我々は、オリバーBrüstleおよび幹細胞工学グループ、サポートと貴重な議論のためのボン大学、のすべてのメンバーに感謝。我々は、SDS - PAGEの準備とプロジェクト全体で永続的なサポートのためのザビーネシェンクに感謝。ニコルラスとアンナMagerhansは、優れた技術サポートを提供した。さらに、映画の制作のためにアンドレアスバーとシーラメルテンスに感謝します。この作品は、フォルクスワーゲン財団（AZ I/77864）と教育研究のドイツ省（BMBF、01 GN 0813）からの補助金によって支えられている。

発現ベクターおよび発現の建設： pSESAME - Creを発現ベクターは、標準的なクローニング方法を使用してAvrIIおよびNheI制限部位を介してpSESAMEにCreをコードする断片を挿入することにより構築した。 pSESAMEはヒスチジン - タグ、TAT -ドメイン、NLS配列とCre、略称HTNCreで構成される融合タンパク質をコードしている。 HTNCreの発現のためpSESAME - CreをはTUNER（DE3）pLacIに変換してグリセロールストックを調製した。 <ol><li>一晩培養物は、グリセロールストックから形質転換した細菌をコーティングしたピペットチップを用いて接種した。オーバーナイト培養液は、濃度50μg/ mlの最終濃度で0.5％のグルコース[V / V]とカルベニシリンを補充したLB培地で構成され、37で成長させた℃で16時間C。 </li><li>次の日密栽培オーバーナイト培養液を1〜40の割合で発現培養を接種したと37℃のインキュベーターに入れ、発現培養は100μg/ mlの最終濃度で0.5％のグルコース[V / V]とアンピシリンを添加したTBメディアから成っていた。 </li><li> 1.5のOD 595で発現培養を1時間では0.5 mMのIPTGで誘導した</li><li>その後、細菌はSLA3000ローターで10分間5000rpmで遠心分離によって回収した。 </li><li>バクテリアペレットは-20℃で精製されるまで保存されていました。 </li></ol>細胞透過性のタンパク質の精製： <ol><li>冷凍細菌ペレットを室温で15分間リットルのフラスコの培養液あたり10mLの溶解バッファーに再懸濁した。 </li><li>室温で混合しながら次いで、この懸濁液をさらに15分間に1 mg / mLのリゾチームとインキュベートした。 </li><li> 25 U / mLのベンゾナーゼはその後加え、室温で15分間混合しながらインキュベートした。 </li><li>混合し、氷上で5分間インキュベートしながら電力の45％で0.5秒のパルスで1.5分、1 mLの冷酒石酸塩緩衝液（TSB）氷上で超音波処理した後1mLあたり懸濁液を注意深く加えた。ライセート分画（L）のSDS - PAGEのサンプルを採取した。 </li><li>清澄化ライセート℃で30分間30,000 gで4℃で遠心分離によって得られた水溶性（S）と不溶性画分（I）のSDS - PAGEのサンプルを採取した。 </li><li>上清を新しい50mlファルコンチューブに移し、4℃で1時間の初期発現培養リットル当たり50％のNi - NTAのスラリーを2mLとCのためにして穏やかに混合したれました。 </li><li>懸濁液を重力流EconoPacのカラムに充填（フロースルーフラクション（FT）のSDS - PAGEのサンプルが取られた）と洗浄バッファーの5ベッドボリュームで2回洗浄した。両方の洗浄画分（W1＆W2）のSDS - PAGEのサンプルを採取した。 </li><li> HTNCre含有画分をSDS - PAGE解析のための溶出画分の溶出緩衝液と試料（E）の3ベッドボリュームで溶出された取られた。 </li><li>イミダゾール二回高塩緩衝液に対して溶出画分を透析により除去した。 </li><li>タンパク質溶液は、さらに2回グリセロール緩衝液に対して透析により濃縮した。すべての透析の手順でサンプルへのバッファの比率は少なくとも50あった。この手順は、200および450μMの間で通常の濃度でHTNCreを含むグリセロールストック溶液をもたらした、すなわち発現培養1リットルは、タンパク質の約12ミリグラムになります。 SDS - PAGE解析のためのグリセロールストック（GS）のサンプルを採取した。 HTNCreストック溶液は、マイナス20℃で保存することができます</li></ol><img border="0" src="/files/ftp_upload/1627/1627fig1tmb.jpg" alt="図1" /> 図1：Creリコンビナーゼの精製の ​​過程で収集されたサンプルのSDS - PAGE分析。 Creリコンビナーゼの発現誘導は、ライセート画分における支配的なバンドで示されます。タンパク質の一部が不溶性であるが溶出液とグリセロールストックの分画に見られるようにCREのタンパク質をさらに充実させることができます。 L：ライセート、I：不溶性、S：上清を、FT：フロースルー、W：洗浄、E：溶出液、GS：Gylcerolストック。してください<a href="https://www.jove.com/files/ftp_upload/1627/1627fig1.jpg">ここをクリックして</a>図1の拡大バージョンを参照すること。 マウス胚性幹（ES）細胞へのタンパク質導入： <ol><li>条件付きのβ-ガラクトシダーゼレポーター5コンストラクトを運ぶES細胞は接着細胞の解離のためTrypLE™Expressを使用して、単一の細胞として播種した。 4〜6時間後に細胞を再装着していたと培地を除去した。 </li><li>その後、ES細胞は、16時間HTNCre含有培地でインキュベートした。 <ul><li>グリセロールストックからHTNCre蛋白質の適切な量（10μMに相当）は、ES培地と続いて無菌ろ過（0.22μmの）中に希釈した。 </li></ul></li><li>タンパク質導入後の培地は、通常の増殖培地に戻って変更されました。 </li><li>二日間の細胞をPBSで洗浄し、10分間4％パラホルムアルデヒド（PFA）で固定した後。 </li><li>さらに2X - gal染色を行う前にPBSでtional洗浄ステップが実行されました。 </li><li>固定された細胞は、X - gal染色液6の層で覆い、37℃で一晩インキュベートした。 </li></ol>代表的な結果： 翌日、X - gal染色液を熱望し、細胞を顕微鏡分析のためのPBSの層で覆われていた。組換え細胞の80〜100％がβ-ガラクトシダーゼ活性が判断したマウスES細胞内で観察することができた。

