ここで紹介する研究は、健康のペンシルバニア部、エリソン医学、Vとエメラルドの基礎によってサポートされていました。

1。組換えTcは TERTの発現<ol><li> TEV切断可能なN -末端で合成Tcは TERTプラスミドを含む（DE3）ロゼッタ変換pLysSを細胞六新鮮なプレートから2YTメディアの6リットル（6 × 2、Lはそれぞれの2YTブイヨン1Lを含む三角フラスコをバッフル）を植菌ヒスチジン-タグ。 </li><li> 0.5から0.6のOD 600、220 rpmで振盪しながら37℃で細胞を成長させる。 </li><li> 1mMのIPTGを添加することによりタンパク質の発現を誘導。 </li><li> ° C、遅い150rpmの振とうし30に温度を下げ、そして細胞は4から5時間にわたってタンパク質を発現することができます</li><li> 4℃で遠心分離により細胞を回収℃、30分間3500 rpmで。 </li><li> 25mMのトリス、pH 7.5、10％グリセロール、0.5 MのKCl、15mMのイミダゾール、5mMのβ-メルカプトエタノール、および0.1mMのフェニルメチルスルホニルフルオリド（PMSF）及びベンズアミジンを含有する緩衝液20mlに各1 L細胞ペレットを再懸濁します。 </li><li>フラッシュ凍結は徐々に各ドロップは、液体N 2で衝撃にフリーズするように液体窒素を充填した50ミリリットルのプラスチックコニカルチューブに細胞を滴下して細胞の混合物を再懸濁した。 -80凍結細胞を保存° Cタンパク質精製の準備ができるまで。 </li></ol> 2。 Tcは TERTの精製<ol><li>液体の一貫性に達するまで室温で細胞を融解し、超音波処理のために氷の上に金属製のビーカーに移動します。 </li><li>電力の約51 Wで30秒の停止時間を30秒間隔、2分間氷上で細胞を超音波処理してください。 </li><li>可溶性および不溶性タンパク質画分を分離し、慎重にニッケルアフィ​​ニティークロマトグラフィーのための上清を除去するために20分18,000 rpmで溶解した細胞をスピンダウンします。 </li><li>のNi - NTA（ニッケル - ニトリロ三酢酸）25 mMトリス、pH 7.5、10％グリセロール、0.5 MのKCl、15mMのイミダゾール、5mMのβ-メルカプトエタノール、および0.1mMのPMSF及びベンズアミジンを含むバッファーでカラムを平衡化する。 </li><li>樹脂を介して上清をロードし、フロースルーを収集する。 </li><li>すべての残留と非特異的に結合したタンパク質を除去するために25mMのトリス、pH 7.5、10％グリセロール、500mMのKClを、15mMのイミダゾール、5mMのβ-メルカプトエタノール、および0.1mMのPMSFを含む緩衝液でカラムを洗浄。 </li><li> 15のイミダゾール勾配を用いてNi - NTAレジンの溶出Tcは TERT - 300 mMのとSDS PAGE分析のために3 mlの画分を集めた。 </li><li>どの分画が Tc TERTの蛋白質が含まれているかを判断し、アミコン30kDaの（ミリポア）フィルタにこれらの画分を濃縮するためにPAGE分析を使用してください。 </li><li>ボリュームをさらに精製するために以下10mlにTcは TERTの分画を集中。 </li><li> Tcは TERTは、さらに25 mMトリス、pH 7.5、10％グリセロール、500mMのKClを、および1mMのDTTを含む緩衝液で予め平衡化陽イオン交換カラム（HSポロス、Applied Biosystems社）で精製される。 </li><li> HSポロスのカラム上のタンパク質ミックスをロードし、すべての蛋白質と核酸の汚染物質を除去するために、それを洗う。 </li><li>残りの汚染物質を除去するためにKClを700ミリメートルに続いて500mMのKClを含む緩衝液でカラムを洗浄。 </li><li> 1 M KClをバッファーでHSポロスの列からのTc TERTの蛋白質を溶出させる。この時点で、蛋白質は、純粋な99％以上でなければなりません。 </li><li>スーパーデックス- 200サイジングカラム（サイズ排除クロマトグラフィー - GEヘルスケア）を介して蛋白質を渡すことによって、任意のタンパク質の凝集物を除去、25mMのトリス、pH 7.5、10％グリセロール、500mMのKClを、および1 mM TCEPを含有する緩衝液で予め平衡化。 </li></ol> 3。コクヌストモドキカブトムシの文化<ol><li> T. castaneumは、最初に博士S.ブラウン、生物学、カンザス州立大学の部門別の贈り物として提供されている場所カブトムシ。 </li><li>スタートT.小麦粉の28.5グラムと乾燥パン酵母1.5gを使ってコンテナに百成人カブトムシを追加することによってcastaneumの文化。 </li><li>生殖と成長を可能にするために3〜4週間室温で、暗い、湿気のある環境でカブトムシの文化を保存する。湿度の高い環境を確保するためにカブトムシの記憶領域内に水を入れたビーカーを置きます。 </li><li> RNAの分離の必要に応じて、カブトムシの幼虫を収穫。残りの幼虫は同じ文化の中でそれらを維持、大人に成熟することができます。 </li><li>毎月、彼らの食糧供給を補充するために新鮮な小麦粉や酵母を含む新しいフラスコに最も活発なカブトムシを（数百を超えない）に転送。 </li></ol> （4）T. castaneumトータルRNA分離<ol><li>大人のカブトムシや小麦粉/酵母培養液から、それらを分離することにより培養物から収穫20カブトムシの幼虫（各文化は500から1000まで幼虫の間に含まれている必要があります）。それらを粉砕する前にカブトムシに接続された小麦粉や酵母粒子を除去する。 </li><li>ベンチエリア、モルタル、およびカブトムシを研削する前にリボヌクレアーゼのすべてのトレースを削除するRNaseZap（Ambion社、（株））で、この手順で使用する乳棒を（同様に手袋を扱う考慮）滅菌する。 </li><li>乳鉢と乳棒とtを用いて微粉末に液体N 2中の幼虫を挽く粉は抽出緩衝液が追加されるまで凍結とどまることを保証するために液体N 2を維持しながら、50 ml遠心チューブにパウダーをケース温度。 </li><li>過剰な液体N 2は、粉体から蒸発することができます。液体N 2が蒸発した後、25mMトリス- HClを含む抽出緩衝液200μl（20カブトムシの幼虫あたり）、5mMのβ-メルカプトエタノール、1mMのEGTA、0.1 mMのベンズアミジン、200mMのKClを、10のカブトムシの粉末を均質化％グリセロール、RNasinの10mMイミダゾールおよび20U、pH7.5および30分間氷上でインキュベートする。 </li><li> 4℃で20分間15,000 rpmでホモジネートを遠心° Cは、上清とフラッシュを集めて液体N 2でサンプルを凍結する。必要に応じて、サンプルは-80℃で一晩保存することができます。 </li><li> RNeasyミニキット（キアゲン）を用いてトータルRNAを抽出する。 </li></ol> 5 Triboliumのcastaneumのテロメラーゼの in vitro再構成で <ol><li> T.50μlと組換えHisタグTcは TERTの20μgを混ぜる25mMのトリス- HCl、200mMのKClを、10％グリセロール、5mMのβ-メルカプトエタノール、及び10mMのイミダゾール、pH7.5を含む結合バッファーでcastaneumトータルRNA。 </li><li> 22℃で2時間トータルRNAの混合物° T.のC - TC TERTをインキュベートRNA分解を防ぐために、RNasin阻害剤の存在下でcastaneumライセート 。 </li><li>ライセート不純物や余分なRNAを除去するためのNi - NTAカラムにコンプレックスを清める。 </li></ol> 6。テロメラーゼリピート増幅プロトコール（TRAP）アッセイ<ol><li>のNi - NTA精製T.4μgのを混ぜて50μlの伸長緩衝液中のcastaneumのテロメラーゼが20mMを含むトリス- HCl（pH - 8.3）、7.5 mMのMgCl 2、63 mMの塩化カリウム、0.05％ツイーン20、1mMのEGTA、0.01％BSA、0.5 mMのdNTPsを、1μMのTCAS - TS DNAプライマー（5&#39; - AAGCCGTCGAGCAGAGTC - 3&#39;）、および30℃混合物をインキュベート℃で60分間、再構成されたTでTCAS - TSのDNAプライマーの伸長を可能にするcastaneumのテロメラーゼ。 </li><li>反応混合物にフェノールクロロホルムの等量を追加することにより、細長い一本鎖DNA（ssDNAを）抽出する。エマルジョンが均質になるまで静かに混合物を振る。 </li><li> 4℃で5分間12,000 rpmでサンプルを遠心° Cは、汚染物質からssDNAを分離する。 </li><li> 1.5mlのエッペンドルフチューブにサンプルの上部の水層を移し、10mMのNaOAc（pH5.0）を最終濃度、MgCl 2を1mMの、そして66.4パーセントエタノール、その後-20℃で一晩を沈殿させるために、サンプルを可能にするを追加° C. </li><li> 4℃で30分間15,000 rpmでサンプルをスピン° Cをペレット沈殿のssDNAに。