zinc finger peptide, bound to DNA.</a> J. Mol. Biol. 363, 405-421 (2006).</li></ol>

著者らは、開示することは何もない。

ここでは、細胞透過性のジンクフィンガー（ZIF）ドメインを用いてタンパク質送達のためのステップバイステップのプロトコルが提供される。 ZIFドメインは、融合した、酵素貨物34の活性を低下させない。非修飾タンパク質で観察されるものとほぼ同じ収率でのタンパク質の産生および精製を可能にする。そして伝統的な細胞透過性ペプチドまたはタンパク質導入ドメインシステム?...

高効率と汎用性タンパク質の送達戦略は、多くの基礎研究および治療用途のために重要である。細胞への精製されたタンパク質の直接送達は、これを達成するための最も安全で簡単な方法のいずれかを表す。核酸からの遺伝子発現に依存する戦略とは異なり1,2、3-5タンパク質の送達は、挿入突然変異の危険をもたらすことはないとは無関係である細胞の転写/翻訳機構と即効することができます。しかし、タンパク質上に細胞透過活性の賦与するための簡単​​で一般化方法の欠如は、日常的に細胞への直接のエントリを混乱させる。細胞内タンパク質の送達を容易にするための現在の方法は、天然6-8または設計された細胞透過性ペプチド、9-12過給導入ドメイン、13,14ナノ粒子15およびリポソーム16ウイルス様粒子17,18発生の使用に基づいている</SUP&gt;とポリマーマイクロスフェア材料。19残念ながら、これらのアプローチの多くは、低細胞取り込み率、20,21安定性が悪く、22不注意細胞型特異性、23低いエンドソーム脱出特性24と毒性によって妨げられている。さらに、25、多くのタンパク質形質導入技術は、配信するタンパク質の生物活性を減らす。14 キメラ制限がプログラム可能なのCys 2 -His 2ジンクフィンガーDNA結合タンパク質とFokI制限エンドヌクレアーゼ26-28の切断ドメインからなるエンドヌクレアーゼ- -本質的に無細胞である私たちの研究室では、以前ジンクフィンガーヌクレアーゼ（ZFN）のタンパク質があることを実証した透過性。29この驚くべき細胞透過活性はカスタム設計ジンクフィンガードメイン、ターゲットゲノムENのための強力なツールとして浮上しているDNA結合プラットフォーム固有の性質であることが示されたgineering、30-32およびタンパク質表面の6つの正に荷電した残基のコンステレーションの結果であると考えられる。実際、c-Junの及びN-DEKを含むいくつかのDNA結合タンパク質は、細胞膜を通過する先天的能力を有することが示されている。 図33は、さらに最近では、我々の研究室では、これらの結果に展開され、亜鉛の細胞透過活性のことを実証指（ZIF）ドメインは、細胞内のタンパク質送達のために活用することができた。従来の多くの細胞透過性ペプチド送達システムを超えた効率性を取り込むために導いた特定のタンパク質の貨物に一次元または二本指をZIFドメインのどちらかの遺伝子融合。34最も顕著、ZIF媒介送達が融合した、酵素貨物の活性を危うくし、促進しませんでした細胞質ゾルデリバリーの高レベル。まとめると、これらの知見は、タンパク質の効率的かつ容易な送達を促進するためのZIFドメインの可能性を実証し、潜在的により多様な種類のマクロ細胞内への分子、。 ここでは、哺乳動物細胞におけるタンパク質の送達のためのZIF技術を実装する方法の詳細なステップバイステップのプロトコルが提供される。我々は以前に一次元、二次元、三、四速、五起因αヘリックスDNA結合残基のそれぞれの置換を、DNAに結合する能力を欠いている6フィンガーZIFドメインのスイートを構築したが、セル34（ 図1）にタンパク質を送達することができる。二本指ZIFドメインを使用してHeLa細胞にエメラルドGFP（EmGFP）の産生および形質導入が記載されている。このプロトコルは、 大腸菌での可溶性発現が可能なほぼすべてのタンパク質、ほぼ任意の哺乳動物細胞型に拡張可能である。期待される結果を提供し、このシステムのパフォーマンスを最大化するための戦略もまた、議論されている。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>XmaI</td>
			<td>New England Biolabs</td>
			<td>R0180L</td>
			<td></td>
		</tr>
		<tr>
			<td>SacI</td>
			<td>New England Biolabs</td>
			<td>R0156L</td>
			<td></td>
		</tr>
		<tr>
			<td>Expand High Fidelity PCR system</td>
			<td>Roche</td>
			<td>11759078001</td>
			<td></td>
		</tr>
		<tr>
			<td>dNTPs</td>
			<td>New England Biolabs</td>
			<td>N0446S</td>
			<td></td>
		</tr>
		<tr>
			<td>4-20% Tris-Glycine Mini protein gels, 1.5 mm, 10 wells</td>
			<td>Life Technologies</td>
			<td>EC6028BOX</td>
			<td></td>
		</tr>
		<tr>
			<td>2x Laemmli Sample Buffer</td>
			<td>BioRad</td>
			<td>161-0737</td>
			<td></td>
		</tr>
		<tr>
			<td>T4 DNA Ligase</td>
			<td>Life Technologies</td>
			<td>15224-017</td>
			<td></td>
		</tr>
		<tr>
			<td>BL21 (DE3) Competent E. coli</td>
			<td>New England Biolabs</td>
			<td>C2527I</td>
			<td></td>
		</tr>
		<tr>
			<td>IPTG</td>
			<td>Thermo Scientific</td>
			<td>R0391</td>
			<td></td>
		</tr>
		<tr>
			<td>Zinc Chloride</td>
			<td>Sigma-Aldrich</td>
			<td>208086-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>Kanamycin Sulfate</td>
			<td>Fisher Scientific</td>
			<td>BP906-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G8270-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris Base</td>
			<td>Fisher Scientific</td>
			<td>BP152-25</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Sigma-Aldrich</td>
			<td>S9888-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>DTT</td>
			<td>Fisher Scientific</td>
			<td>PR-V3151&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>PMSF</td>
			<td>Thermo Scientific</td>
			<td>36978</td>
			<td></td>
		</tr>
		<tr>
			<td>Ni-NTA Agarose Resin</td>
			<td>QIAGEN</td>
			<td>30210</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Sigma-Aldrich</td>
			<td>G5516-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Imidazole</td>
			<td>Sigma-Aldrich</td>
			<td>I5513-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>Amicon Ultra-15 Centrifugal Filter Units</td>
			<td>EMO Millipore</td>
			<td>UFC900324</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Life Technologies</td>
			<td>11966-025</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Life Technologies</td>
			<td>10437-028</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibiotic-Antimycotic&#160;</td>
			<td>Life Technologies</td>
			<td>15240-062</td>
			<td></td>
		</tr>
		<tr>
			<td>24-Well Flat Bottom Plate</td>
			<td>Sigma-Aldrich</td>
			<td>CLS3527-100EA</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-Lysine</td>
			<td>Sigma-Aldrich</td>
			<td>P7280</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS, No Calcium, No Magnesium</td>
			<td>Life Technologies</td>
			<td>21600010</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparan Sulfate</td>
			<td>Sigma-Aldrich</td>
			<td>H4777</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Life Technologies</td>
			<td>25300054</td>
			<td></td>
		</tr>
		<tr>
			<td>Hela cells</td>
			<td>ATCC</td>
			<td>CCL-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Nano Drop ND-1000 spectrophotometer&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAquick PCR Purification Kit</td>
			<td>QIAGEN</td>
			<td>28104</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAquick Gel Extraction Kit</td>
			<td>QIAGEN</td>
			<td>28704</td>
			<td></td>
		</tr>
	</tbody>
</table>