<ol>
	<li>Gump, J. M., Dowdy, S. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TAT+transduction:+the+molecular+mechanism+and+therapeutic+prospects.">TAT transduction: the molecular mechanism and therapeutic prospects.</a> Trends Mol Med. 13 (10), 443-448 (2007).</li><li>Edenhofer, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+transduction+revisited:+novel+insights+into+the+mechanism+underlying+intracellular+delivery+of+proteins.">Protein transduction revisited: novel insights into the mechanism underlying intracellular delivery of proteins.</a> Curr Pharm Des. 14 (34), 3628-3636 (2008).</li><li>Branda, C. S., Dymecki, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Talking+about+a+revolution:+The+impact+of+site-specific+recombinases+on+genetic+analyses+in+mice.">Talking about a revolution: The impact of site-specific recombinases on genetic analyses in mice.</a> Dev Cell. 6 (1), 7-28 (2004).</li><li>Nolden, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Site-specific+recombination+in+human+embryonic+stem+cells+induced+by+cell-permeant+Cre+recombinase.">Site-specific recombination in human embryonic stem cells induced by cell-permeant Cre recombinase.</a> Nat Methods. 3 (6), 461-467 (2006).</li><li>Zhang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inducible+site-directed+recombination+in+mouse+embryonic+stem+cells.">Inducible site-directed recombination in mouse embryonic stem cells.</a> Nucleic Acids Res. 24 (4), 543-548 (1996).</li><li>Peitz, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+purification+of+cell-permeant+Cre+and+germline+transmission+after+transduction+into+mouse+embryonic+stem+cells.">Enhanced purification of cell-permeant Cre and germline transmission after transduction into mouse embryonic stem cells.</a> Genesis. 45 (8), 508-517 (2007).</li><li>Bosnali, M., Edenhofer, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+transducible+versions+of+transcription+factors+Oct4+and+Sox2.">Generation of transducible versions of transcription factors Oct4 and Sox2.</a> Biol Chem. 389 (7), 851-861 (2008).</li><li>Landry, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Runx+genes+are+direct+targets+of+Scl/Tal1+in+the+yolk+sac+and+fetal+liver.">Runx genes are direct targets of Scl/Tal1 in the yolk sac and fetal liver.</a> Blood. 111 (6), 3005-3014 (2008).</li></ol>

