Name: telomerase activity in the various regions of mouse brain: non-radioactive telomerase repeat amplification protocol (trap) assay
Uploaded: 2014-09-02T13:00:00.000+00:00
Duration: 10 min 14 s
Description: Watch this Scientific Journal Video about Telomerase Activity in the Various Regions of Mouse Brain: Non-Radioactive Telomerase Repeat Amplification Protocol TRAP Assay at JoVE.com

This work was partially supported by B.G. Negev technology and Linda Powers's contribution.

動物実験は、ベングリオン大学（IL-39-08-2010）における動物実験のための倫理委員会によって承認された。 1マウス脳タンパク質抽出<ol><li>各氷上で5ミリリットルリンゲル液と場所のチューブを含む15ミリリットルの3つのチューブを準備します。 </li><li>頸椎脱臼即時断頭続い10-20秒間 - （化学フードを使う注意）イソフルラン麻酔を使用して、マウスを生け贄に捧げる。 </li><li>頭蓋骨から脳を取り出し、脳組織への損傷を防ぐために、氷冷リンゲル液で数秒間すすぐ。 </li><li>外科用メスを使用して、前頭皮質から小脳（CR）と脳幹（BS）を分離。次に、脳幹から小脳を分離および前頭皮質から前頭葉（FL）をカット。前頭葉から嗅球を削除します。指定された15ミリリットルのチューブに3脳領域を配置します。 </li><li>各チューブ内の組織を均質化する。 FRを追加ESHの氷冷リンゲル液、各チューブは、800×gで7分間、同じボリュームや遠心分離機が含まれています。 </li><li>上清を除去し、各チューブにCHAPS溶解バッファー100μlを加える。 21 Gの針を1mlシリンジに溶液を数回（〜10）を渡すことで、よく混ぜ、そして30分間、氷上でインキュベートする。 </li><li> 13000×gで30分間、新しいマイクロ遠心チューブと遠心分離機に各チューブの内容を転送します。新しいマイクロ遠心チューブに上清を移し、以前に確立された方法を用いてタンパク質濃度を決定。 -80℃で10μlの一定分量におけるタンパク質抽出物を保管してください。 </li></ol> 2楽器のTRAPアッセイの準備、反応溶液、およびタンパク質希釈の計算<ol><li>タンパク質抽出物希釈液とテロメラーゼ反応バッファー用のラベル微量遠心管の適切な数を用意してください。汚染を防ぐためにフィルターチップを使用して、プロトコルのすべてのピペット操作を実行します。 </li&gt; <li> TRAP反応ミックス10倍、TSプライマー、のdNTP、UPWとタンパク質抽出物（試薬および試薬濃度のための材料の表を参照してください）​​：氷上で以下の手段を解凍する。 </li><li>超純水（UPW）を使用して、を1μg/μlの最終濃度までタンパク質抽出物の溶液を希釈する。 </li></ol> 3テロメラーゼ反応<ol><li> 2μlのTRAPミックス（10倍）、1μlのTSプライマー（0.1μg）を、1μlのdNTPを（10 mM）をし、15μlの超純水：マイクロ遠心チューブに以下を追加することによって、テロメラーゼ反応ミックスを調製する。複数のサンプルについては、サンプル数、1を加えたことによって、前述のボリュームを掛けます。 </li><li>各反応管に反応ミックス19μLを加え、20μlの最終容量のために各チューブに希釈したタンパク質抽出物を1μl（1μgのタンパク質）を追加します。 </li><li> 45分間30℃に予熱した水浴中で反応チューブを配置します。 </li></ol> 4 PCR反応<ol><li>フィフティーンマイルACXプライマー、チタンのTaqバッファー、UPW：nのテロメラーゼ反応の終了前に、次の解決策を解凍。 </li><li> 2.5μlのチタンのTaqバッファー（10倍）、1μlのACXプライマー（0.1μg）を、2μlの（ICが使用されている1μl）を超純水と0.5μlのチタンTaqポリメラーゼ：マイクロ遠心チューブに以下を追加することで、PCR反応ミックスを調製する（50X）。複数のサンプルのために、試料を加えた2の数によって、上記のボリュームを掛けます。 </li><li>各PCRチューブにPCRミックス6μlのを追加し、26μlの最終容量のために各チューブにテロメラーゼ反応（20μL）のボリューム全体を追加します。 </li><li>のPCRサーモサイクラーにPCRチューブを挿入し、次のプログラムを実行します。 - 90°C - 2分。 - 94℃ - 30秒*。 - 60℃ - 30秒*。 - 72℃ - 45秒​​。 - 72℃ - 2分。 -20℃で保存PCR産物。 * = 34サイクル</li></ol> 5。アガロースミニGELの準備、実行サンプル、およびゲル染色<ol><li> 25ミリリットルチルド電気泳動バッファー（TBE）と緩衝液の2〜4倍のボリュームであり、ゆっくりと高解像度（HR）アガロースの粉末1.125グラムで溶液が急速に撹拌している間に振りかけるビーカーに攪拌棒を追加します。 。攪拌棒を取り外し、換気のための小さな穴にプラスチック製のラップやピアスを入れたビーカーをカバーしています。 3分間の「低」の電源をマイクロ波中、ビーカーを加熱した後、「ハイ」の電源に切り替え、こぼれる原因にさせないように注意しながら短パルス内の溶液を加熱する。加熱により作成されたフォームが数秒後に大きな気泡となって消滅する際、アガロースの準備ができていることに注意してください。 </li><li>ゲルは室温で固化した後、ゲルは、使用前に4℃で20分間冷やしできるように、水平ミニゲル装置にゲルをキャスト。 </li><li> TRAPサンプル25μlを（20μlのTRAPのPCR産物と5μlの6×ゲルローディング色素）を各レーンにロードし、11で実行4℃の低温室での3時間0 V。 </li><li> 15μlの10,000倍の核酸染色、0.1MのNaCl（0.29グラム）で50mlの再蒸留水（DDW）の原液を添加することにより、ソリューションの作業核酸染色の3倍を準備します。穏やかに振盪しながら20分間ゲルを染色。 </li><li> TRAPラダーDNAバンドを表示するには、302 nmの波長のUVトランスイルミネーターを使用してください。 </li></ol>