direct protein delivery to mammalian cells using cell-permeable cys2-his2 zinc-finger domains

Due to their modularity and ability to be reprogrammed to recognize a wide range of DNA sequences, Cys2-His2 zinc-finger DNA-binding domains have emerged as useful tools for targeted genome engineering. Like many other DNA-binding proteins, zinc-fingers also possess the innate ability to cross cell membranes. We recently demonstrated that this intrinsic cell-permeability could be leveraged for intracellular protein delivery. Genetic fusion of zinc-finger motifs leads to efficient transport of protein and enzyme cargo into a broad range of mammalian cell types. Unlike other protein transduction technologies, delivery via zinc-finger domains does not inhibit enzyme activity and leads to high levels of cytosolic delivery. Here a detailed step-by-step protocol is presented for the implementation of zinc-finger technology for protein delivery into mammalian cells. Key steps for achieving high levels of intracellular zinc-finger-mediated delivery are highlighted and strategies for maximizing the performance of this system are discussed.

二本指ZIF-EmGFP融合タンパク質はE.で発現させることができる大腸菌 &gt; 95％の均質性及び高収率で（&gt; 25 mg / ml）で（ 図2）。一般的には、1次元と2本指のZIF融合タンパク質は、野生型非修飾タンパク質のものとほぼ同じ量で製造することができる。しかし、いくつかの文脈において、5員環及び6フィンガーZIF融合タンパク質は、下流の用途のために十分に高い収率で?...

Watch this Scientific Journal Video about Direct Protein Delivery to Mammalian Cells Using Cell-permeable Cys2-His2 Zinc-finger Domains at JoVE.com

Direct Protein Delivery to Mammalian Cells Using Cell-permeable Cys2-His2 Zinc-finger Domains

ジンクフィンガードメインは、本質的に細胞透過性及び広範囲の哺乳類細胞型へのタンパク質の送達を媒介することができる。ここでは、細胞内タンパク質の送達のためのジンクフィンガーの技術を実施するための詳細なステップバイステップのプロトコルが提供される。

細胞透過性のCysを用いて哺乳動物細胞への直接タンパク質送達<sub&gt; 2</sub&gt; -His<sub&gt; 2</sub&gt;ジンクフィンガードメイン

Due to their modularity and ability to be reprogrammed to recognize a wide range of DNA sequences, Cys2-His2 zinc-finger DNA-binding domains have emerged ...

direct-protein-delivery-to-mammalian-cells-using-cell-permeable-cys2

Research

JoVE Journal

Biology

Direct Protein Delivery to Mammalian Cells Using Cell-permeable Cys2-His2 Zinc-finger Domains

9.8K ビュー.  The Scripps Research Institute. ジンクフィンガードメインは、本質的に細胞透過性及び広範囲の哺乳類細胞型へのタンパク質の送達を媒介することができる。ここでは、細胞内タンパク質の送達のためのジンクフィンガーの技術を実施するための詳細なステップバイステップのプロトコルが提供される。 

ビデオ: Direct Protein Delivery to Mammalian Cells Using Cell-permeable Cys2-His2 Zinc-finger Domains