Creを融合タンパク質の精製プロセス中にそれは遠心分離の前に氷冷TBS緩衝液の添加を省略することが重要です。そうしないとCreリコンビナーゼは、グリセロールのバッファ内に沈殿する傾向がある。 溶出画分は、融合タンパク質、追加の溶出緩衝液の高濃度に起因する混濁になるために表示されている場合は解決策が再びクリアされるまで追加する必要があります。 Creを融合タンパク質の10μMのアプリケーションは、通常80〜100％の組換えの効率が低下します。 ES細胞用培地の主要な成分であるウシ胎児血清（FCS）が強くタンパク質導入を阻害する。したがって、Creリコンビナーゼの高濃度は、使用する必要がありました。無血清条件下での作業時に以下の蛋白質（0.5 - 2μM）は、同様の組換えの効率を達成するために使用することができます。 手でpSESAMEベクター系では、ある者は、Oct4とSox2の7とScl/Tal1 8などなどの転写因子を含む他のタンパク質へのタンパク質導入の手法を適用することができます。

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<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>TUNER (DE3) pLacI</td>
<td>&nbsp;</td>
<td>Novagen</td>
<td>70625</td>
<td>&nbsp;</td>
<tr>
<td>Glycerol</td>
<td>&nbsp;</td>
<td>Carl Roth</td>
<td>3783.2</td>
<td>&nbsp;</td>
<tr>
<td>Na2HPO4</td>
<td>&nbsp;</td>
<td>Roth</td>
<td>T876.1</td>
<td>&nbsp;</td>
<tr>
<td>Trizma Base</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>T1503</td>
<td>&nbsp;</td>
<tr>
<td>HCl</td>
<td>&nbsp;</td>
<td>Roth</td>
<td>4625.1</td>
<td>&nbsp;</td>
<tr>
<td>Imidazol</td>
<td>&nbsp;</td>
<td>Roth</td>
<td>X998.4</td>
<td>&nbsp;</td>
<tr>
<td>NaCl</td>
<td>&nbsp;</td>
<td>Roth</td>
<td>9265.2</td>
<td>&nbsp;</td>
<tr>
<td>Yeast Extract</td>
<td>&nbsp;</td>
<td>Roth</td>
<td>2363.4</td>
<td>&nbsp;</td>
<tr>
<td>Trypton/Pepton</td>
<td>&nbsp;</td>
<td>Roth</td>
<td>8952.4</td>
<td>&nbsp;</td>
<tr>
<td>K2HPO4 </td>
<td>&nbsp;</td>
<td>Roth</td>
<td>P749.2</td>
<td>&nbsp;</td>
<tr>
<td>KH2PO4</td>
<td>&nbsp;</td>
<td>Roth</td>
<td>3904.1</td>
<td>&nbsp;</td>
<tr>
<td>Ampicillin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A9518</td>
<td>&nbsp;</td>
<tr>
<td>Carbenicillin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>6344.2</td>
<td>&nbsp;</td>
<tr>
<td>HEPES</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>H3375</td>
<td>&nbsp;</td>
<tr>
<td>Lysozyme</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>62971</td>
<td>&nbsp;</td>
<tr>
<td>Benzonase</td>
<td>&nbsp;</td>
<td>Novagen</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>L-Tartaric acid, disodium salt </td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>50% Ni-NTA slurry</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>R901-15</td>
<td>&nbsp;</td>
<tr>
<td>EconoPac columns</td>
<td>&nbsp;</td>
<td>Biorad</td>
<td>732-1010</td>
<td>&nbsp;</td>
<tr>
<td>Sterile filter 0,22&mu;m</td>
<td>&nbsp;</td>
<td>Whatman</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Paraformaldehyde (PFA)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>LB medium</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Yeast extract, Trypton/Pepton, NaCl</td>
</tr>
<tr>
<td>TB medium</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Yeast extract, Trypton/Pepton, Glycerol, K2HPO4, KH2PO4</td>
</tr>
<tr>
<td>Lysis Buffer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>50 mM Na2HPO4, 5 mM Tris, pH 7.8</td>
</tr>
<tr>