<ol>
	<li>Urquidi, V., Tarin, D., Goodison, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+telomerase+in+cell+senescence+and+oncogenesis.">Role of telomerase in cell senescence and oncogenesis.</a> Annu Rev Med. 51, 65-79 (2000).</li><li>Cong, Y., Shay, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Actions+of+human+telomerase+beyond+telomeres.">Actions of human telomerase beyond telomeres.</a> Cell Res. 18, 725-732 (2008).</li><li>Tichon, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxidative+stress+protection+by+novel+telomerase+activators+in+mesenchymal+stem+cells+derived+from+healthy+and+diseased+individuals.">Oxidative stress protection by novel telomerase activators in mesenchymal stem cells derived from healthy and diseased individuals.</a> Curr.Mol. Med. 13, 1010-1022 (2013).</li><li>Lannilli, F., Zalfa, F., Gartner, A., Bagni, C., Dotti, C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytoplasmic+TERT+associates+to+RNA+granules+in+fully+mature+neurons:+Role+in+the+translational+control+of+the+cell+cycle+inhibitor+p15INK4B.">Cytoplasmic TERT associates to RNA granules in fully mature neurons: Role in the translational control of the cell cycle inhibitor p15INK4B.</a> PloS one. 8, e66602 (2013).</li><li>De Jesus, B. B., Blasco, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potential+of+telomerase+activation+in+extending+health+span+and+logevity.">Potential of telomerase activation in extending health span and logevity.</a> Curr opinion in Cell Biology. 24, 1-5 (2012).</li><li>Blackburn, E., Collins, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Telomerase:+An+RNP+enzyme+synthesizes+DNA.">Telomerase: An RNP enzyme synthesizes DNA.</a> Cold Spring Harb Perspect Biol. 3, 1-9 (2011).</li><li>Klapper, W., Shin, T., Mattson, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+regulation+of+telomerase+activity+and+TERT+expression+during+brain+development+in+mice.">Differential regulation of telomerase activity and TERT expression during brain development in mice.</a> J. Neurosci. Res. 64, 252-260 (2001).</li><li>Caporaso, G. L., Lim, D. A., Alvarez-Buylla, A., Chao, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Telomerase+activity+in+the+subventricular+zone+of+adult+mice.">Telomerase activity in the subventricular zone of adult mice.</a> Mol. Cell Neurosci. 23, 693-702 (2003).</li><li>Eitan, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+Telomerase-increasing+compound+in+mouse+brain+delays+the+onset+of+amyotrophic+lateral+sclerosis.">Novel Telomerase-increasing compound in mouse brain delays the onset of amyotrophic lateral sclerosis.</a> EMBO Mol. Med. 4, 313-329 (2012).</li><li>Kim, N. W., Wu, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+quantification+and+characterization+of+telomerase+activity+by+the+telomeric+repat+amplification+protocol+(TRAP).">Advances in quantification and characterization of telomerase activity by the telomeric repat amplification protocol (TRAP).</a> Nuc. Acid Res. 25, 2595-2597 (1997).</li></ol>

The authors declare that they have no competing financial interests.