Long-term Imaging Mammalian Cells using Wide-Field Microscopy

Actually what I'm gonna do, mount it in here first and then swap out You. So now I'm gonna aspirate try and be as gentle as possible.Okay. A few seconds just holding.Okay.Alright.So the important features of our lifestyle imaging grid are that, so it's an inverted scope, which means the objective, the samples here in the objective is underneath. So the objective is pointing up and the whole scope, or practically the whole, practically the whole scope, including the objectives and the sample are at 37 degrees. So everything in this plastic box is at 37 degrees.And right on top of the stage we have an air and CO2 mix. That is 5%CO2. So the idea is that the sample is actually in a cell, it's like a cell incubator.And so we have, this is our mixer for the air and the CO2 and, and it gets humidified and heated and then piped onto the, the sample on the stage.Okay. And today we're just gonna be doing fluorescence one channel GFP fluorescence. This is the hose that the CO2 and 5%CO2 goes in and it just fits into the stage holder.That's important. And then we can close these, take these down, and then we go to our first position. And what I'm gonna do is go to the first field, which is my control sample, and I'll pick positions based on what I see on the screen.So I just make sure I actually look into the microscope in each new, well, because the focal plane for each of the wells is different, the, the dish is not perfectly flat on the bottom. So you will need to make fine adjustments when you move from well to the well. So right now I'm on the first, well, what I'll do is I'll focus, so I just hit, I just told, I just turned on the light, the transmitted light for the microscope.And I have make sure all the light is going to the eye pieces. And so I just look in, make sure I get a nice clean field. And then what I'll do is turn that light off, turn the fluorescence light on, make sure the cells that I'm looking at look healthy and they're not apoptotic.And, and I turn that off. And what I do next is send, so now I'm gonna start picking positions, which means I have to te send the light path to the camera. This is the camera right here.So I tell the microscope to send all the camera, they don't see anything in the eye pieces. What I'll do is in the software controlling the microscope, I'll choose the right filter set, which for our purposes is psy. And then set an exposure time.And I know these cells very well and I know that they take a 0.2 second exposure time. So I tell, that's what I tell the software to do. And what I'll do is hit focus.This is, and it's basically just alive. The, A light will turn on and you can, oh, I don't know if you can see cells, but the reason this is a good field is because there's, all the cells are, even in terms of their fluorescence, There are no apoptotic cells. Apoptotic cells tend to be very bright and there's no dirt or schmutz in the field.And you can see that they're actually, this is a metaphase cell and that's a metaphase cell, that's a cell that's probably just exited mitosis. And I can just tell that because it looks like those are, those two sets of chromatin are very nicely oriented to each other. Like they just exited an face.So that's a good field. So, and now that I know that the exposure time and the filter set is correct, and this is gonna be software specific, I'll go and I'll take positions and now the software has memorized the X, Y, Z position of that field. And then when I do pick another field, it's, and I'll, so when I'm picking, when I'm picking new fields, but in the same, well, I'll use the, I'll move the i'll, I'll turn the shutter, I'll open the shutter and I'll look to see what I see on the screen and I'll pick fields based on that versus looking in the field.So I don't see any of my tonics in there. But again, even nicely centered, these cells will go through my PS I continue, here's a good example. Here's a good example of what I would avoid that, see how bright that is?That's very bright. That's probably remnants of an apoptotic cell. And the reason I would avoid that is because since it's so bright, when the software auto focuses, it will autofocus just on that and it won't be in the same plane as all these other cells.And so I won't get any information from that field. That looks nice. There's a, this is a pro metaphase right here, That's A metaphase.So, and I'm gonna take three positions per well. So that was three positions and now I'm going to go to my next Well, so you can, so what I will probably do is take images every 10 minutes. So it's a compromise.Lifestyle imaging is always a compromise, right? You have to image, you have to keep the cells healthy, especially if you're interested in mitosis. So you have to minimize their exposure to the fluorescent line.And, but you wanna get information. So you have to take images, you have to, so you have to compromise, you have to make a choice about what you want to capture, what kind of information you wanna capture. For our purposes, mitosis tends to take about an hour.So if we're taking 10 minute intervals, that's pretty good. We see prophase, we see metaphase, we see anaphase, and we can, we can see when there are, there are treatments or drugs or siRNAs that affect those specific cell