<td>Tartaric Salt Buffer (TSB)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>PTB containing 2 M L-Tartaric acid, disodium salt, and 20 mM Imidazol</td>
</tr>
<tr>
<td>Washing Buffer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>PTB, 500 mM NaCl, 15 mM Imidazol</td>
</tr>
<tr>
<td>Elution Buffer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>PTB, 500 mM NaCl, 250 mM Imidazol</td>
</tr>
<tr>
<td>High Salt Buffer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>600 mM NaCl, 20 mM HEPES, pH 7.4</td>
</tr>
<tr>
<td>Gylcerol Buffer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>50% glycerol, 500 mM NaCl, 20 mM HEPES, pH 7.4</td>
</tr>
<tr>
<td>TrypLE&trade; Express</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>ESGRO (LIF)</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>NEAA</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>11140035</td>
<td>&nbsp;</td>
<tr>
<td>L-Glutamin</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>25030024</td>
<td>&nbsp;</td>
<tr>
<td>&beta;-Mercaptoethanol</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>31350010</td>
<td>&nbsp;</td>
<tr>
<td>DMEM</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>11960044</td>
<td>&nbsp;</td>
<tr>
<td>PBS</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Fetal Calf Serum (FCS)</td>
<td>&nbsp;</td>
<td>PAA</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>X-Gal staining solution:</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>4 mM K3(FeIII(CN)6), 4 mM K4(FeII(CN)6), 2mM MgCl2 0.4 mg/mL X-Gal solved in PBS</td>
</tr>
<tr>
<td>K3(FeIII(CN)6)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P-3367</td>
<td>&nbsp;</td>
<tr>
<td>K4 (FeII(CN)6)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P-9387</td>
<td>&nbsp;</td>
<tr>
<td>MgCl2</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>M8266</td>
<td>&nbsp;</td>
<tr>
<td>X-Gal</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>B4252</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

engineering cell-permeable protein

タンパク質導入法は、哺乳動物細胞への生物学的活性物質の直接配信が可能になります[レビューのために1,2を参照してください]。この1つは、いわゆる細胞透過性ペプチド（CPPの）の転位能力を利用することができるため、また、タンパク質導入ドメイン（PTDs）として指定。ヒト免疫不全ウイルス1型（HIV - 1）タット（転写のトランス活性化因子）タンパク質は、広く使用されているから派生したTAT - CPP。正に帯電したTATは、それによってエンドサイトーシスまたは/およびダイレクト膜貫通2で細胞膜の障壁を克服する細胞透過性を促進する。核局在化シグナルとの組み合わせで（NLS）の融合タンパク質は核展示機能を入力することができます。私たちのビデオプレゼンテーションは、細胞透過性タンパク質のエンジニアリング、建設、生産とDNA修飾酵素Creの細胞透過性のバージョンのアプリケーションのための例示として、示しています。 CREは、in vitroおよびin vivoでの哺乳類細胞には34塩基対のloxP部位を認識し、再結合することができる部位特異的リコンビナーゼである。そのためのCre / loxP部位システムが広く、条件付きで生きている細胞3,4のゲノムに突然変異を誘発するために使用されます。細胞へのアクティブなCreリコンビナーゼの配信は、しかし、制限を表します。 我々は、目的遺伝子の直接挿入を可能にし、急速に便利と標準化された方法でベクトル内で使用される別のドメインとタグのクローンを作成するためのプラットフォームを提供するpSESAMEベクターシステムを、説明します。別のタグの再配置は、より高い収率と良好な溶解性を達成する可能性を提供する融合タンパク質の生化学的特性を変更することが示されている。我々は、で、大腸菌から組換え細胞透過性のタンパク質を発現し、精製する方法を示しています。組換えのCreタンパク質の機能は、最終的にその細胞内リコンビナーゼの活性を評価することにより、細胞培養で検証されます。

Watch this Scientific Journal Video about Engineering Cell-permeable Protein at JoVE.com

Engineering Cell-permeable Protein

タンパク質導入は、細胞に生物学的に活性なタンパク質の直接配信を可能にします。このようなDNAトランスフェクションまたはウイルス形質導入などの従来の方法とは対照的に、この非侵襲的なパラダイムは、細胞毒性および永久的な遺伝子修飾による発癌性形質転換のリスクを回避滴定可能な方法で高効率な細胞の操作が可能になります。

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 ...

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JoVE Journal

Biology

14.4K ビュー.  University of Bonn - Life & Brain Center and Hertie Foundation. タンパク質導入は、細胞に生物学的に活性なタンパク質の直接配信を可能にします。このようなDNAトランスフェクションまたはウイルス形質導入などの従来の方法とは対照的に、この非侵襲的なパラダイムは、細胞毒性および永久的な遺伝子修飾による発癌性形質転換のリスクを回避滴定可能な方法で高効率な細胞の操作が可能になります。

ビデオ: Engineering Cell-permeable Protein

Experimental Approaches to Tissue Engineering

組織工学への実験的アプローチ

生物学

Micro-scale Engineering for Cell Biology

細胞生物学のためのマイクロスケールのエンジニアリング

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

クーマシー有機溶媒および酢酸のないG - 250でゲル中のタンパク質の染色

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

Cellular Encapsulation in 3D Hydrogels for Tissue Engineering 

The 3D encapsulation of cells within hydrogels represents an increasingly important and popular technique for culturing cells and towards the development of constructs for tissue engineering. This environment better mimics what cells observe in vivo, compared to standard tissue culture, due to the tissue-like properties and 3D environment. Synthetic polymeric hydrogels are water-swollen networks that can be designed to be stable or to degrade through hydrolysis or proteolysis as new tissue is deposited by encapsulated cells. A wide variety of polymers have been explored for these applications, such as poly(ethylene glycol) and hyaluronic acid. Most commonly, the polymer is functionalized with reactive groups such as methacrylates or acrylates capable of undergoing crosslinking through various mechanisms. In the past decade, much progress has been made in engineering these microenvironments - e.g., via the physical or pendant covalent incorporation of biochemical cues - to improve viability and direct cellular phenotype, including the differentiation of encapsulated stem cells (Burdick et al.).
The following methods for the 3D encapsulation of cells have been optimized in our and other laboratories to maximize cytocompatibility and minimize the number of hydrogel processing steps. In the following protocols (see Figure 1 for an illustration of the procedure), it is assumed that functionalized polymers capable of undergoing crosslinking are already in hand; excellent reviews of polymer chemistry as applied to the field of tissue engineering may be found elsewhere (Burdick et al.) and these methods are compatible with a range of polymer types. Further, the Michael-type addition (see Lutolf et al.) and light-initiated free radical (see Elisseeff et al.) mechanisms focused on here constitute only a small portion of the reported crosslinking techniques. Mixed mode crosslinking, in which a portion of reactive groups is first consumed by addition crosslinking and followed by a radical mechanism, is another commonly used and powerful paradigm for directing the phenotype of encapsulated cells (Khetan et al., Salinas et al.).

Cellular Encapsulation in 3D Hydrogels for Tissue Engineering

We present protocols for the 3-dimensional (3D) encapsulation of cells within synthetic hydrogels. The encapsulation procedure is outlined for two commonly used methods of crosslinking (michael-type addition and light-initiated free radical mechanisms), as well as a number of techniques for assessing encapsulated cell behavior.

組織工学のための3Dハイドロゲルの細胞のカプセル化

The 3D encapsulation of cells within hydrogels represents an increasingly important and popular technique for culturing cells and towards the development ...

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 ...

Imaging Protein-protein Interactions in vivo

Protein-protein interactions are a hallmark of all essential cellular processes. However, many of these interactions are transient, or energetically weak, preventing their identification and analysis through traditional biochemical methods such as co-immunoprecipitation. In this regard, the genetically encodable fluorescent proteins (GFP, RFP, etc.) and their associated overlapping fluorescence spectrum have revolutionized our ability to monitor weak interactions in vivo using F&ouml;rster resonance energy transfer (FRET)1-3. Here, we detail our use of a FRET-based proximity assay for monitoring receptor-receptor interactions on the endothelial cell surface.

Imaging Protein-protein Interactions in vivo

This protocol describes how to image protein-protein interactions using a FRET-based proximity assay.

イメージングタンパク質間相互作用 in vivoで</em

Protein-protein interactions are a hallmark of all essential cellular processes. However, many of these interactions are transient, or energetically weak, ...

Automated Hydrophobic Interaction Chromatography Column Selection for Use in Protein Purification

In contrast to other chromatographic methods for purifying proteins (e.g. gel filtration, affinity, and ion exchange), hydrophobic interaction chromatography (HIC) commonly requires experimental determination (referred to as screening or "scouting") in order to select the most suitable chromatographic medium for purifying a given protein 1. The method presented here describes an automated approach to scouting for an optimal HIC media to be used in protein purification.
HIC separates proteins and other biomolecules from a crude lysate based on differences in hydrophobicity. Similar to affinity chromatography (AC) and ion exchange chromatography (IEX), HIC is capable of concentrating the protein of interest as it progresses through the chromatographic process. Proteins best suited for purification by HIC include those with hydrophobic surface regions and able to withstand exposure to salt concentrations in excess of 2 M ammonium sulfate ((NH4)2SO4). HIC is often chosen as a purification method for proteins lacking an affinity tag, and thus unsuitable for AC, and when IEX fails to provide adequate purification. Hydrophobic moieties on the protein surface temporarily bind to a nonpolar ligand coupled to an inert, immobile matrix. The interaction between protein and ligand are highly dependent on the salt concentration of the buffer flowing through the chromatography column, with high ionic concentrations strengthening the protein-ligand interaction and making the protein immobile (i.e. bound inside the column) 2. As salt concentrations decrease, the protein-ligand interaction dissipates, the protein again becomes mobile and elutes from the column. Several HIC media are commercially available in pre-packed columns, each containing one of several hydrophobic ligands (e.g. S-butyl, butyl, octyl, and phenyl) cross-linked at varying densities to agarose beads of a specific diameter 3. Automated column scouting allows for an efficient approach for determining which HIC media should be employed for future, more exhaustive optimization experiments and protein purification runs 4.
The specific protein being purified here is recombinant green fluorescent protein (GFP); however, the approach may be adapted for purifying other proteins with one or more hydrophobic surface regions. GFP serves as a useful model protein, due to its stability, unique light absorbance peak at 397 nm, and fluorescence when exposed to UV light 5. Bacterial lysate containing wild type GFP was prepared in a high-salt buffer, loaded into a Bio-Rad DuoFlow medium pressure liquid chromatography system, and adsorbed to HiTrap HIC columns containing different HIC media. The protein was eluted from the columns and analyzed by in-line and post-run detection methods. Buffer blending, dynamic sample loop injection, sequential column selection, multi-wavelength analysis, and split fraction eluate collection increased the functionality of the system and reproducibility of the experimental approach.