TRAPアッセイを介したマウスの脳内のテロメラーゼ活性の分析4の手順で構成されます。脳組織2の特定の領域からの1）タンパク質抽出）テロメラーゼによるTSプライマー伸長 - PCR法4によるテロメラーゼ反応生成物のテロメラーゼ反応3）増幅高分解能アガロースゲルを用いてPCR産物を）分離する。 敏感なPCR産物の検出とDNAラダーのPAGE分離のためのdCTP年代放射性の使用：?...

テロメラーゼは、テロメラーゼからなるリボ核タンパク質逆転写酵素（TERT）、テロメラーゼの触媒サブユニット、およびRNA成分（TERC）である。テロメラーゼの標準的な役割は、ゲノムの完全性を促進するため、テロメア末端に反復配列（TTAGGG）を添加することによりテロメアの適切な長さを維持することであり、細胞増殖1。それはさまざまな損傷剤2により誘導されるアポトーシスに対する耐性を付与する、つまり追加の役割は、細胞内でTERTに起因した、酸化的ストレス3からのヒト間葉系幹細胞を保護し、肯定的なRNAをTIA1するその協会が完全に分化したニューロンにおける生存促進の役割を果たしている顆粒4。 酵素発現および活性レベルは緊密に非分裂細胞5,6で規制されている間TERTは、分裂細胞および癌のほとんどの種類の中で、主に体細胞で発現され、アクティブにされている。研究は、rの中でそれを示しているTERT mRNAが成人期7に低いレベルに維持しながらodent脳は、テロメラーゼ活性は、生後10日目では検出不可能になる。他の研究では、成人のサブ脳室帯、嗅球、海馬内のテロメラーゼ活性を示し、そして大人の小脳および皮質8。 、注射したマウスSOD1トランスジェニックにおける筋萎縮性側索硬化症の発症および進行を遅延 - 私たちは、最近、脳および脊髄におけるテロメラーゼ発現および活性を増加させる新規化合物は、N-メチル-D-アスパラギン酸（NMDA）における神経保護効果を発揮することを実証マウスおよびこれらのマウス9の脊髄における運動ニューロンの生存を増加させた。完全ニューロンおよび脳におけるテロメラーゼの生存促進の役割を理解するためには、脳のさまざまな領域におけるテロメラーゼ活性の検出のための単純かつ比較的敏感な高速アッセイを開発することが不可欠である。 テロメアREPEAT増幅プロトコール（TRAP）は、テロメラーゼの標準的な活性とポリメラーゼ連鎖反応（PCR）を組み合わせるよく知られた高感度のアッセイである。このアッセイでは、テロメラーゼはTTAGGGがテロメラーゼ基質へのTSリバースプライマー用いて6塩基対（bp）のDNAラダー生成するPCR増幅に続いて（TSプライマー）、オリゴヌクレオチド、繰り返さ付加する。 - ACXを32 P標識dCTPを者が検出するために使用されるPCR産物の量が少ない。 DNAラダーは、ポリアクリルアミドゲル電気泳動（PAGE）を配列決定することにより解決される。どちらのDNAバンドの強度とDNAラダーの長さは、活性酵素分子の量とその処理能力はそれぞれ10を反 ​​映している。 この伝統的なアッセイは、いくつかの主要な難しさを保持します（一晩どちらの場合も）シーケンシングPAGEおよびゲル走行時の消費量と放射性製品のフィルム露光を処理するために大規模なハード放射性物質への暴露の危険性。 </p&gt; このプロトコルは、改良された非放射性アガロースミニゲルに基づくアッセイを提供しています。 DNA 6 bpラダーは4.5％高分解能アガロースゲルを用いて解決され、検出は高感度の核酸染色を用いて達成される。高分解能アガロースミニゲルを用いて、敏感な核酸染色アッセイを扱うことは容易提供の組み合わせは、放射能への暴露の危険性を排除し、大幅に数時間から3日全体のアッセイ時間を短縮する。