cycle stages. So for our purposes, that's enough, but we always want more.We always want more positions or finer time and interval or something like that. So it's a compromise. So, and then I'll hit start.And so our software, it does an autofocusing pass on the first pass. So once I hit start, it should start auto autofocusing and you'll see the shutter open, open and close between auto focusing. So I'll hit start.So that's the shutter opening and closing. The Z motor is engaging and it's actually trying to find the plane with the most contrast, which will be the most in focus. You can actually see on the screen going in and out of finding the best plane of focus that might be worrisome.So what it does is it, it drives the Z motor to find the best, the most in focus plane focus, well the most in image. And then it acquires picture there and then it learns that Z position. And then we'll use that Z position for the next 12 passes.And then we'll do the auto focusing pass again. What Kind of experiments do they do? Okay, so our lab is mostly interested in understanding what regulates mitic progression, or at least I'm most interested in understanding that.So with live cell imaging and especially our multi position, long-term imaging scope, you can take, you can film cells for two or three days entering anding mitosis, and you can image multiple rounds of mitosis so you can understand. And then you can put drugs or RNAs or any kind of perturbations on the cells that you would like to study. So you can understand how, how those drugs or, or genes are, are important in regulating mitotic progression.Because it's not fixed cell work and live cell work, you can understand what, when you need to have a perturbation to affect mitosis or what stage of mitosis is affected. So it's really powerful in that respect. What, what are the main technical elements of this experiments?What are the possible problems? Well, the biggest problem besides getting the rig set up, getting the rig set up is not trivial. A lot of people have the rig, but just making sure it works well for your application.The financial element is not, you know, is not small. So there's the setting up of the apparatus and then, and then once you gather the data, there's a real data analysis issue. It takes these movie, if, so, if you're gathering 40 movies each with 30 cells, so you have 40 in 40 fields that you just filmed and each of those fields has 30 cells, then you have to sit down with each one of those movies and look at each one of those cells in that movie.So it takes a really long time to do data analysis. And that's, so it takes a week to analyze data that it took you two days to collect. So there's a, that's a very big obstacle in terms of gathering lots of data.So it's not, while it's high throughput data collection, it's not high throughput analysis. So what would you make to make this experiment really high throughput? So our lab is in the process of developing a program that does the data analysis automatically.And it's a pretty decent program. It's not perfect. The human eye is always better, but it's getting there.So What are the interesting application of this experiment? Maybe other fields you can mention? Besides cell division?You mean for the, oh, for the live cell imaging? Yes.Well, you can do cell motility assays. You can, depending on the, the microscopy that's involved, you can, you know, look at how cells, you could look at tumor development if you really wanted to.But there, you, you're, if as long as you have the right imaging set up, you can, you're pretty much not limited in terms of anything. Our scope happens to be just a wide field and we use 20 by 20 x objective. So it's a pretty low resolution set up, but that's sufficient for what we need to see.So there are, but there definitely aren't, you wouldn't be limited. Anything that has any dynamic kind of experiment, which is biology, it would be, would really benefit from lifestyle imaging. So calcium imaging, although you couldn't do it on our apparatus, that's a live cell.You definitely need live cell imaging for that. Like I said, patient assays, motility assays, those kinds of things would really benefit from live cell imaging. Would you like to tell a few words about your previous experience?How long do you work on these techniques? What do you you work on before? Wow.So in my PhD I didn't work.I did a lot of confocal microscopy, but no lifestyle imaging. And so during my postdoc I was, I helped Dr.King get this apparatus that, that we just used to set up these fragment, get that up and running. And, and so during my postdoc, I had a lot of experience with both widefield with TimeLapse imaging on our scope, which is the widefield and also TimeLapse imaging with confocal microscopy.And I would say that it's really powerful. You get a lot of information, but it's not easy to get working perfectly. So it really requires a lot of attention and a lot of attention to details at every little step of the process.And, and then you still have problems. So it's a tough application, but you can really get, if, if it's what you need to have to, to study your problem, your scientific problem, then it, it's invaluable in that respect, but it's tough. So find someone who knows what they're doing and use them.That's my advice. So good.

