Name: direct protein delivery to mammalian cells using cell-permeable cys2-his2 zinc-finger domains
Uploaded: 2015-03-25T15:00:00
Description: Watch this Scientific Journal Video about Direct Protein Delivery to Mammalian Cells Using Cell-permeable Cys2-His2 Zinc-finger Domains at JoVE.com

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</str...

<ol>
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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 ...

Research

JoVE Journal

Biology

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

Long-term Imaging Mammalian Cells using Wide-Field Microscopy

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

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

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

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 with CompoZr Custom Zinc Finger Nucleases ZFNs

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

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

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

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

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

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 ...