<table><tbody><tr><td>TRAP Mix (10x)</td><td></td><td></td><td>Made manually, mix the following in UPW: 630 mM KCl, 200 mM Tris-HCl pH 8.2, 10 mM EDTA, 15 mM MgCl&lt;sub&gt;2&lt;/sub&gt;, 1 mg/ml BSA, 0.5% TWEEN (purchased from Sigma). Aliquots are frozen at -20 &amp;deg;C.</td></tr><tr><td>Ringer&#039;s solution</td><td></td><td></td><td>Made manually, mix the following in DDW: 124 mM NaCl, 3 mM KCl, 1.25 mM NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;&amp;middot;H&lt;sub&gt;2&lt;/sub&gt;O, 2 mM MgSO&lt;sub&gt;4&lt;/sub&gt;, 26 mM NaHCO&lt;sub&gt;3&lt;/sub&gt;, 1.802 g/L D-(+)-glucose anhydrous.</td></tr><tr><td>Isoflurane</td><td>Minrad INC.</td><td>NDC 60307-110-10</td><td>Handle with care in a chemical hood</td></tr><tr><td>dNTPs (10 mM)</td><td>Sigma</td><td>D7295</td><td></td></tr><tr><td>TS primer (5&#039;-AATCCGTCGAGCAGAGTT-3&#039;)</td><td>Sigma</td><td></td><td>DNA oligos orderd in DRY format and UPW added according to requierd concentration</td></tr><tr><td>ACX primer (5&#039;-GCGCGGCTTACCCTTACCCTTACCCTAACC-3&#039;)</td><td>Sigma</td><td></td><td>DNA oligos orderd in DRY format and UPW added according to requierd concentration</td></tr><tr><td>Titanium Taq Polymerase/Buffer</td><td>Clontech</td><td>S1792/S1793</td><td></td></tr><tr><td>High Resolution agarose</td><td>Sigma</td><td>A4718</td><td>Any high quality high-resolution agarose may be sutible</td></tr><tr><td>Mini-gel apparatus</td><td>Bio-Rad</td><td>170-4466</td><td></td></tr><tr><td>Nucleic Acid Stain (GEL-RED)</td><td>Biotium</td><td>41003</td><td></td></tr><tr><td>IC primer (5&#039;-ATCGCTTCTCGGCCTTTT-3&#039;)</td><td>Sigma</td><td></td><td>DNA oligos orderd in DRY format and UPW added according to requierd concentration</td></tr><tr><td>CHAPS lysis buffer</td><td>Cell Signaling Technology</td><td>9852S</td><td>Add PMSF (protease inhibitor) to a final concentration of 1 mM</td></tr><tr><td>Ultra Pure Water (UPW)</td><td>Biological Industries</td><td>01-866-1B</td><td></td></tr><tr><td>CD-1 mice</td><td>HARLAN Laboratories INC.</td><td></td><td></td></tr></tbody></table>

telomerase activity in the various regions of mouse brain: non-radioactive telomerase repeat amplification protocol (trap) assay

テロメラーゼ、リボ核タンパク質は、テロメアの長さを維持し、したがって、ゲノムの完全性、増殖および寿命を促進するための責任があります。さらに、テロメラーゼは、酸化ストレスからミトコンドリアを保護し、そのようなニューロンのような非有糸分裂活性の高い細胞の生存のために重要である可能性を示唆し、アポトーシスに対する耐性を付与する。私たちは、以前にさまざまなマウスの脳領域におけるテロメラーゼ活性および発現を増加させ、酸化ストレスからの運動ニューロンの細胞を保護する新規なテロメラーゼ活性剤の能力を実証した。これらの結果は、テロメラーゼは、さまざまな病変からのニューロンの保護に関与しているという考えを強化する。脳内のテロメラーゼの役割を強調するために、ここでは男性と女性のマウス脳におけるテロメラーゼの活性、年齢への依存を比較します。 TRAPアッセイは、さまざまな組織または細胞株においてテロメラーゼ活性を検出するための標準的な方法である。ここでは、ANALYを実証標準的なTRAPアッセイの改変が続くCHAPS溶解緩衝液を用いてタンパク質抽出を非変性によるマウス脳の三つの領域におけるテロメラーゼ活性のSIS。 この2工程アッセイでは、内因性テロメラーゼはTTAGGG 6 bpの反復（テロメラーゼ反応）を添加することにより、特定のテロメラーゼ基質（TSプライマー）を伸長する。テロメラーゼ反応生成物は、6 bpの増分のDNAラダーを作成するPCR反応により増幅される。 DNAラダーの分析は、高感度の核酸染色で染色し、続いて4.5％高分解能アガロースゲル電気泳動によって行われる。 32 Pは、DNAの検出およびDNAラダーを解決するためのポリアクリルアミドゲル電気泳動のために放射性のdCTPのラベル付け活用する従来のTRAPアッセイと比較すると、このプロトコルは、マウス脳におけるテロメラーゼ活性を評価する能力を実証するためのTRAPアッセイを節約非毒性時間を提供telomerasの相違を検出さまざまな雌雄マウスの脳領域での電子の活動。

全細胞タンパク質抽出物の場合は1、3ヶ月の年齢で3 CD-1メスと3 CD-1雄マウスに由来し、前頭葉（フロリダ州）、脳幹（BS）と小脳（CR）から調製した改変されたTRAPアッセイおよび放射性TRAPアッセイの古い2ヶ月歳の時に3 CD-1雄マウス。テロメラーゼ活性は、改変されたTRAPアッセイを用いて抽出物1μgのタンパク質において検出され、2μgのタンパク質は、放射性TRAPアッセイのために抽出した。放...