広視野顕微鏡を用いて長期的なイメージング哺乳類細胞

生物学

Trypsinizing and Subculturing Mammalian Cells

As cells reach confluency, they must be subcultured or passaged. Failure to subculture confluent cells results in reduced mitotic index and eventually in cell death. The first step in subculturing is to detach cells from the surface of the primary culture vessel by trypsinization or mechanical means. The resultant cell suspension is then subdivided, or reseeded, into fresh cultures. Secondary cultures are checked for growth and fed periodically, and may be subsequently subcultured to produce tertiary cultures. The time between passaging of cells varies with the cell line and depends on the growth rate.

As cells reach confluency, they must be subcultured or passaged. This video will demonstrate a procedure for subculturing both adherent and suspension cells.

哺乳動物細胞をトリプシン処理し、継代培養

As cells reach confluency, they must be subcultured or passaged. Failure to subculture confluent cells results in reduced mitotic index and eventually in ...

Engineering Cell-permeable Protein

The protein transduction technique enables the direct delivery of biologically active material into mammalian cells [for review see 1,2]. For this one can make use of the translocating ability of so-called cell penetrating peptides (CPPs), also designated as protein transduction domains (PTDs). The TAT-CPP derived from the human immunodeficiency virus type 1 (HIV-1) Tat (trans-activator of transcription) protein has been widely used. The positively charged TAT promotes cell permeability thereby overcoming the barriers of the cellular membrane by endocytosis or/and direct membrane penetration2. In combination with a nuclear localization signal (NLS) fusion proteins are able to enter the nucleus exhibiting functionality. Our video presentation demonstrates, as an exemplification for the engineering of cell-permeable proteins, the construction, production and application of a cell-permeable version of the DNA-modifying enzyme Cre.
Cre is a site-specific recombinase that is able to recognize and recombine 34 base pair loxP sites in mammalian cells in vitro and in vivo. Therefore the Cre/loxP system is widely used to conditionally induce mutations in the genome of living cells3,4. The delivery of active Cre recombinase to cells, however, represents a limitation.
We describe the pSESAME vector system, which allows a direct insertion of the gene-of-interest and provides a platform to rapidly clone different domains and tags used within the vector in a convenient and standardized manner. Rearranging of the different tags has been shown to modify the biochemical properties of the fusion proteins providing a possibility to achieve higher yield and better solubility. We demonstrate how to express and purify recombinant cell-permeant proteins in and from E. coli. The functionality of the recombinant Cre protein is finally validated in cell culture by assessing its intracellular recombinase activity.