ピペッターを使用してソリューションをデカントし、サンプルから、残りのNaOAc及びMgCl 2を洗浄するためにサンプルに70％エタノール200μlを加える。再ペレットサンプルに同じ速度と温度で5分間サンプルを遠心分離します。 </li><li>ピペッターを使用してEtOH溶液をデカントし、エッペンドルフチュー​​ブが3分間、室温で開いてインキュベートすることにより、エタノールの残りのトレースを削除。サンプル中のエタノールの存在を持つことは、PCR増幅のステップに干渉します。 </li><li> 10mMトリス- HCl（pH8）に、50mMの塩化カリウム、2mMのMgCl 2、100μMのdNTPを（dATPを含むPCR反応緩衝液50μlのssDNAのペレットを（テロメラーゼ細長い製品とTCA - TSプライマーを含む）再懸濁しdGTPおよびdTTP）、10μM[32 P] dCTPを、1μMのTCAS - CXのプライマー（5&#39; - GTGTGACCTGACCTGACC - 3&#39;）および1.25 UのTaq DNAポリメラーゼ。 </li><li> PCRは、12％ポリアクリルアミドゲル上に表示されるように、テロメラーゼの伸長製品（94℃30秒、50℃30秒、72℃で1分間の29サイクル）を増幅する。 </li><li>前のPCRサンプルを実行するために、30分間44.5 mMトリス、44.5 mMのホウ酸、1mMのEDTA、pHが8を含むトリス - ホウ酸- EDTA（TBE）を実行してバッファーを持つ12％のゲルを事前に実行してください。事前に実行し、PCR反応でゲルをロードした後、摂氏または氷上で4℃で1.5〜2時間、185ボルトで泳動する。 </li><li>ゲルを乾燥させ、活動を可視化する蛍光体イメージングプレートを使用してください。 </li></ol> 7。代表的な結果： サイズ排除クロマトグラフィー後の精製Tcは TERTの例を図2に示されています。精製後の濃縮タンパク質を12％SDS -ポリアクリルアミドゲル上で実行されており（図2A）、純粋な99％以上であることが示されている。サイズ排除（S200）クロマトグラムは、純度や精製されたタンパク質試料からの凝集性の欠如を証明する（図2B）が用意されています。最終的な収率が変化するものの、すべての精製工程が完了した後に5 mgのタンパク質の収量は、通常、得られる。 再構成されたTの代表的なのTRAPアッセイ以前Mitchellらによって報告されたcastaneumのテロメラーゼを図3に示されています。12。テロメラーゼの製品の段階的なラダーはしかし、レーン1で見ることができます彼らは、レーン2とそれぞれ3内に含まれるRNase処理テロメラーゼとTERT活性部​​位変異体（D251A）サンプルの両方には存在しない。 <img src="/files/ftp_upload/2799/2799fig1.jpg" alt="図1"/> 図1。組換えTcは TERTの再構成に必要な手順のフローチャート 。最初に、組換えTcは TERTは、E.で過剰発現され大腸菌 。タンパク質は、ニッケル、陽イオン交換、サイズ排除クロマトグラフィーによって精製する。 T. castaneumのカブトムシは、家の中で培養され、その幼虫は、トータルRNAを分離するために使用されています。精製Tcは TERTとトータルRNAを再構成するテロメラーゼRNP複合体を混合し、その後のTRAPアッセイは、RNP複合体が活性テロメラーゼであることを確認するために使用されます。 <img src="/files/ftp_upload/2799/2799fig2.jpg" alt="図2"/> 図2。 Tcは TERTの蛋白質のサイズ排除クロマトグラムとSDS PAGE分析。サイズ排除クロマトグラフィー後のTcは TERTの蛋白質の（A）12％SDS -ポリアクリルアミドゲル。それが高い等電点（pI - 9.8）があるため、Tcは TERTの蛋白質（70 kDa）はSDS PAGEゲル上で高速に移行されます。Tcは TERTの蛋白質の（B）サイズ排除クロマトグラム（スーパーデックスS200）。 <img src="/files/ftp_upload/2799/2799fig3.jpg" alt="図3"/> 図3。 in vitroでのTRAPアッセイは、コクヌストモドキのテロメラーゼは、Mitchellらから適応再構成12。レーン1：野生型Tcは TERTとカブトムシの幼虫から単離した全RNA。レーン2：野生型Tcは TERTとRNaseは、幼虫のトータルRNAと細胞抽出物を処理した。レーン3：活性部位変異体のTc TERT（D251A）とカブトムシの幼虫の合計の幼虫のRNA。