Watch this Scientific Journal Video about Telomerase Activity in the Various Regions of Mouse Brain: Non-Radioactive Telomerase Repeat Amplification Protocol TRAP Assay at JoVE.com

Telomerase Activity in the Various Regions of Mouse Brain: Non-Radioactive Telomerase Repeat Amplification Protocol TRAP Assay

Telomerase is expressed in the neonatal brain and also in distinct regions of the adult brain. We present a non-toxic time saving TRAP assay for the analysis of telomerase activity in various regions of the mouse brain and detection of differences in telomerase activity between male and female mouse brains.

マウス脳のさまざまな領域におけるテロメラーゼ活性：非放射性テロメラーゼリピート増幅プロトコル（TRAP）アッセイ

Telomerase, a ribonucleoprotein, is responsible for maintaining the telomere length and therefore promoting genomic integrity, proliferation, and ...

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JoVE Journal

Neuroscience

Telomerase Activity in the Various Regions of Mouse Brain: Non-Radioactive Telomerase Repeat Amplification Protocol (TRAP) Assay

14.6K ビュー.  Ben-Gurion University of the Negev. Telomerase is expressed in the neonatal brain and also in distinct regions of the adult brain. We present a non-toxic time saving TRAP assay for the analysis of telomerase activity in various regions of the mouse brain and detection of differences in telomerase activity between male and female mouse brains.

ビデオ: Telomerase Activity in the Various Regions of Mouse Brain: Non-Radioactive Telomerase Repeat Amplification Protocol TRAP Assay

Semi-quantitative Detection of RNA-dependent RNA Polymerase Activity of Human Telomerase Reverse Transcriptase Protein

Human telomerase reverse transcriptase (TERT) is the catalytic subunit of telomerase, and it elongates telomere through RNA-dependent DNA polymerase activity. Although TERT is named as a reverse transcriptase, structural and phylogenetic analyses of TERT demonstrate that TERT is a member of right-handed polymerases, and relates to viral RNA-dependent RNA polymerases (RdRPs) as well as viral reverse transcriptase. We firstly identified RdRP activity of human TERT that generates complementary RNA stand to a template non-coding RNA and contributes to RNA silencing in cancer cells. To analyze this non-canonical enzymatic activity, we developed RdRP assay with recombinant TERT in 2009, thereafter established in vitro RdRP assay for endogenous TERT. In this manuscript, we describe the latter method. Briefly, TERT immune complexes are isolated from cells, and incubated with template RNA and rNTPs including radioactive rNTP for RdRP reaction. To eliminate single-stranded RNA, reaction products are treated with RNase I, and the final products are analyzed with polyacrylamide gel electrophoresis. Radiolabeled RdRP products can be detected by autoradiography after overnight exposure.

Human telomerase reverse transcriptase (TERT) synthesizes not only telomeric DNA but also double-stranded RNA through RNA-dependent RNA polymerase activity. Here, we describe a newly established assay to detect RNA-dependent RNA polymerase activity of endogenous TERT.

テロメラーゼ逆転写酵素蛋白質の RNA 依存 RNA ポリメラーゼ活性の半定量的な検出

Human telomerase reverse transcriptase (TERT) is the catalytic subunit of telomerase, and it elongates telomere through RNA-dependent DNA polymerase ...

遺伝学

High Sensitivity 5-hydroxymethylcytosine Detection in Balb/C Brain Tissue

DNA hydroxymethylation is a long known modification of DNA, but has recently become a focus in epigenetic research. Mammalian DNA is enzymatically modified at the 5th carbon position of cytosine (C) residues to 5-mC, predominately in the context of CpG dinucleotides. 5-mC is amenable to enzymatic oxidation to 5-hmC by the Tet family of enzymes, which are believed to be involved in development and disease. Currently, the biological role of 5-hmC is not fully understood, but is generating a lot of interest due to its potential as a biomarker. This is due to several groundbreaking studies identifying 5-hydroxymethylcytosine in mouse embryonic stem (ES) and neuronal cells. Research techniques, including bisulfite sequencing methods, are unable to easily distinguish between 5-mC and 5-hmC . A few protocols exist that can measure global amounts of 5-hydroxymethylcytosine in the genome, including liquid chromatography coupled with mass spectrometry analysis or thin layer chromatography of single nucleosides digested from genomic DNA. Antibodies that target 5-hydroxymethylcytosine also exist, which can be used for dot blot analysis, immunofluorescence, or precipitation of hydroxymethylated DNA, but these antibodies do not have single base resolution.In addition, resolution depends on the size of the immunoprecipitated DNA and for microarray experiments, depends on probe design. Since it is unknown exactly where 5-hydroxymethylcytosine exists in the genome or its role in epigenetic regulation, new techniques are required that can identify locus specific hydroxymethylation. The EpiMark 5-hmC and 5-mC Analysis Kit provides a solution for distinguishing between these two modifications at specific loci. The EpiMark 5-hmC and 5-mC Analysis Kit is a simple and robust method for the identification and quantitation of 5-methylcytosine and 5-hydroxymethylcytosine within a specific DNA locus. This enzymatic approach utilizes the differential methylation sensitivity of the isoschizomers MspI and HpaII in a simple 3-step protocol. Genomic DNA of interest is treated with T4-BGT, adding a glucose moeity to 5-hydroxymethylcytosine. This reaction is sequence-independent, therefore all 5-hmC will be glucosylated; unmodified or 5-mC containing DNA will not be affected. This glucosylation is then followed by restriction endonuclease digestion. MspI and HpaII recognize the same sequence (CCGG) but are sensitive to different methylation states. HpaII cleaves only a completely unmodified site: any modification (5-mC, 5-hmC or 5-ghmC) at either cytosine blocks cleavage. MspI recognizes and cleaves 5-mC and 5-hmC, but not 5-ghmC. The third part of the protocol is interrogation of the locus by PCR. As little as 20 ng of input DNA can be used. Amplification of the experimental (glucosylated and digested) and control (mock glucosylated and digested) target DNA with primers flanking a CCGG site of interest (100-200 bp) is performed. If the CpG site contains 5-hydroxymethylcytosine, a band is detected after glucosylation and digestion, but not in the non-glucosylated control reaction. Real time PCR will give an approximation of how much hydroxymethylcytosine is in this particular site. In this experiment, we will analyze the 5-hydroxymethylcytosine amount in a mouse Babl/C brain sample by end point PCR.

The EpiMark 5-hmC and 5-mC Analysis Kit can be used to analyze and quantitate 5-methylcytosine and 5-hydroxymethylcytosine within a spe cific locus. The kit distinguishes 5-mC from 5-hmC by the addition of glucose to the hydroxyl group of 5-hmC via an enzymatic reaction utilizing &beta;-glucosyltransferase (T4-BGT). When 5-hmC occurs In the context of CCGG, this modification converts a cleavable MspI site to a non-cleavable site.

のBalb / Cの脳組織の高感度5 hydroxymethylcytosine検出

DNA hydroxymethylation is a long known modification of DNA, but has recently become a focus in epigenetic research.   Mammalian DNA is enzymatically ...

神経科学

Quantitative Analysis of Alternative Pre-mRNA Splicing in Mouse Brain Sections Using RNA In Situ Hybridization Assay

Alternative splicing (AS) occurs in more than 90% of human genes. The expression pattern of an alternatively spliced exon is often regulated in a cell type-specific fashion. AS expression patterns are typically analyzed by RT-PCR and RNA-seq using RNA samples isolated from a population of cells. In situ examination of AS expression patterns for a particular biological structure can be carried out by RNA in situ hybridization (ISH) using exon-specific probes. However, this particular use of ISH has been limited because alternative exons are generally too short to design exon-specific probes. In this report, the use of BaseScope, a recently developed technology that employs short antisense oligonucleotides in RNA ISH, is described to analyze AS expression patterns in mouse brain sections. Exon 23a of neurofibromatosis type 1 (Nf1) is used as an example to illustrate that short exon-exon junction probes exhibit robust hybridization signals with high specificity in RNA ISH analysis on mouse brain sections. More importantly, signals detected with exon inclusion- and skipping-specific probes can be used to reliably calculate the percent spliced in values of Nf1 exon 23a expression in different anatomical areas of a mouse brain. The experimental protocol and calculation method for AS analysis are presented. The results indicate that BaseScope provides a powerful new tool to assess AS expression patterns in situ.

Quantitative Analysis of Alternative Pre-mRNA Splicing in Mouse Brain Sections Using RNA In Situ Hybridization Assay

An in situ hybridization (ISH) protocol that uses short antisense oligonucleotides to detect alternative pre-mRNA splicing patterns in mouse brain sections is described.

代替の Pre-mrna スプライシング RNA In Situハイブリダイゼーション法によるマウス脳のセクションでの定量的解析

Alternative splicing (AS) occurs in more than 90% of human genes. The expression pattern of an alternatively spliced exon is often regulated in a cell ...

Generation of Cancer Cell Clones to Visualize Telomeric Repeat-containing RNA TERRA Expressed from a Single Telomere in Living Cells

Telomeres are transcribed, giving rise to telomeric repeat-containing long noncoding RNAs (TERRA), which have been proposed to play important roles in telomere biology, including heterochromatin formation and telomere length homeostasis. Recent findings revealed that TERRA molecules also interact with internal chromosomal regions to regulate gene expression in mouse embryonic stem (ES) cells. In line with this evidence, RNA fluorescence in situ hybridization (RNA-FISH) analyses have shown that only a subset of TERRA transcripts localize at chromosome ends. A better understanding of the dynamics of TERRA molecules will help define their function and mechanisms of action. Here, we describe a method to label and visualize single-telomere TERRA transcripts in cancer cells using the MS2-GFP system. To this aim, we present a protocol to generate stable clones, using the AGS human stomach cancer cell line, containing MS2 sequences integrated at a single subtelomere. Transcription of TERRA from the MS2-tagged telomere results in the expression of MS2-tagged TERRA molecules that are visualized by live-cell fluorescence microscopy upon co-expression of a MS2 RNA-binding protein fused to GFP (MS2-GFP). This approach enables researchers to study the dynamics of single-telomere TERRA molecules in cancer cells, and it can be applied to other cell lines.