Protein transduction enables the direct delivery of biologically active proteins into cells. In contrast to conventional methods such as DNA transfection or viral transduction this non-invasive paradigm allows highly efficient cellular manipulation in a titratable manner circumventing cellular toxicity and the risk of oncogenic transformation by permanent genetic modification.

エンジニアリング細胞透過性のタンパク質

The protein transduction technique enables the direct delivery of biologically active material into mammalian cells [for review see 1,2]. For this one can ...

Using the GELFREE 8100 Fractionation System for Molecular Weight-Based Fractionation with Liquid Phase Recovery

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based fractionation. The system is comprised of single-use, 8-sample capacity cartridges and a benchtop GELFREE Fractionation Instrument. During separation, a constant voltage is applied between the anode and cathode reservoirs, and each protein mixture is electrophoretically driven from a loading chamber into a specially designed gel column gel. Proteins are concentrated into a tight band in a stacking gel, and separated based on their respective electrophoretic mobilities in a resolving gel. As proteins elute from the column, they are trapped and concentrated in liquid phase in the collection chamber, free of the gel. The instrument is then paused at specific time intervals, and fractions are collected using a pipette. This process is repeated until all desired fractions have been collected. If fewer than 8 samples are run on a cartridge, any unused chambers can be used in subsequent separations.
This novel technology facilitates the quick and simple separation of up to 8 complex protein mixtures simultaneously, and offers several advantages when compared to previously available fractionation methods. This system is capable of fractionating up to 1mg of total protein per channel, for a total of 8mg per cartridge. Intact proteins over a broad mass range are separated on the basis of molecular weight, retaining important physiochemical properties of the analyte. The liquid phase entrapment provides for high recovery while eliminating the need for band or spot cutting, making the fractionation process highly reproducible1.

The accompanying video describes the use of the GELFREE 8100 Fractionation System, which partitions complex protein samples on the basis of molecular weight and recovers the fractions in liquid phase. The video describes how the technology works, how it is used, and provides resultant data, with polyacrylamide gel electrophoresis analysis of fractionated bovine liver homogenate.

液相の回復と分子量ベース画用GELFREE 8100分別システムの使用

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based ...

Genome Editing with CompoZr Custom Zinc Finger Nucleases (ZFNs)

Genome editing is a powerful technique that can be used to elucidate gene function and the genetic basis of disease. Traditional gene editing methods such as chemical-based mutagenesis or random integration of DNA sequences confer indiscriminate genetic changes in an overall inefficient manner and require incorporation of undesirable synthetic sequences or use of aberrant culture conditions, potentially confusing biological study. By contrast, transient ZFN expression in a cell can facilitate precise, heritable gene editing in a highly efficient manner without the need for administration of chemicals or integration of synthetic transgenes.
Zinc finger nucleases (ZFNs) are enzymes which bind and cut distinct sequences of double-stranded DNA (dsDNA). A functional CompoZr ZFN unit consists of two individual monomeric proteins that bind a DNA "half-site" of approximately 15-18 nucleotides (see Figure 1). When two ZFN monomers "home" to their adjacent target sites the DNA-cleavage domains dimerize and create a double-strand break (DSB) in the DNA.1 Introduction of ZFN-mediated DSBs in the genome lays a foundation for highly efficient genome editing. Imperfect repair of DSBs in a cell via the non-homologous end-joining (NHEJ) DNA repair pathway can result in small insertions and deletions (indels). Creation of indels within the gene coding sequence of a cell can result in frameshift and subsequent functional knockout of a gene locus at high efficiency.2 While this protocol describes the use of ZFNs to create a gene knockout, integration of transgenes may also be conducted via homology-directed repair at the ZFN cut site.
The CompoZr Custom ZFN Service represents a systematic, comprehensive, and well-characterized approach to targeted gene editing for the scientific community with ZFN technology. Sigma scientists work closely with investigators to 1) perform due diligence analysis including analysis of relevant gene structure, biology, and model system pursuant to the project goals, 2) apply this knowledge to develop a sound targeting strategy, 3) then design, build, and functionally validate ZFNs for activity in a relevant cell line. The investigator receives positive control genomic DNA and primers, and ready-to-use ZFN reagents supplied in both plasmid DNA and in-vitro transcribed mRNA format. These reagents may then be delivered for transient expression in the investigator&#x2019;s cell line or cell type of choice. Samples are then tested for gene editing at the locus of interest by standard molecular biology techniques including PCR amplification, enzymatic digest, and electrophoresis. After positive signal for gene editing is detected in the initial population, cells are single-cell cloned and genotyped for identification of mutant clones/alleles.