<ol>
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利害の衝突は宣言されません。

ここで紹介する方法はT.の触媒サブユニットの大量の製造を可能にする構造的および生化学的研究のための水溶性、アクティブなフォームでcastaneumのテロメラーゼTERT。 Tcは TERTの過剰発現の方法は、上記のものから、温度や細胞密度の微妙な変化に敏感で、飛躍的にタンパク質の発現レベルに影響を与える可能性があります。具体的には、光学密度が600nmで0.5に到達する前...

<table border="1"><tbody><tr><th>試薬の名前</th><th>会社</th><th>カタログ番号</th></tr><tr><td>ロゼッタ（DE3）pLysSを細胞</td><td> Novagen社</td><td> 70956 </td></tr><tr><td> 2YTスープ</td><td> Teknova </td><td> Y0215 </td></tr><tr><td> IPTG </td><td>ゴールドのバイオテクノロジー</td><td> I2481C </td></tr><tr><td> MISONIXソニケーター3000 </td><td> Qsonica、LLC。 </td><td></td></tr><tr><td> AKTA清浄FPLC </td><td> GEライフサイエンス</td><td></td></tr><tr><td>のNi - NTA Superflowレジン</td><td>キアゲン</td><td> 30410 </td></tr><tr><td>アミコンウルトラ- 15遠心フィルターデバイス</td><td>ミリポア</td><td> UFC903008 </td></tr><tr><td> POROS 50 HS強陽イオン交換パッキ​​ング</td><td>アプライドバイオシステムズ</td><td> 1-3359-06 </td></tr><tr><td> POROS 50 HQ強陽イオン交換パッキ​​ング</td><td>アプライドバイオシステムズ</td><td> 1-2559-06 </td></tr><tr><td>スーパーデックス200 300分の10サイズ排除カラム</td><td> GEライフサイエンス</td><td> 17-5175-01 </td></tr><tr><td>フェノール：クロロホルム：10mMのトリス、pHを8、1mMのEDTAとイソアミル25:24:1 </td><td>シグマ</td><td> P3803 - 100mLの</td></tr><tr><td> RNaseZap </td><td>アンビオン</td><td> AM9780 </td></tr><tr><td>組換えRnasinのリボヌクレアーゼインヒビター</td><td>プロメガ</td><td> N251B </td></tr><tr><td> RNeasyミニキット</td><td>キアゲン</td><td> 74104 </td></tr><tr><td> DNAオリゴヌクレオチド</td><td>統合されたDNAの技術</td><td></td></tr></tbody></table>

in vitro reconstitution of the active t. castaneum telomerase

テロメラーゼの触媒サブユニットを分離するための努力、TERTは、構造的な研究のための十分な量で、10年以上の限られた成果しかされています。ここで、我々は組換えコクヌストモドキTERT（Tcは TERT）の単離とアクティブなTの再構成のための方法を提示in vitroでcastaneumのテロメラーゼリボ核タンパク質（RNP）複合体。 テロメラーゼは、エンドツーエンドの融合や劣化から守るために役立つ線形染色体2の3&#39;末端にテロメアと呼ばれる短いDNAの繰り返しを、追加する特殊な逆転写酵素1です。 DNA複製の後、短いセグメントは3番染色体の端にとテロメラーゼなしで失われ、細胞は最終的に彼らのヘイフリック制限を4に達するまで分割し続けます。さらに、テロメラーゼは、成人のほとんどの体細胞5に休止状態ですが、がん細胞では細胞の不死7を促進する6アクティブになります。 酵素とTERTがテロメア8,9を合成するために使用するテンプレートが含まれている不可欠なRNA成分（TER）の触媒サブユニットを含むタンパク質のサブユニット（TERT）、：最小限のテロメラーゼの酵素は、2つのコアコンポーネントで構成されています。前の2008年まで、個々のテロメラーゼのドメイン用の唯一の構造は10,11を解決していた。この分野で大きなブレークスルーは、アクティブな12の結晶構造の決定、T.の触媒サブユニットから来たcastaneumのテロメラーゼ、Tcは TERT 1。 ここで、我々は、構造的および生化学的研究のためのアクティブな、水溶性のTcの TERT、およびテロメラーゼ活性のアッセイのためのin vitroでのテロメラーゼRNP複合体の再構成を大量に製造する方法を提示する。使用した実験方法の概要を図1に示されています。

Watch this Scientific Journal Video about In vitro Reconstitution of the Active T. castaneum Telomerase at JoVE.com

In vitro Reconstitution of the Active T. castaneum Telomerase

テロメラーゼの触媒サブユニットを分離するための努力、TERTは、構造的な研究のための十分な量で、10年以上の限られた成果しかされています。ここで、我々は組換えコクヌストモドキTERT（単離するための方法を提示<em&gt; TC</em&gt; TERT）とアクティブの再構成<em&gt; T. castaneum</em&gt;テロメラーゼリボ核タンパク質（RNP）複合体<em&gt; in vitroで</em&gt;。

アクティブT.の試験管内再構成でcastaneumのテロメラーゼ

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more ...

in-vitro-reconstitution-of-the-active-t-castaneum-telomerase

Research

JoVE Journal

Biology

In vitro Reconstitution of the Active T. castaneum Telomerase

11.6K ビュー.  University of Pennsylvania. テロメラーゼの触媒サブユニットを分離するための努力、TERTは、構造的な研究のための十分な量で、10年以上の限られた成果しかされています。ここで、我々は組換えコクヌストモドキTERT（単離するための方法を提示 TC TERT）とアクティブの再構成 T. castaneumテロメラーゼリボ核タンパク質（RNP）複合体 in vitroで。

ビデオ: In vitro Reconstitution of the Active T. castaneum Telomerase

Quantification of &gamma;H2AX Foci in Response to Ionising Radiation

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA lesions with respect to survival and preservation of genomic integrity. An early response to the induction of DSBs is phosphorylation of the H2A histone variant, H2AX, at the serine-139 residue, in the highly conserved C-terminal SQEY motif, forming &gamma;H2AX1. Following induction of DSBs, H2AX is rapidly phosphorylated by the phosphatidyl-inosito 3-kinase (PIKK) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)2. Typically, only a few base-pairs (bp) are implicated in a DSB, however, there is significant signal amplification, given the importance of chromatin modifications in DNA damage signalling and repair. Phosphorylation of H2AX mediated predominantly by ATM spreads to adjacent areas of chromatin, affecting approximately 0.03% of total cellular H2AX per DSB2,3. This corresponds to phosphorylation of approximately 2000 H2AX molecules spanning ~2 Mbp regions of chromatin surrounding the site of the DSB and results in the formation of discrete &gamma;H2AX foci which can be easily visualized and quantitated by immunofluorescence microscopy2. The loss of &gamma;H2AX at DSB reflects repair, however, there is some controversy as to what defines complete repair of DSBs; it has been proposed that rejoining of both strands of DNA is adequate however, it has also been suggested that re-instatement of the original chromatin state of compaction is necessary4-8. The disappearence of &gamma;H2AX involves at least in part, dephosphorylation by phosphatases, phosphatase 2A and phosphatase 4C5,6. Further, removal of &gamma;H2AX by redistribution involving histone exchange with H2A.Z has been implicated7,8. Importantly, the quantitative analysis of &gamma;H2AX foci has led to a wide range of applications in medical and nuclear research. Here, we demonstrate the most commonly used immunofluorescence method for evaluation of initial DNA damage by detection and quantitation of &gamma;H2AX foci in &gamma;-irradiated adherent human keratinocytes9.

Quantification of &#947;H2AX Foci in Response to Ionising Radiation

Quantification of DNA double-strand streaks using &gamma;H2AX formation as a molecular marker has become an invaluable tool in radiation biology. Here we demonstrate the use of an immunofluorescence assay for quantification of &gamma;H2AX foci after exposure of cells to radiation.

電離放射線への応答でγH2AX病巣の定量化

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA ...

生物学

Evaluation of the Spatial Distribution of &gamma;H2AX following Ionizing Radiation

An early molecular response to DNA double-strand breaks (DSBs) is phosphorylation of the Ser-139 residue within the terminal SQEY motif of the histone H2AX1,2. This phosphorylation of H2AX is mediated by the phosphatidyl-inosito 3-kinase (PI3K) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)3. The phosphorylated form of H2AX, referred to as &gamma;H2AX, spreads to adjacent regions of chromatin from the site of the DSB, forming discrete foci, which are easily visualized by immunofluorecence microscopy3. Analysis and quantitation of &gamma;H2AX foci has been widely used to evaluate DSB formation and repair, particularly in response to ionizing radiation and for evaluating the efficacy of various radiation modifying compounds and cytotoxic compounds4.
Given the exquisite specificity and sensitivity of this de novo marker of DSBs, it has provided new insights into the processes of DNA damage and repair in the context of chromatin. For example, in radiation biology the central paradigm is that the nuclear DNA is the critical target with respect to radiation sensitivity. Indeed, the general consensus in the field has largely been to view chromatin as a homogeneous template for DNA damage and repair. However, with the use of &gamma;H2AX as molecular marker of DSBs, a disparity in &gamma;-irradiation-induced &gamma;H2AX foci formation in euchromatin and heterochromatin has been observed5-7. Recently, we used a panel of antibodies to either mono-, di- or tri- methylated histone H3 at lysine 9 (H3K9me1, H3K9me2, H3K9me3) which are epigenetic imprints of constitutive heterochromatin and transcriptional silencing and lysine 4 (H3K4me1, H3K4me2, H3K4me3), which are tightly correlated actively transcribing euchromatic regions, to investigate the spatial distribution of &gamma;H2AX following ionizing radiation8. In accordance with the prevailing ideas regarding chromatin biology, our findings indicated a close correlation between &gamma;H2AX formation and active transcription9. Here we demonstrate our immunofluorescence method for detection and quantitation of &gamma;H2AX foci in non-adherent cells, with a particular focus on co-localization with other epigenetic markers, image analysis and 3D-modeling.

Evaluation of the Spatial Distribution of &#947;H2AX following Ionizing Radiation

Microscopic analysis of &gamma;H2AX foci, which form following the phosphorylation of H2AX at Ser-139 in response to DNA double-strand breaks, has become an invaluable tool in radiation biology. Here we used an antibody to mono-methylated histone H3 at lysine 4 as an epigenetic marker of actively transcribing euchromatin, to evaluate the spatial distribution of radiation-induced &gamma;H2AX formation within the nucleus.

電離放射線は、次のγH2AXの空間分布の評価

An early molecular response to DNA double-strand breaks (DSBs) is phosphorylation of the Ser-139 residue within the terminal SQEY motif of the histone ...

Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

Schwann cells are one of the commonly used cells in repair strategies following spinal cord injuries. Schwann cells are capable of supporting axonal regeneration and sprouting by secreting growth factors 1,2 and providing growth promoting adhesion molecules 3 and extracellular matrix molecules 4. In addition they myelinate the demyelinated axons at the site of injury 5.
However following transplantation, Schwann cells do not migrate from the site of implant and do not intermingle with the host astrocytes 6,7. This results in formation of a sharp boundary between the Schwann cells and astrocytes, creating an obstacle for growing axons trying to exit the graft back into the host tissue proximally and distally. Astrocytes in contact with Schwann cells also undergo hypertrophy and up-regulate the inhibitory molecules 8-13.
In vitro assays have been used to model Schwann cell-astrocyte interactions and have been important in understanding the mechanism underlying the cellular behaviour.
These in vitro assays include boundary assay, where a co-culture is made using two different cells with each cell type occupying different territories with only a small gap separating the two cell fronts. As the cells divide and migrate, the two cellular fronts get closer to each other and finally collide. This allows the behaviour of the two cellular populations to be analyzed at the boundary. Another variation of the same technique is to mix the two cellular populations in culture and over time the two cell types segregate with Schwann cells clumped together as islands in between astrocytes together creating multiple Schwann-astrocyte boundaries.
The second assay used in studying the interaction of two cell types is the migration assay where cellular movement can be tracked on the surface of the other cell type monolayer 14,15. This assay is commonly known as inverted coverslip assay. Schwann cells are cultured on small glass fragments and they are inverted face down onto the surface of astrocyte monolayers and migration is assessed from the edge of coverslip.
Both assays have been instrumental in studying the underlying mechanisms involved in the cellular exclusion and boundary formation. Some of the molecules identified using these techniques include N-Cadherins 15, Chondroitin Sulphate proteoglycans(CSPGs) 16,17, FGF/Heparin 18, Eph/Ephrins19.
This article intends to describe boundary assay and migration assay in stepwise fashion and elucidate the possible technical problems that might occur.

Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

This article intends to describe in stepwise fashion the commonly used in vitro assays used in studying Schwann cell-asrtocyte interactions.

使用シュワン-アストロサイト相互作用の解析インビトロアッセイ

Schwann cells are one of the commonly used cells in repair strategies following spinal cord injuries. Schwann cells are capable of supporting axonal ...

Telomere Length and Telomerase Activity; A Yin and Yang of Cell Senescence

Telomeres are repeating DNA sequences at the tip ends of the chromosomes that are diverse in length and in humans can reach a length of 15,000 base pairs. The telomere serves as a bioprotective mechanism of chromosome attrition at each cell division. At a certain length, telomeres become too short to allow replication, a process that may lead to chromosome instability or cell death. Telomere length is regulated by two opposing mechanisms: attrition and elongation. Attrition occurs as each cell divides. In contrast, elongation is partially modulated by the enzyme telomerase, which adds repeating sequences to the ends of the chromosomes. In this way, telomerase could possibly reverse an aging mechanism and rejuvenates cell viability. These are crucial elements in maintaining cell life and are used to assess cellular aging. In this manuscript we will describe an accurate, short, sophisticated and cheap method to assess telomere length in multiple tissues and species. This method takes advantage of two key elements, the tandem repeat of the telomere sequence and the sensitivity of the qRT-PCR to detect differential copy numbers of tested samples. In addition, we will describe a simple assay to assess telomerase activity as a complementary backbone test for telomere length.

An accurate, short, sophisticated and cheap method is described that assesses telomere length in multiple tissues and species using qRT-PCR. In addition, we will describe a simple assay to assess telomerase activity as a complementary backbone test for telomere length.

テロメア長とテロメラーゼ活性、細胞の老化の陰陽

Telomeres  are repeating DNA sequences at the tip ends of the chromosomes that are diverse  in length and in humans can reach a length of 15,000 base ...

Rapid Generation of Amyloid from Native Proteins In vitro

Proteins carry out crucial tasks in organisms by exerting functions elicited from their specific three dimensional folds. Although the native structures of polypeptides fulfill many purposes, it is now recognized that most proteins can adopt an alternative assembly of beta-sheet rich amyloid. Insoluble amyloid fibrils are initially associated with multiple human ailments, but they are increasingly shown as functional players participating in various important cellular processes. In addition, amyloid deposited in patient tissues contains nonproteinaceous components, such as nucleic acids and glycosaminoglycans (GAGs). These cofactors can facilitate the formation of amyloid, resulting in the generation of different types of insoluble precipitates. By taking advantage of our understanding how proteins misfold via an intermediate stage of soluble amyloid precursor, we have devised a method to convert native proteins to amyloid fibrils in vitro. This approach allows one to prepare amyloid in large quantities, examine the properties of amyloid generated from specific proteins, and evaluate the structural changes accompanying the conversion.

Rapid Generation of Amyloid from Native Proteins In vitro

Proteins can either adopt a native structure or misfold into insoluble amyloid. Conditions that favor the misfolding pathway lead to the formation of different types of amyloid fibrils. The methods described here allow rapid conversion of native proteins into amyloid in vitro.

天然のタンパク質からアミロイドの迅速な生成<em&gt;インビトロ</em

Proteins carry out crucial tasks in organisms by exerting functions elicited from their specific three dimensional folds. Although the native structures ...

Analysis of RNA Processing Reactions Using Cell Free Systems: 3' End Cleavage of Pre-mRNA Substrates in vitro

The 3&#8217; end of mammalian mRNAs is not formed by abrupt termination of transcription by RNA polymerase II (RNPII). Instead, RNPII synthesizes precursor mRNA beyond the end of mature RNAs, and an active process of endonuclease activity is required at a specific site. Cleavage of the precursor RNA normally occurs 10-30 nt downstream from the consensus polyA site (AAUAAA) after the CA dinucleotides. Proteins from the cleavage complex, a multifactorial protein complex of approximately 800 kDa, accomplish this specific nuclease activity. Specific RNA sequences upstream and downstream of the polyA site control the recruitment of the cleavage complex. Immediately after cleavage, pre-mRNAs are polyadenylated by the polyA polymerase (PAP) to produce mature stable RNA messages.
Processing of the 3&#8217; end of an RNA transcript may be studied using cellular nuclear extracts with specific radiolabeled RNA substrates. In sum, a long 32P-labeled uncleaved precursor RNA is incubated with nuclear extracts in vitro, and cleavage is assessed by gel electrophoresis and autoradiography. When proper cleavage occurs, a shorter 5&#8217; cleaved product is detected and quantified. Here, we describe the cleavage assay in detail using, as an example, the 3&#8217; end processing of HIV-1 mRNAs.

Analysis of RNA Processing Reactions Using Cell Free Systems: 3' End Cleavage of Pre-mRNA Substrates in vitro

RNA polymerase II synthesizes a precursor RNA that extends beyond the 3' end of the mature mRNA. The end of the mature RNA is generated cotranscriptionally, at a site dictated by RNA sequences, via the endonuclease activity of the cleavage complex. Here, we detail the method to study cleavage reactions in vitro.

事前のmRNA基質の3 &#39;末端切断：無細胞系を用いたRNAプロセシング反応の解析<em&gt; in vitroで</em

The 3&#8217; end of mammalian mRNAs is not formed by abrupt termination of transcription by RNA polymerase II (RNPII). Instead, RNPII synthesizes ...

Reconstitution Of &#946;-catenin Degradation In Xenopus Egg Extract

Xenopus laevis egg extract is a well-characterized, robust system for studying the biochemistry of diverse cellular processes. Xenopus egg extract has been used to study protein turnover in many cellular contexts, including the cell cycle and signal transduction pathways1-3. Herein, a method is described for isolating Xenopus egg extract that has been optimized to promote the degradation of the critical Wnt pathway component, &beta;-catenin. Two different methods are described to assess &beta;-catenin protein degradation in Xenopus egg extract. One method is visually informative ([35S]-radiolabeled proteins), while the other is more readily scaled for high-throughput assays (firefly luciferase-tagged fusion proteins). The techniques described can be used to, but are not limited to, assess &beta;-catenin protein turnover and identify molecular components contributing to its turnover. Additionally, the ability to purify large volumes of homogenous Xenopus egg extract combined with the quantitative and facile readout of luciferase-tagged proteins allows this system to be easily adapted for high-throughput screening for modulators of &beta;-catenin degradation.

Reconstitution Of &#946;-catenin Degradation In Xenopus Egg Extract

A method is described for analyzing protein degradation using radiolabeled and luciferase-fusion proteins in Xenopus egg extract and its adaptation for high-throughput screening for small molecule modulators of protein degradation.

β-カテニンの分解にの再構成<em&gt;アフリカツメガエル</em&gt;卵抽出

Xenopus laevis egg extract is a well-characterized, robust system for studying the biochemistry of diverse cellular processes. Xenopus egg extract has ...

Production of Haploid Zebrafish Embryos by In Vitro Fertilization

The zebrafish has become a mainstream vertebrate model that is relevant for many disciplines of scientific study. Zebrafish are especially well suited for forward genetic analysis of developmental processes due to their external fertilization, embryonic size, rapid ontogeny, and optical clarity &#8211; a constellation of traits that enable the direct observation of events ranging from gastrulation to organogenesis with a basic stereomicroscope. Further, zebrafish embryos can survive for several days in the haploid state. The production of haploid embryos in vitro is a powerful tool for mutational analysis, as it enables the identification of recessive mutant alleles present in first generation (F1) female carriers following mutagenesis in the parental (P) generation. This approach eliminates the necessity to raise multiple generations (F2, F3, etc.) which involves breeding of mutant families, thus saving the researcher time along with reducing the needs for zebrafish colony space, labor, and the husbandry costs. Although zebrafish have been used to conduct forward screens for the past several decades, there has been a steady expansion of transgenic and genome editing tools. These tools now offer a plethora of ways to create nuanced assays for next generation screens that can be used to further dissect the gene regulatory networks that drive vertebrate ontogeny. Here, we describe how to prepare haploid zebrafish embryos. This protocol can be implemented for novel future haploid screens, such as in enhancer and suppressor screens, to address the mechanisms of development for a broad number of processes and tissues that form during early embryonic stages.

Production of Haploid Zebrafish Embryos by In Vitro Fertilization

The zebrafish is a powerful model system for developmental biology and human disease research due to their genetic similarity with higher vertebrates. This protocol describes a methodology to create haploid zebrafish embryos that can be utilized for forward screen strategies to identify recessive mutations in genes essential for early embryogenesis.

半数ゼブラフィッシュ胚の生成により<em&gt;体外</em&gt;受精

The zebrafish has become a mainstream vertebrate model that is relevant for many disciplines of scientific study. Zebrafish are especially well suited for ...

In Vitro Reconstitution of Light-harvesting Complexes of Plants and Green Algae

In plants and green algae, light is captured by the light-harvesting complexes (LHCs), a family of integral membrane proteins that coordinate chlorophylls and carotenoids. In vivo, these proteins are folded with pigments to form complexes which are inserted in the thylakoid membrane of the chloroplast. The high similarity in the chemical and physical properties of the members of the family, together with the fact that they can easily lose pigments during isolation, makes their purification in a native state challenging. An alternative approach to obtain homogeneous preparations of LHCs was developed by Plumley and Schmidt in 19871, who showed that it was possible to reconstitute these complexes in vitro starting from purified pigments and unfolded apoproteins, resulting in complexes with properties very similar to that of native complexes. This opened the way to the use of bacterial expressed recombinant proteins for in vitro reconstitution. The reconstitution method is powerful for various reasons: (1) pure preparations of individual complexes can be obtained, (2) pigment composition can be controlled to assess their contribution to structure and function, (3) recombinant proteins can be mutated to study the functional role of the individual residues (e.g., pigment binding sites) or protein domain (e.g., protein-protein interaction, folding). This method has been optimized in several laboratories and applied to most of the light-harvesting complexes. The protocol described here details the method of reconstituting light-harvesting complexes in vitro currently used in our laboratory, and examples describing applications of the method are provided.

In Vitro Reconstitution of Light-harvesting Complexes of Plants and Green Algae

This protocol details the reconstitution of light-harvesting complexes in vitro. These integral membrane proteins coordinate chlorophylls and carotenoids and are responsible for harvesting light in higher plants and green algae.

植物や緑藻の光捕集複合体のin vitro再構成では、

In plants and green algae, light is captured by the light-harvesting complexes (LHCs), a family of integral membrane proteins that coordinate chlorophylls ...

Larval RNA Interference in the Red Flour Beetle, Tribolium castaneum

The red flour beetle, Tribolium&#160;castaneum, offers a repertoire of experimental tools for genetic and developmental studies, including a fully annotated genome sequence, transposon-based transgenesis, and effective RNA interference (RNAi). Among these advantages, RNAi-based gene knockdown techniques are at the core of Tribolium research. T. castaneum show a robust systemic RNAi response, making it possible to perform RNAi at any life stage by simply injecting double-stranded RNA (dsRNA) into the beetle&#8217;s body cavity.
In this report, we provide an overview of our larval RNAi technique in T. castaneum. The protocol includes (i) isolation of the proper stage of T. castaneum larvae for injection, (ii) preparation for the injection setting, and (iii) dsRNA injection. Larval RNAi is a simple, but powerful technique that provides us with quick access to loss-of-function phenotypes, including multiple gene knockdown phenotypes as well as a series of hypomorphic phenotypes. Since virtually all T. castaneum tissues are susceptible to extracellular dsRNA, the larval RNAi technique allows researchers to study a wide variety of tissues in diverse contexts, including the genetic basis of organismal responses to the outside environment. In addition, the simplicity of this technique stimulates more student involvement in research, making T. castaneum an ideal genetic system for use in a classroom setting.

Larval RNA Interference in the Red Flour Beetle, Tribolium castaneum

RNA interference (RNAi)-based gene knockdown techniques are at the core of Tribolium research. Here, we provide an overview of our larval RNAi technique in Tribolium castaneum. Larval RNAi is a simple, but powerful technique that provides quick access to loss-of-function phenotypes, allowing researchers to study gene functions in diverse contexts.