Here, we present a protocol to generate cancer cell clones containing a MS2 sequence tag at a single subtelomere. This approach, relying on the MS2-GFP system, enables visualization of the endogenous transcripts of telomeric repeat-containing RNA (TERRA) expressed from a single telomere in living cells.

生きている細胞の単一テロメアから表現されるテロメア繰り返し含む RNA テラを視覚化するがん細胞クローンの生成

Telomeres are transcribed, giving rise to telomeric repeat-containing long noncoding RNAs (TERRA), which have been proposed to play important roles in ...

Droplet Digital TRAP (ddTRAP): Adaptation of the Telomere Repeat Amplification Protocol to Droplet Digital Polymerase Chain Reaction

The telomere repeat amplification protocol (TRAP) is the most widely used assay to detect telomerase activity within a given a sample. The polymerase chain reaction (PCR)-based method allows for robust measurements of enzyme activity from most cell lysates. The gel-based TRAP with fluorescently labeled primers limits sample throughput, and the ability to detect differences in samples is restricted to two fold or greater changes in enzyme activity. The droplet digital TRAP, ddTRAP, is a highly sensitive approach that has been modified from the traditional TRAP assay, enabling the user to perform a robust analysis on 96 samples per run and obtain absolute quantification of the DNA (telomerase extension products) input within each PCR. Therefore, the newly developed ddTRAP assay overcomes the limitations of the traditional gel-based TRAP assay and provides a more efficient, accurate, and quantitative approach to measuring telomerase activity within laboratory and clinical settings.

Droplet Digital TRAP ddTRAP: Adaptation of the Telomere Repeat Amplification Protocol to Droplet Digital Polymerase Chain Reaction

We successfully converted the standard telomere repeat amplification protocol (TRAP) assay to be employed in droplet digital polymerase chain reactions. This new assay, called ddTRAP, is more sensitive and quantitative, allowing for better detection and statistical analysis of telomerase activity within various human cells.

液滴デジタルトラップ (ddTRAP): 液滴デジタルポリメラーゼ連鎖反応へのテロメア反復増幅プロトコルの適応

The telomere repeat amplification protocol (TRAP) is the most widely used assay to detect telomerase activity within a given a sample. The polymerase ...

がん研究

非放射性化学発光検出によるテロメア長の定量化

Optimization of Performance Parameters of the TAGGG Telomere Length Assay

Telomeres are repetitive sequences which are present at chromosomal ends; their shortening is a characteristic feature of human somatic cells. Shortening occurs due to a problem with end replication and the absence of the telomerase enzyme, which is responsible for maintaining telomere length. Interestingly, telomeres also shorten in response to various internal physiological processes, like oxidative stress and inflammation, which may be impacted due to extracellular agents like pollutants, infectious agents, nutrients, or radiation. Thus, telomere length serves as an excellent biomarker of aging and various physiological health parameters. The TAGGG telomere length assay kit is used to quantify average telomere lengths using the telomere restriction fragment (TRF) assay and is highly reproducible. However, it is an expensive method, and because of this, it is not employed routinely for large sample numbers. Here, we describe a detailed protocol for an optimized and cost-effective measurement of telomere length using Southern blots or TRF analysis and non-radioactive chemiluminescence-based detection.

著者スポットライト:TAGGG Telomere Length Assay の性能パラメータの最適化(英語)  

Here, we describe in detail the protocol for quantifying telomere length using non-radioactive chemiluminescent detection, with a focus on the optimization of various performance parameters of the TAGGG telomere length assay kit, such as buffer quantities and probe concentrations.

TAGGGテロメア長アッセイの性能パラメータの最適化

Telomeres are repetitive sequences which are present at chromosomal ends; their shortening is a characteristic feature of human somatic cells. Shortening ...

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Rapid Genotyping of Animals Followed by Establishing Primary Cultures of Brain Neurons

High-resolution analysis of the morphology and function of mammalian neurons often requires the genotyping of individual animals followed by the analysis of primary cultures of neurons. We describe a set of procedures for: labeling newborn mice to be genotyped, rapid genotyping, and establishing low-density cultures of brain neurons from these mice. Individual mice are labeled by tattooing, which allows for long-term identification lasting into adulthood. Genotyping by the described protocol is fast and efficient, and allows for automated extraction of nucleic acid with good reliability. This is useful under circumstances where sufficient time for conventional genotyping is not available, e.g., in mice that suffer from neonatal lethality. Primary neuronal cultures are generated at low density, which enables imaging experiments at high spatial resolution. This culture method requires the preparation of glial feeder layers prior to neuronal plating. The protocol is applied in its entirety to a mouse model of the movement disorder DYT1 dystonia (&#916;E-torsinA knock-in mice), and neuronal cultures are prepared from the hippocampus, cerebral cortex and striatum of these mice. This protocol can be applied to mice with other genetic mutations, as well as to animals of other species. Furthermore, individual components of the protocol can be used for isolated sub-projects. Thus this protocol will have wide applications, not only in neuroscience but also in other fields of biological and medical sciences.

We describe procedures for labeling and genotyping newborn mice and generating primary neuronal cultures from them. The genotyping is rapid, efficient and reliable, and allows for automated nucleic-acid extraction. This is especially useful for neonatally lethal mice and their cultures that require prior completion of genotyping.

脳神経細胞の初代培養の確立が続く動物の急速なジェノタイピング

High-resolution analysis of the morphology and function of mammalian neurons often requires the genotyping of individual animals followed by the analysis ...

Utilizing Murine Inducible Telomerase Alleles in the Studies of Tissue Degeneration/Regeneration and Cancer

Telomere dysfunction-induced loss of genome integrity and its associated DNA damage signaling and checkpoint responses are well-established drivers that cause tissue degeneration during ageing. Cancer, with incidence rates greatly increasing with age, is characterized by short telomere lengths and high telomerase activity. To study the roles of telomere dysfunction and telomerase reactivation in ageing and cancer, the protocol shows how to generate two murine inducible telomerase knock-in alleles 4-Hydroxytamoxifen (4-OHT)-inducible TERT-Estrogen Receptor (mTERT-ER) and Lox-Stopper-LoxTERT (LSL-mTERT). The protocol describes the procedures to induce telomere dysfunction and reactivate telomerase activity in mTERT-ER and LSL-mTERT mice in vivo. The representative data show that reactivation of telomerase activity can ameliorate the tissue degenerative phenotypes induced by telomere dysfunction. In order to determine the impact of telomerase reactivation on tumorigenesis, we generated prostate tumor model G4 PB-Cre4 PtenL/L p53L/L LSL-mTERTL/L and thymic T-cell lymphoma model G4 Atm-/- mTERTER/ER. The representative data show that telomerase reactivation in the backdrop of genomic instability induced by telomere dysfunction can greatly enhance tumorigenesis. The protocol also describes the procedures used to isolate neural stem cells (NSCs) from mTERT-ER and LSL-mTERT mice and reactivate telomerase activity in NSCs in vitro. The representative data show that reactivation of telomerase can enhance the self-renewal capability and neurogenesis in vitro. Finally, the protocol describes the procedures for performing telomere FISH (Fluorescence In Situ Hybridization) on both mouse FFPE (Formalin Fixed and Paraffin Embedded) brain tissues and metaphase chromosomes of cultured cells.

Telomere and telomerase play essential roles in ageing and tumorigenesis. The goal of this protocol is to show how to generate two murine inducible telomerase knock-in alleles and how to utilize them in the studies of tissue degeneration/regeneration and cancer.

組織の変性/再生と癌の研究におけるマウス誘導テロメラーゼ対立遺伝子を利用

Telomere dysfunction-induced loss of genome integrity and its associated DNA damage signaling and checkpoint responses are well-established drivers that ...

マウス脳のさまざまな領域におけるテロメラーゼ活性：非放射性テロメラーゼリピート増幅プロトコル（TRAP）アッセイ

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テロメラーゼ逆転写酵素蛋白質の RNA 依存 RNA ポリメラーゼ活性の半定量的な検出

のBalb / Cの脳組織の高感度5 hydroxymethylcytosine検出

代替の Pre-mrna スプライシング RNA In Situハイブリダイゼーション法によるマウス脳のセクションでの定量的解析

生きている細胞の単一テロメアから表現されるテロメア繰り返し含む RNA テラを視覚化するがん細胞クローンの生成

液滴デジタルトラップ (ddTRAP): 液滴デジタルポリメラーゼ連鎖反応へのテロメア反復増幅プロトコルの適応

TAGGGテロメア長アッセイの性能パラメータの最適化

TAGGGテロメア長アッセイの性能パラメータの最適化

TAGGGテロメア長アッセイの性能パラメータの最適化

脳神経細胞の初代培養の確立が続く動物の急速なジェノタイピング

組織の変性/再生と癌の研究におけるマウス誘導テロメラーゼ対立遺伝子を利用