Genome Editing with CompoZr Custom Zinc Finger Nucleases ZFNs

The CompoZr Custom Zinc-Finger Nuclease (ZFN) Service enables precise genome editing in any organism or cell line at any locus defined by the user. This article describes the process for the design, manufacture, validation and implementation of the CompoZr Custom ZFN Service.

CompoZrカスタムジンクフィンガーヌクレアーゼによるゲノム編集（ZFNs）

Genome editing is a powerful technique that can be used to elucidate gene function and the genetic basis of disease. Traditional gene editing methods such ...

siRNA Screening to Identify Ubiquitin and Ubiquitin-like System Regulators of Biological Pathways in Cultured Mammalian Cells

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that regulates diverse biological pathways including the hypoxia response, proteostasis, the DNA damage response and transcription.&#160; To better understand how UBLs regulate pathways relevant to human disease, we have compiled a human siRNA &#8220;ubiquitome&#8221; library consisting of 1,186 siRNA duplex pools targeting all known and predicted components of UBL system pathways. This library can be screened against a range of cell lines expressing reporters of diverse biological pathways to determine which UBL components act as positive or negative regulators of the pathway in question.&#160; Here, we describe a protocol utilizing this library to identify ubiquitome-regulators of the HIF1A-mediated cellular response to hypoxia using a transcription-based luciferase reporter.&#160; An initial assay development stage is performed to establish suitable screening parameters of the cell line before performing the screen in three stages: primary, secondary and tertiary/deconvolution screening. &#160;The use of targeted over whole genome siRNA libraries is becoming increasingly popular as it offers the advantage of reporting only on members of the pathway with which the investigators are most interested.&#160; Despite inherent limitations of siRNA screening, in particular false-positives caused by siRNA off-target effects, the identification of genuine novel regulators of the pathways in question outweigh these shortcomings, which can be overcome by performing a series of carefully undertaken control experiments.

Here, we describe a methodology to perform a targeted siRNA &#8220;ubiquitome&#8221; screen to identify novel ubiquitin and ubiquitin-like regulators of the HIF1A-mediated cellular response to hypoxia.&#160; This can be adapted to any biological pathway where a robust read out of reporter activity is available.

培養哺乳動物細胞内の生物学的経路のユビキチンおよびユビキチン様システムレギュレータを識別するために、siRNAのスクリーニング

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that ...

Identification of Protein Interaction Partners in Mammalian Cells Using SILAC-immunoprecipitation Quantitative Proteomics

Quantitative proteomics combined with immuno-affinity purification, SILAC immunoprecipitation, represent a powerful means for the discovery of novel protein:protein interactions. By allowing the accurate relative quantification of protein abundance in both control and test samples, true interactions may be easily distinguished from experimental contaminants. Low affinity interactions can be preserved through the use of less-stringent buffer conditions and remain readily identifiable. This protocol discusses the labeling of tissue culture cells with stable isotope labeled amino acids, transfection and immunoprecipitation of an affinity tagged protein of interest, followed by the preparation for submission to a mass spectrometry facility. This protocol then discusses how to analyze and interpret the data returned from the mass spectrometer in order to identify cellular partners interacting with a protein of interest. As an example this technique is applied to identify proteins binding to the eukaryotic translation initiation factors: eIF4AI and eIF4AII.

SILAC immunoprecipitation experiments represent a powerful means for discovering novel protein:protein interactions. By allowing the accurate relative quantification of protein abundance in both control and test samples, true interactions may be easily distinguished from experimental contaminants, and low affinity interactions preserved through use of less-stringent buffer conditions.

SILAC免疫沈降定量的プロテオミクスを用いて哺乳動物細胞におけるタンパク質相互作用パートナーの同定

Quantitative proteomics combined with immuno-affinity purification, SILAC immunoprecipitation, represent a powerful means for the discovery of novel ...