An automated method for identifying suitable hydrophobic interaction chromatography (HIC) media to be used in the process of protein purification is presented. The method utilizes a medium-pressure liquid chromatography system including automated buffer blending, dynamic sample loop injection, sequential column selection, multi-wavelength analysis, and split fraction eluate collection.

タンパク質の精製で使用するための自​​動化された疎水性相互作用クロマトグラフィーカラムの選択

In contrast to other chromatographic methods for purifying proteins (e.g. gel filtration, affinity, and ion exchange), hydrophobic interaction ...

Protein WISDOM: A Workbench for In silico De novo Design of BioMolecules

The aim of de novo protein design is to find the amino acid sequences that will fold into a desired 3-dimensional structure with improvements in specific properties, such as binding affinity, agonist or antagonist behavior, or stability, relative to the native sequence. Protein design lies at the center of current advances drug design and discovery. Not only does protein design provide predictions for potentially useful drug targets, but it also enhances our understanding of the protein folding process and protein-protein interactions. Experimental methods such as directed evolution have shown success in protein design. However, such methods are restricted by the limited sequence space that can be searched tractably. In contrast, computational design strategies allow for the screening of a much larger set of sequences covering a wide variety of properties and functionality. We have developed a range of computational de novo protein design methods capable of tackling several important areas of protein design. These include the design of monomeric proteins for increased stability and complexes for increased binding affinity.
To disseminate these methods for broader use we present Protein WISDOM (<a href="http://www.proteinwisdom.org" target="_blank">http://www.proteinwisdom.org</a>), a tool that provides automated methods for a variety of protein design problems. Structural templates are submitted to initialize the design process. The first stage of design is an optimization sequence selection stage that aims at improving stability through minimization of potential energy in the sequence space. Selected sequences are then run through a fold specificity stage and a binding affinity stage. A rank-ordered list of the sequences for each step of the process, along with relevant designed structures, provides the user with a comprehensive quantitative assessment of the design. Here we provide the details of each design method, as well as several notable experimental successes attained through the use of the methods.

Protein WISDOM: A Workbench for In silico De novo Design of BioMolecules

We developed computational de novo protein design methods capable of tackling several important areas of protein design. To disseminate these methods we present Protein WISDOM, an online tool for protein design (<a href="http://www.proteinwisdom.org" target="_blank">http://www.proteinwisdom.org</a>). Starting from a structural template, design of monomeric proteins for increased stability and complexes for increased binding affinity can be performed.

プロテインWISDOM：ベンチ用<em&gt;インシリコで</em&gt;<em&gt;デノボ</em生体分子の&gt;デザイン

The aim of de novo protein design is to find the amino acid sequences that will fold into a desired 3-dimensional structure with improvements in specific ...

Mouse Genome Engineering Using Designer Nucleases

Transgenic mice carrying site-specific genome modifications (knockout, knock-in) are of vital importance for dissecting complex biological systems as well as for modeling human diseases and testing therapeutic strategies. Recent advances in the use of designer nucleases such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 system for site-specific genome engineering open the possibility to perform rapid targeted genome modification in virtually any laboratory species without the need to rely on embryonic stem (ES) cell technology. A genome editing experiment typically starts with identification of designer nuclease target sites within a gene of interest followed by construction of custom DNA-binding domains to direct nuclease activity to the investigator-defined genomic locus. Designer nuclease plasmids are in vitro transcribed to generate mRNA for microinjection of fertilized mouse oocytes. Here, we provide a protocol for achieving targeted genome modification by direct injection of TALEN mRNA into fertilized mouse oocytes.

Designer nucleases such as zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) can be used to modify the genome of mouse preimplantation embryos by triggering both the nonhomologous end joining (NHEJ) and homologous recombination (HR) pathways. These advances enable the rapid generation of mice with precise genetic modifications.

デザイナーヌクレアーゼを用いてマウスゲノムエンジニアリング

Transgenic mice carrying site-specific genome modifications (knockout, knock-in) are of vital importance for dissecting complex biological systems as well ...

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 ...