Zinc-finger Nuclease Enhanced Gene Targeting in Human Embryonic Stem Cells

One major limitation with current human embryonic stem cell (ESC) differentiation protocols is the generation of heterogeneous cell populations.&#160;These cultures contain the cells of interest, but are also contaminated with undifferentiated ESCs, non-neural derivatives and other neuronal subtypes.&#160; This limits their use in in vitro and in vivo applications, such as in vitro modeling for drug discovery or cell replacement therapy. To help overcome this, reporter cell lines, which offer a means to visualize, track and isolate cells of interest, can be engineered.&#160;However, to achieve this in human embryonic stem cells via conventional homologous recombination is extremely inefficient.&#160;This protocol describes targeting of the Pituitary homeobox 3 (PITX3) locus in human embryonic stem cells using custom designed zinc-finger nucleases, which introduce site-specific double-strand DNA breaks, together with a PITX3-EGFP-specific DNA donor vector. Following the generation of the PITX3 reporter cell line, it can then be differentiated using published protocols for use in studies such as in vitro Parkinson&#8217;s disease modeling or cell replacement therapy.

Reporter cell lines offer a means to visualize, track and isolate cells of interest from heterogeneous populations.&#160;However, gene-targeting using conventional homologous recombination in human embryonic stem cells is extremely inefficient. Herein, we describe targeting CNS midbrain specific transcription factor PITX3 locus with EGFP using zinc-finger nuclease enhanced homologous recombination.

ヒト胚性幹細胞におけるターゲティングジンクフィンガーヌクレアーゼ遺伝子の強化

One major limitation with current human embryonic stem cell (ESC) differentiation protocols is the generation of heterogeneous cell ...

Examination of Proteins Bound to Nascent DNA in Mammalian Cells Using  BrdU-ChIP-Slot-Western Technique

Histone deacetylases 1 and 2 (HDAC1,2) localize to the sites of DNA replication. In the previous study, using a selective inhibitor and a genetic knockdown system, we showed novel functions for HDAC1,2 in replication fork progression and nascent chromatin maintenance in mammalian cells. Additionally, we used a BrdU-ChIP-Slot-Western technique that combines chromatin immunoprecipitation (ChIP) of bromo-deoxyuridine (BrdU)-labeled DNA with slot blot and Western analyses to quantitatively measure proteins or histone modification associated with nascent DNA.
Actively dividing cells were treated with HDAC1,2 selective inhibitor or transfected with siRNAs against Hdac1 and Hdac2 and then newly synthesized DNA was labeled with the thymidine analog bromodeoxyuridine (BrdU). The BrdU labeling was done at a time point when there was no significant cell cycle arrest or apoptosis due to the loss of HDAC1,2 functions. Following labeling of cells with BrdU, chromatin immunoprecipitation (ChIP) of histone acetylation marks or the chromatin-remodeler was performed with specific antibodies. BrdU-labeled input DNA and the immunoprecipitated (or ChIPed) DNA was then spotted onto a membrane using the slot blot technique and immobilized using UV. The amount of nascent DNA in each slot was then quantitatively assessed using Western analysis with an anti-BrdU antibody. The effect of loss of HDAC1,2 functions on the levels of newly synthesized DNA-associated histone acetylation marks and chromatin remodeler was then determined by normalizing the BrdU-ChIP signal obtained from the treated samples to the control samples.

In this protocol, we describe a novel BrdU-ChIP-Slot-Western technique to examine proteins and histone modifications associated with newly synthesized or nascent DNA.

BrdUでのChIP-スロット西洋技術を用いて哺乳動物細胞中で新生DNAに結合したタンパク質の検討

Histone deacetylases 1 and 2 (HDAC1,2) localize to the sites of DNA replication. In the previous study, using a selective inhibitor and a genetic ...

A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex signaling mechanisms underlying filopodial initiation, elongation and subsequent stabilization or retraction, it is crucial to determine the spatio-temporal protein activity in these dynamic structures. To analyze protein function in filopodia, we recently developed a semi-automated tracking algorithm that adapts to filopodial shape-changes, thus allowing parallel analysis of protrusion dynamics and relative protein concentration along the whole filopodial length. Here, we present a detailed step-by-step protocol for optimized cell handling, image acquisition and software analysis. We further provide instructions for the use of optional features during image analysis and data representation, as well as troubleshooting guidelines for all critical steps along the way. Finally, we also include a comparison of the described image analysis software with other programs available for filopodia quantification. Together, the presented protocol provides a framework for accurate analysis of protein dynamics in filopodial protrusions using image analysis software.

We present a software solution for semi-automated tracking of relative protein concentration along the length of dynamic cellular protrusions.

動的細胞突起におけるソフトウェア支援トラッキングのためのグラフィカルユーザインタフェース

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex ...