著者らは、確認応答がありません。

1コンカテマー<ol><li>再懸 ​​濁プライマーFWとRVが分子生物学グレードのH 2 O中10μg/μlの濃度に（プライマー配列のための材料の表を参照）その後、1.5mlのマイクロチューブにFwの5μlのとRVのプライマーの5μLを混ぜる。 </li><li> 15分間の正しいアニーリング温度（ここで説明するプライマーについて75℃）に加熱することによりアニールプライマーミックス。室温（RT）で冷却するベンチにしておきます。のddH 2 Oに溶解させることにより、このインキュベーション中に60％PEG8000を準備</li><li> 10倍のポリヌクレオチドキナーゼ（PNK）緩衝液、H 2 O27μlの、およびプライマーミックスへPNKの3μlと10μlの60％PEG8000（30％の最終濃度を達成するために）の50を添加する。 2時間37℃でインキュベートする。 </li><li> RTでクールダウンし、DNAリガーゼ緩衝液11μlを加える。次に、DNAリガーゼの3 Uを追加し、37℃で一晩インキュベートする。 </li></ol> 2 DNA精製と分析<ol><li>連結混合量を増加させるために、H 2 Oを300μlを加える。注：フェノールの毒性によるドラフト内で進みます。クロロホルム蒸気。 </li><li>フェノール1容量（400μlの）追加：クロロホルムを激しく振とうすることにより混合します。 </li><li>卓上マイクロにおける1分間最高速度で遠心。水性および溶剤の層が上に水層と2層の間の沈殿したタンパク質の白い層で分離されます。 </li><li>慎重なピペッティングにより新しいチューブに上層を移し（中央の転送を回避するため、白色層） </li><li>繰り返します2.2と2.3倍以上または全く沈殿したタンパク質は、遠心分離後がなくなるまで繰り返します。 </li><li>クロロホルムの1容量（400μl）を添加し、ステップ2.3から2.4を繰り返します。 </li><li> 100％予冷2容量のエタノール（800μl）を添加し、少なくとも1時間、または一晩-20℃でインキュベートする。注：このステップは、DNAコンカテマーを沈殿させる。 </li><li>最高速度と吸引Supeのでスピンダウンrnatant。 70％エタノール1mlを添加することにより、一回洗い、ドラフト内で空気乾燥させ。 </li><li>エンドトキシンを含まないのddH 2 O50μlの再懸濁DNA注：エンドトキシンを含まない試薬の使用は、細胞およびin vivoでの DNA-特異的先天性免疫応答の生成に重要である。 </li><li>新しいチューブにコンカテマーDNAを2μlのアリコートを取り出し、1ミリリットルDNAゲルローディングバッファーを追加します。 </li><li> （オーブン容量の電源に適したまたは時間）で2分間、電子レンジでフルパワーで加熱することによりTAE緩衝液中の1％アガロースを溶解する。 </li><li>鋳造装置にゲルを注ぎ、蛍光DNAキレート色素を4μlを追加、コームを挿入し、設定するためのゲルで約20分間、室温で残す。 </li><li>一度設定し、コームを削除し、TAE緩衝液を含む電気泳動槽にゲルを置く。ロードウェルにDNAサンプルやサイズマーカーの3μlの。 </li><li>定電流で90 Vで30〜40分間、ゲル電気泳動を実行します。 Visualiぜ紫外光下でDNAのコンカテマー化（ 図1A）を観察した。 </li><li> UV吸光度分光法によって試料の残りの部分でDNA濃度を測定する。 DNAを再懸濁し、200〜300ナノメートルの吸光度スペクトルを測定するために使用した同じのddH 2 Oブランク分析計。注：DNA、Aは吸光度で濃度がランベルト·ベールの法則を用いて計算することができ、A =イー·のc * lを、吸光係数をメール、およびlは光路の距離。 DNAを測定するために260nmにおける吸光度を測定し、0.027の吸光係数（μg/ ml）をすべきである-1 cm -1で使用される濃度。光路距離、lは、分光光度計に依存して変化する。濃度を測定しながら、DNA純度の指標として280分の260 nmの吸光度比をメモします。 2.00上記のこの比率の値が推奨されます。 </li></ol> VIに二本鎖DNAコンカテマーの3フェTRO <ol><li>組織培養ディッシュまたはプレートにおける種子細胞は、刺激の時点で70％コンフルエントであった。 </li><li> 1-10μgの/ mlの最終濃度を用いて細胞を刺激するために必要なDNAの量を計算します。 DNAの必要な質量は、以下のXと表記されます。その後、DNAの2×5 = 10μgの質量が必要とされるの5μg/ mlのDNA濃度で培地2mlで6ウェルプレートの1ウェル中の細胞を刺激した場合の例である。 </li><li>適切なサイズの滅菌チューブに無血清培地50 * Xを添加する。ピペットを慎重媒体の中心に、チューブの側面に触れることなく、陽イオン性脂質トランスフェクション試薬の2 * Xμlの5分間のままにしておきます。 </li><li>チューブとボルテックスを簡単にDNAののXμgのを追加します。少なくとも20分間RTでのままにします。 in vitroでのトランスフェクションのために細胞質DNAセンシング経路を刺激するために細胞に直接この混合物を追加します。 </li></ol>二本鎖DNAコンカテマー私の4トランスフェクションnは生体内<ol><li> 1μgのDNAを、2μlの脂質トランスフェクション試薬、及び刺激されるマウスの耳あたり18μlの無血清培地を用いて3.1から3.5で説明したように、トランスフェクションミックスを調製する。無菌の30 G針で無菌20μlのハミルトンシリンジ、キャップを用意し、DNAトランスフェクションミックスをロードします。 </li><li> 1〜2％イソフルランを使用して、C57BL / 6マウスを麻酔。必要に応じて、運動の損失と一定の呼吸数によってピンチ反射試験による麻酔を確認してください。麻酔中に動物の目を監視し、必要に応じて過度の乾燥を避けるために軟膏を使用しています。 </li><li>無菌技術を使用して、耳の耳介に注意して、液体のシングル「バブル」が耳の皮膚層の間に形成されているように、トランスフェクション混合物10μlを注入する。注：、親指や人差し指にゴム製のシンブルを使用して、その上に耳を伸ばして、針が耳介の皮膚の最初の層を貫通して確保するために鋭角に注入します。 </li><li>手綱troduceマウスが戻ってケージに、定期的にポスト麻酔回復を監視します。麻酔したマウスの過剰な冷却を避けるために、加熱ランプを使用してください。彼らは胸骨横臥位を維持するのに十分な意識を取り戻したまでは無人マウスを放置しないでください。 </li><li>フラッシュ2分間液体窒素中に浸漬することにより微小遠心管全体耳介をフリーズ。 </li></ol>定量的PCRによるDNA刺激のサンプルと分析5。処理（定量PCR） <ol><li>細胞培養から：ピペットで吸引メディアと廃棄物を捨てる。 </li><li>滅菌PBSを1mlを加える。 5.1および5.2を二回繰り返し、5.3に進みます</li><li>耳組織から：無菌セラミック乳棒と乳鉢を用いて液体窒素下で組織を挽く無菌スパチュラを用いてマイクロチューブにアース組織を転送し、5.4に進みます</li><li> 350μlの溶解緩衝液を加えて、市販のRNA抽出キットプロトコールを進める。抽出キット精製からの溶出RNA40μlののddH 2 Oの列注：商業RNA抽出キット（材料/機器の表を参照してください）​​非常によく動作します。他の技術は、標準以下の結果でテストされている。 </li><li> （2.13節）上記のDNAについて説明したように、UV分光法によりRNAを定量化。注：RNAの吸光係数は0.025（μg/ ml）を-1 cm -1である。 </li><li>オリゴ1μlの（dT）プライマー、10mMのdNTPミックス1μlの、および250μgのRNAを滅菌したPCRチューブに追加します。無菌のddH 2 Oで11μlにボリュームを構成する</li><li> 65℃で5分間インキュベートする。その後、4μlのファーストストランドバッファー、1μlの0.1Mのジチオスレイトールを追加、0.25μlをチューブに転写酵素および1.75μLのddH 2 Oを逆転。 15分間72℃、続いて1時間50℃でインキュベートする。のddH 2 Oを用いて、5：必要に応じて、cDNA産物1を希釈</li><li>定量的PCRのために、2μlののddH 2 O、5μlの定量PCRマスターミックス、および1を2μlのcDNAを使用してフォワード10μMのそれぞれをμlでと希望の遺伝子を増幅するためのプライマー株式を反転させます。 96ウェルプレート中で混合し、定量的PCR機（ 図1Bおよび1C）を用いて分析する。 </li></ol>

<ol>
	<li>Yoshida, H., Okabe, Y., Kawane, K., Fukuyama, H., Nagata, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lethal+anemia+caused+by+interferon-beta+produced+in+mouse+embryos+carrying+undigested+DNA.">Lethal anemia caused by interferon-beta produced in mouse embryos carrying undigested DNA.</a> Nat. Immunol. 6, 49-56 (2005).</li><li>Ishikawa, H., Ma, Z., Barber, G. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=STING+regulates+intracellular+DNA-mediated,+type+I+interferon-dependent+innate+immunity.">STING regulates intracellular DNA-mediated, type I interferon-dependent innate immunity.</a> Nature. 461, 788-792 (2009).</li><li>Ishii, K. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TANK-binding+kinase-1+delineates+innate+and+adaptive+immune+responses+to+DNA+vaccines.">TANK-binding kinase-1 delineates innate and adaptive immune responses to DNA vaccines.</a> Nature. 451, 725-729 (2008).</li><li>Stetson, D. B., Medzhitov, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recognition+of+cytosolic+DNA+activates+an+IRF3-dependent+innate+immune+response.">Recognition of cytosolic DNA activates an IRF3-dependent innate immune response.</a> Immunity. 24, 93-103 (2006).</li><li>Pichlmair, A., Reise Sousa, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Innate+recognition+of+viruses.">Innate recognition of viruses.</a> Immunity. 27, 370-383 (2007).</li><li>Unterholzner, L., 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=IFI16+is+an+innate+immune+sensor+for+intracellular+DNA.">IFI16 is an innate immune sensor for intracellular DNA.</a> Nat. Immunol. 11, 997-1004 (2010).</li><li>Ferguson, B. J., Mansur, D. S., Peters, N. E., Ren, H., Smith, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA-PK+is+a+DNA+sensor+for+IRF-3-dependent+innate+immunity.">DNA-PK is a DNA sensor for IRF-3-dependent innate immunity.</a> Elife. 1, e00047 (2012).</li><li>Sun, L., Wu, J., Du, F., Chen, X., Chen, Z. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyclic+GMP-AMP+synthase+is+a+cytosolic+DNA+sensor+that+activates+the+type+I+interferon+pathway.">Cyclic GMP-AMP synthase is a cytosolic DNA sensor that activates the type I interferon pathway.</a> Science. 339, 786-791 (2013).</li><li>Honda, K., Taniguchi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IRFs:+master+regulators+of+signalling+by+Toll-like+receptors+and+cytosolic+pattern-recognition+receptors.">IRFs: master regulators of signalling by Toll-like receptors and cytosolic pattern-recognition receptors.</a> Nat. Rev. Immunol. 6, 644-658 (2006).</li><li>Karayel, 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=The+TLR-independent+DNA+recognition+pathway+in+murine+macrophages:+Ligand+features+and+molecular+signature.">The TLR-independent DNA recognition pathway in murine macrophages: Ligand features and molecular signature.</a> Eur. J. Immunol. 39, 1929-1936 (2009).</li></ol>

The authors have nothing to disclose.

ここで説明するプロトコルは、広範囲の細胞型およびin vivoで再現性のある結果を持つ細胞質ゾル先天性免疫応答を刺激する二本鎖DNAを生成するために使用される。使用される主な基質試薬は合成オリゴヌクレオチドであるので、代わりのDNA配列または化学修飾を使用するためのこのシステムの柔軟性がある。それは、例えば、蛍光標識または局在化を追跡したり、他の生体分子との相...

DNAは、真核生物は、特定の区画に保つ全ての生物や細胞の重要な構成要素である。自然免疫系の1つの機能は、異物が物理的、外部障壁の違反を介して生物に導入されたときそのような区画化が故障したとき、または識別することである。人体では、正しい区画化を逃れることが少量のDNAを分解する手助けするために存在するタンパク質の数があります; DNA分解酵素I、II、およびIIIは、それぞれ、血漿、エンドソーム、および細胞質ゾル中のDNAを分解する。しかし、DNA分解酵素IIを欠くマウスは、自然免疫系は、外核DNAに応答することを示唆しているインターフェロン1の過剰産生に起因する重度の貧血で死ぬことが示されている。この応答はあっても、Toll様受容体シグナル伝達能力の完全に欠損しているマウスでは発生します。多くの研究は現在、インターフェロン遺伝子（STING）2およびTANK結合キナーゼ1（TBの刺激装置を示しているK1）3は、細胞質中のDNAの検出およびこのシグナル伝達経路は、インターフェロン調節因子（IRF）-3 4を活性化することに関与している。サイトカイン、ケモカイン、およびインターフェロン遺伝子のその後のIRF3依存性転写は、病原体や危険関連分子パターン（PAMPまたはDAMP）5のいずれかに先天性免疫応答の特徴である。 細胞内の核酸センシング経路を研究するための欲求が、他の先天性免疫応答を活性化することなく細胞内にDNAまたはRNAを導入することによって細胞を刺激するための堅牢かつ再現性のある方法を開発する必要性につながっている。 STING / TBK1 / IRF3経路を刺激することのできる一つの方法は、そのようなDNA-PK、IFI16、およびCGAS 6-8のようなDNAセンサによってアクセスすることができる細胞質内に核酸を送達するためにカチオン性脂質トランスフェクション試薬を使用することによるものである。 現在effのを可能にするように理解されているDNAの特性他の経路の刺激なしの細胞質先天性免疫応答のicient活性化は、その長さ、質量、純度であり、およびCpGの欠如は4,7,9をモチーフ。合成オリゴヌクレオチドは、それらがDNA配列は、純粋な化学種として最適化して調製することを可能にするように先天性免疫応答を生成するために非常に適している。 45塩基対の免疫刺激DNA（ISD）配列は、この目的に適しは、CpGのないシーケンスであるとステットソンら 4により同定された。この二本鎖DNAシーケンスは、そうでなければランダムであり、CpGモチーフの欠如を超えていない特定の機能を持っていません。私たちは、有意に長いストランドにISDオリゴヌクレオチドをconcatemerizingによる刺激7の大きさを増大させることを見出した。コンカテマーISDの生成は、細胞質先天性免疫応答を刺激するために有用である。ここでは、この試薬 ​​を調製するためのプロトコルを提示し、それがDNA にセンシング経路を活性化する方法を試験管内だけでなく、in vivoで 。 注：すべての動物実験が承認制度プロトコルや動物のケアのガイドラインの下で行われている。

<table border="0" cellpadding="0" cellspacing="0" width="1066">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:295px;">Forward Primer</td>
			<td style="width:221px;">Integrated DNA technologies</td>
			<td style="width:144px;">n/a</td>
			<td style="width:407px;">TACAGATCTACTAGTGATCTATGACTGATCTGTACATGATCTACA</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:295px;">Reverse Primer</td>
			<td style="width:221px;">Integrated DNA technologies</td>
			<td style="width:144px;">n/a</td>
			<td>TGTAGATCATGTACAGATCAGTCATAGATCACTAGTAGATCTGTA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">PEG8000</td>
			<td style="width:221px;">Sigma-Aldrich</td>
			<td style="width:144px;">P-4463</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Molecular biology grade water</td>
			<td style="width:221px;">Sigma-Aldrich</td>
			<td style="width:144px;">W4502-1L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">T4 Polynucleotide Kinase</td>
			<td style="width:221px;">Promega</td>
			<td style="width:144px;">M4101</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">T4 DNA Ligase</td>
			<td style="width:221px;">Promega</td>
			<td style="width:144px;">M1801</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Phenol:Chloroform</td>
			<td style="width:221px;">Fisher Chemical</td>
			<td style="width:144px;">BPE1752p-400</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Chloroform</td>
			<td style="width:221px;">Fisher Chemical</td>
			<td style="width:144px;">C/4960/PB08</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Ethanol</td>
			<td style="width:221px;">Sigma-Aldrich</td>
			<td style="width:144px;">32221-2.5L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">DNA gel loading buffer</td>
			<td style="width:221px;">New England biolabs</td>
			<td style="width:144px;">B7021S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Agarose</td>
			<td style="width:221px;">Bioline</td>
			<td style="width:144px;">BIO-41025</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Optimem</td>
			<td style="width:221px;">Life Technologies</td>
			<td style="width:144px;">11058021</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TransIT-LT1&#160;</td>
			<td style="width:221px;">Mirus Bioscience</td>
			<td style="width:144px;">MIR 2300</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Hamilton Syringe</td>
			<td style="width:221px;">Hamilton</td>
			<td style="width:144px;">80901 Model 1705</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">30G needles&#160;</td>
			<td style="width:221px;">BD bioscience</td>
			<td style="width:144px;">304000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">SYBR Safe DNA gel stain</td>
			<td style="width:221px;">Life Technologies</td>
			<td style="width:144px;">S33102</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Rneasy Plus Mini Kit</td>
			<td style="width:221px;">Qiagen</td>
			<td style="width:144px;">74134</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Oligo(dT) primer</td>
			<td style="width:221px;">Thermo Scientific</td>
			<td style="width:144px;">SO131</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">dNTP mix</td>
			<td style="width:221px;">Fermentas</td>
			<td style="width:144px;">R0192</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Super Script III reverse transcriptase</td>
			<td style="width:221px;">Life Technologies</td>
			<td align="right" style="width:144px;">56575</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">First strand cDNA synthesis buffer</td>
			<td style="width:221px;">Life Technologies</td>
			<td style="width:144px;">y02321</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">SybrGreen qPCR Fast master mix</td>
			<td style="width:221px;">Applied Biosystems</td>
			<td align="right" style="width:144px;">4385612</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">cxcl10 murine forward primer&#160;</td>
			<td style="width:221px;">Integrated DNA technologies</td>
			<td style="width:144px;">n/a</td>
			<td>ACTGCATCCATATCGATGAC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">cxcl10 murine reverse primer</td>
			<td style="width:221px;">Integrated DNA technologies</td>
			<td style="width:144px;">n/a</td>
			<td>TTCATCGTGGCAATGATCTC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">il6 murine forward primer&#160;</td>
			<td style="width:221px;">Integrated DNA technologies</td>
			<td style="width:144px;">n/a</td>
			<td>GTAGCTATGGTACTCCAGAAGAC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">il6 murine forward primer&#160;</td>
			<td style="width:221px;">Integrated DNA technologies</td>
			<td style="width:144px;">n/a</td>
			<td>GTAGCTATGGTACTCCAGAAGAC</td>
		</tr>
	</tbody>
</table>

stimulation of cytoplasmic dna sensing pathways in vitro and in vivo

効率的に先天性免疫応答を刺激するために、DNAは、十分な長さと純度でなければならない。私たちは、必要な特性を持つ二本鎖DNA（dsDNAのは）センシング経路が安価かつ容易に生成することができ細胞質DNAを刺激する手法を提案する。 （CpGモチーフを欠く）、短い合成オリゴヌクレオチドのコンカテマー化によって、二本鎖DNAは、細胞質DNAセンシング経路を活性化するのに十分な長さであるように生成することができる。このプロトコルは、効率的なライゲーションが起こるための環境を提供し、ポリエチレングリコール（PEG）の存在下でオリゴヌクレオチドの平滑末端ライゲーションを伴う。二本鎖DNAコンカテマーを、標準的なトランスフェクションプロトコールによるインビトロでの先天性免疫応答をシミュレートするために、フェノール/クロロホルム抽出により精製後、使用することができる。このDNAはまた、例えば、マウスの耳介に皮内注射することによってインビボでの先天性免疫を刺激するために使用することができる。バイコンカテマー化プロセスとその後の刺激プロトコルを標準化する、自然免疫系の信頼性と再現性の活性化は、製造することができる。

<html>以下の結果は、コンカテマーDNAを比較的容易に生成され、細胞およびマウスにおける強固な先天性免疫応答を刺激することができることができることを示している。 PEG8000を用いて、それが明らかに発生しているDNAの長さはかなり長く、CXCL10の転写を誘導することも同様に可能であることが分かる。アガロースゲル分析によって見られるようにPEG8000を含むサンプルのために、DNAの長さが1...

Watch this Scientific Journal Video about Stimulation of Cytoplasmic DNA Sensing Pathways In Vitro and In Vivo at JoVE.com

Stimulation of Cytoplasmic DNA Sensing Pathways In Vitro and In Vivo

プロトコルの目的は、細胞およびインビボで細胞質DNAセンシング経路を刺激するための最適な方法を使用することである。これは、平滑末端ライゲーションの間に長い、二本鎖DNAの生成を改善することによって達成される。細胞またはマウスは、その後、脂質トランスフェクション試薬を用いてトランスフェクトされる。

細胞質DNAセンシング経路の刺激<em&gt;体外</em&gt;と<em&gt;インビボ</em

In order to efficiently stimulate an innate immune response, DNA must be of sufficient length and purity. We present a method where double stranded DNA ...

stimulation-of-cytoplasmic-dna-sensing-pathways-in-vitro-and-in-vivo

Research

JoVE Journal

Biology

Stimulation of Cytoplasmic DNA Sensing Pathways In Vitro and In Vivo

8.8K ビュー.  University of Cambridge. プロトコルの目的は、細胞およびインビボで細胞質DNAセンシング経路を刺激するための最適な方法を使用することである。これは、平滑末端ライゲーションの間に長い、二本鎖DNAの生成を改善することによって達成される。細胞またはマウスは、その後、脂質トランスフェクション試薬を用いてトランスフェクトされる。 

ビデオ: Stimulation of Cytoplasmic DNA Sensing Pathways In Vitro and In Vivo

In vitro and in vivo Bioluminescence Reporter Gene Imaging of Human Embryonic Stem Cells

The discovery of human embryonic stem cells (hESCs) has dramatically increased the tools available to medical scientists interested in regenerative medicine. However, direct injection of hESCs, and cells differentiated from hESCs, into living organisms has thus far been hampered by significant cell death, teratoma formation, and host immune rejection. Understanding the in vivo hESC behavior after transplantation requires novel imaging techniques to longitudinally monitor hESC localization, proliferation, and viability. Molecular imaging has given investigators a high-throughput, inexpensive, and sensitive means for tracking in vivo cell proliferation over days, weeks, and even months. This advancement has significantly increased the understanding of the spatio-temporal kinetics of hESC engraftment, proliferation, and teratoma-formation in living subjects.

A major advance in molecular imaging has been the extension of noninvasive reporter gene assays from molecular and cellular biology into in vivo multi-modality imaging platforms. These reporter genes, under control of engineered promoters and enhancers that take advantage of the host cell s transcriptional machinery, are introduced into cells using a variety of vector and non-vector methods. Once in the cell, reporter genes can be transcribed either constitutively or only under specific biological or cellular conditions, depending on the type of promoter used. Transcription and translation of reporter genes into bioactive proteins is then detected with sensitive, noninvasive instrumentation (e.g., CCD cameras) using signal-generating probes such as D-luciferin.

To avoid the need for excitatory light to track stem cells in vivo as is required for fluorescence imaging, bioluminescence reporter gene imaging systems require only an exogenously administered probe to induce light emission. Firefly luciferase, derived from the firefly Photinus pyralis, encodes an enzyme that catalyzes D-luciferin to the optically active metabolite, oxyluciferin. Optical activity can then be monitored with an external CCD camera. Stably transduced cells that carry the reporter construct within their chromosomal DNA will pass the reporter construct DNA to daughter cells, allowing for longitudinal monitoring of hESC survival and proliferation in vivo. Furthermore, because expression of the reporter gene product is required for signal generation, only viable parent and daughter cells will create bioluminescence signal; apoptotic or dead cells will not. 

In this video, the specific materials and methods needed for tracking stem cell proliferation and teratoma formation with bioluminescence imaging will be described.

With the growing interest in stem cell therapies, molecular imaging techniques are ideal for monitoring stem cell behavior after transplantation. Luciferase reporter genes have enabled non-invasive, repetitive assessment of cell survival, location, and proliferation in vivo. This video will demonstrate how to track hESC proliferation in a living mouse.

in vitroおよびヒト胚性幹細胞のin vivoでの生物発光レポーター遺伝子イメージングの

The discovery of human embryonic stem cells (hESCs) has dramatically increased the tools available to medical scientists interested in regenerative ...

生物学

DNA Transfection of Mammalian Skeletal Muscles using In Vivo Electroporation

A growing interest in cell biology is to express transgenically modified forms of essential proteins (e.g. fluorescently tagged constructs and/or mutant variants) in order to investigate their endogenous distribution and functional relevance. An interesting approach that has been implemented to fulfill this objective in fully differentiated cells is the in vivo transfection of plasmids by various methods into specific tissues such as liver1, skeletal muscle2,3, and even the brain4. We present here a detailed description of the steps that must be followed in order to efficiently transfect genetic material into fibers of the flexor digitorum brevis (FDB) and interosseus (IO) muscles of adult mice using an in vivo electroporation approach. The experimental parameters have been optimized so as to maximize the number of muscle fibers transfected while minimizing tissue damages that may impair the quality and quantity of the proteins expressed in individual fibers. We have verified that the implementation of the methodology described in this paper results in a high yield of soluble proteins, i.e. EGFP and ECFP3, calpain, FKBP12, &beta;2a-DHPR, etc. ; structural proteins, i.e. minidystrophin and &alpha;-actinin; and membrane proteins, i.e. &alpha;1s-DHPR, RyR1, cardiac Na/Ca2+ exchanger , NaV1.4 Na channel, SERCA1, etc., when applied to FDB, IO and other muscles of mice and rats. The efficient expression of some of these proteins has been verified with biochemical3 and functional evidence5. However, by far the most common confirmatory approach used by us are standard fluorescent microscopy and 2-photon laser scanning microscopy (TPLSM), which permit to identify not only the overall expression, but also the detailed intracellular localization, of fluorescently tagged protein constructs. The method could be equally used to transfect plasmids encoding for the expression of proteins of physiological relevance (as shown here), or for interference RNA (siRNA) aiming to suppress the expression of normally expressed proteins (not tested by us yet). It should be noted that the transfection of FDB and IO muscle fibers is particularly relevant for the investigation of mammalian muscle physiology since fibers enzymatically dissociated from these muscles are currently one of the most suitable models to investigate basic mechanisms of excitability and excitation-contraction coupling under current or voltage clamp conditions2,6-8.

DNA Transfection of Mammalian Skeletal Muscles using In Vivo Electroporation

We describe detailed procedures for the efficient transfection of plasmid DNA into the fibers of foot muscles of live mice using electroporation and the subsequent visualization of protein expression using fluorescence microscopy.

使用して哺乳類の骨格筋のDNAトランスフェクションインビボでのエレクトロポレーション

 A growing interest in cell biology is to express transgenically modified forms of essential proteins (e.g. fluorescently tagged constructs and/or mutant ...

In vivo and in vitro Studies of Adaptor-clathrin Interaction

A major endocytic pathway initiates with the formation of clathrin-coated vesicles (CCVs) that transport cargo from the cell surface to endosomes1-6. CCVs are distinguished by a polyhedral lattice of clathrin that coats the vesicle membrane and serves as a mechanical scaffold. Clathrin coats are assembled during vesicle formation from individual clathrin triskelia , the soluble form of clathrin composed of three heavy and three light chain subunits7,8. Because the triskelion does not have the ability to bind to the membrane directly, clathrin-binding adaptors are critical to link the forming clathrin lattice to the membrane through association with lipids and/or membrane proteins9. Adaptors also package transmembrane protein cargo, such as receptors, and can interact with each other and with other components of the CCV formation machinery9.
Over twenty clathrin adaptors have been described, several are involved in clathrin mediated endocytosis and others localize to the trans Golgi network or endosomes9. With the exception of HIP1R (yeast Sla2p), all known clathrin adaptors bind to the N-terminal -propeller domain of the clathrin heavy chain9. Clathrin adaptors are modular proteins consisting of folded domains connected by unstructured flexible linkers. Within these linker regions, short binding motifs mediate interactions with the clathrin N-terminal domain or other components of the vesicle formation machinery9. Two distinct clathrin-binding motifs have been defined: the clathrin-box and the W-box9. The consensus clathrin-box sequence was originally defined as L[L/I][D/E/N][L/F][D/E]10 but variants have been subsequently discovered11. The W-box conforms to the sequence PWxxW (where x is any residue).
Sla1p (Synthetic Lethal with Actin binding protein-1) was originally identified as an actin associated protein and is necessary for normal actin cytoskeleton structure and dynamics at endocytic sites in yeast cells12. Sla1p also binds the NPFxD endocytic sorting signal and is critical for endocytosis of cargo bearing the NPFxD signal13,14. More recently, Sla1p was demonstrated to bind clathrin through a motif similar to the clathrin box, LLDLQ, termed a variant clathrin-box (vCB), and to function as an endocytic clathrin adaptor15. In addition, Sla1p has become a widely used marker for the endocytic coat in live cell fluorescence microscopy studies16. Here we use Sla1p as a model to describe approaches for adaptor-clathrin interaction studies. We focus on live cell fluorescence microscopy, GST-pull down, and co-immunoprecipitation methods.

In vivo and in vitro Studies of Adaptor-clathrin Interaction

Clathrin-mediated endocytosis depends on adaptor proteins that coordinate cargo selection and clathrin coat assembly. Here we describe procedures to study adaptor-clathrin physical interaction and live cell imaging approaches using as a model the yeast endocytic adaptor protein Sla1p.

in vivoおよびアダプター、クラスリンの相互作用のin vitro試験

A major endocytic pathway initiates with the formation of clathrin-coated vesicles (CCVs) that transport cargo from the cell surface to endosomes1-6. CCVs ...

Visualization of MG53-mediated Cell Membrane Repair Using in vivo and in vitro Systems

Repair of acute injury to the cell membrane is an elemental process of normal cellular physiology, and defective membrane repair has been linked to many degenerative human diseases. The recent discovery of MG53 as a key component of the membrane resealing machinery allows for a better molecular understanding of the basic biology of tissue repair, as well as for potential translational applications in regenerative medicine. Here we detail the experimental protocols for exploring the in vivo function of MG53 in repair of muscle injury using treadmill exercise protocols on mouse models, for testing the ex vivo membrane repair capacity by measuring dye entry into isolated muscle fibers, and for monitoring the dynamic process of MG53-mediated vesicle trafficking and cell membrane repair in cultured cells using live cell confocal microscopy.

Visualization of MG53-mediated Cell Membrane Repair Using in vivo and in vitro Systems

Described here are protocols used to visualize the dynamic process of MG53-mediated cell membrane repair in whole animals and at the cellular level. These methods can be applied to investigate the cell biology of plasma membrane resealing and regenerative medicine.

使用してMG53を介した細胞膜修復の可視化 in vivoでと in vitroでシステム

Repair of acute injury to the cell membrane is an elemental process of normal cellular physiology, and defective membrane repair has been linked to many ...

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

Apoptosis, or programmed cell death, is a conserved and highly regulated pathway by which cells die1. Apoptosis can be triggered when cells encounter a wide range of cytotoxic stresses. These insults initiate signaling cascades that ultimately cause the release of cytochrome c from the mitochondrial intermembrane space to the cytoplasm2. The release of cytochrome c from mitochondria is a key event that triggers the rapid activation of caspases, the key cellular proteases which ultimately execute cell death3-4.
The pathway of apoptosis is regulated at points upstream and downstream of cytochrome c release from mitochondria5. In order to study the post-mitochondrial regulation of caspase activation, many investigators have turned to direct cytoplasmic microinjection of holocytochrome c (heme-attached) protein into cells6-9. Cytochrome c is normally localized to the mitochondria where attachment of a heme group is necessary to enable it to activate apoptosis10-11. Therefore, to directly activate caspases, it is necessary to inject the holocytochrome c protein instead of its cDNA, because while the expression of cytochrome c from cDNA constructs will result in mitochondrial targeting and heme attachment, it will be sequestered from cytosolic caspases. Thus, the direct cytosolic microinjection of purified heme-attached cytochrome c protein is a useful tool to mimic mitochondrial cytochrome c release and apoptosis without the use of toxic insults which cause cellular and mitochondrial damage.
In this article, we describe a method for the microinjection of cytochrome c protein into cells, using mouse embryonic fibroblasts (MEFs) and primary sympathetic neurons as examples. While this protocol focuses on the injection of cytochrome c for investigations of apoptosis, the techniques shown here can also be easily adapted for microinjection of other proteins of interest.

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

In this protocol, we describe the direct cytoplasmic microinjection of cytochrome c protein into fibroblasts and primary sympathetic neurons. This technique allows for the introduction of cytochrome c protein into the cytoplasm of cells and mimics the release of cytochrome c from mitochondria, which occurs during apoptosis.

シトクロムの細胞質マイクロインジェクションによるアポトーシスの活性化 C</em

Apoptosis, or programmed cell death, is a conserved and highly regulated pathway by which cells die1.  Apoptosis can be triggered when cells encounter a ...

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the messagefor review see 1. However, it's now widely accepted that synonymous codon usage is the primary cause of non-uniform translational elongation rates1. Synonymous codons are not used with identical frequency. A bias exists in the use of synonymous codons with some codons used more frequently than others2. Codon bias is organism as well as tissue specific2,3. Moreover, frequency of codon usage is directly proportional to the concentrations of cognate tRNAs4. Thus, a frequently used codon will have higher multitude of corresponding tRNAs, which further implies that a frequent codon will be translated faster than an infrequent one. Thus, regions on mRNA enriched in rare codons (potential pause sites) will as a rule slow down ribosome movement on the message and cause accumulation of nascent peptides of the respective sizes5-8. These pause sites can have functional impact on the protein expression, mRNA stability and protein foldingfor review see 9. Indeed, it was shown that alleviation of such pause sites can alter ribosome movement on mRNA and subsequently may affect the efficiency of co-translational (in vivo) protein folding1,7,10,11. To understand the process of protein folding in vivo, in the cell, that is ultimately coupled to the process of protein synthesis it is essential to gain comprehensive insights into the impact of codon usage/tRNA content on the movement of ribosomes along mRNA during translational elongation.
Here we describe a simple technique that can be used to locate major translation pause sites for a given mRNA translated in various cell-free systems6-8. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA. The rationale is that at low-frequency codons, the increase in the residence time of the ribosomes results in increased amounts of nascent peptides of the corresponding sizes. In vitro transcribed mRNA is used for in vitro translational reactions in the presence of radioactively labeled amino acids to allow the detection of the nascent chains. In order to isolate ribosome bound nascent polypeptide complexes the translation reaction is layered on top of 30% glycerol solution followed by centrifugation. Nascent polypeptides in polysomal pellet are further treated with ribonuclease A and resolved by SDS PAGE. This technique can be potentially used for any protein and allows analysis of ribosome movement along mRNA and the detection of the major pause sites. Additionally, this protocol can be adapted to study factors and conditions that can alter ribosome movement and thus potentially can also alter the function/conformation of the protein.

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

A technique to identify translational pause sites on mRNA is described. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA, followed by the size analysis of the nascent chains using a denaturing gel electrophoresis.

リボソーム結合した新生ポリペプチドの分離<em&gt; in vitroで</em&gt; mRNAに沿って並進一時サイトを識別するために

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the ...

Visualizing Cytoplasmic Flow During Single-cell Wound Healing in Stentor coeruleus

Although wound-healing is often addressed at the level of whole tissues, in many cases individual cells are able to heal wounds within themselves, repairing broken cell membrane before the cellular contents leak out. The giant unicellular organism Stentor coeruleus, in which cells can be more than one millimeter in size, have been a classical model organism for studying wound healing in single cells. Stentor cells can be cut in half without loss of viability, and can even be cut and grafted together. But this high tolerance to cutting raises the question of why the cytoplasm does not simply flow out from the size of the cut. Here we present a method for cutting Stentor cells while simultaneously imaging the movement of cytoplasm in the vicinity of the cut at high spatial and temporal resolution. The key to our method is to use a &#34;double decker&#34; microscope configuration in which the surgery is performed under a dissecting microscope focused on a chamber that is simultaneously viewed from below at high resolution using an inverted microscope with a high NA lens. This setup allows a high level of control over the surgical procedure while still permitting high resolution tracking of cytoplasm.

Visualizing Cytoplasmic Flow During Single-cell Wound Healing in Stentor coeruleus

The giant ciliate Stentor coeruleus is a classical system for studying regeneration and wound healing in single cells.&#160;By imaging Stentor cells simultaneously at low and high magnification it is possible to measure cytoplasmic flows before, during, and after wounding.

Although wound-healing is often addressed at the level of whole tissues, in many cases individual cells are able to heal wounds within themselves, ...

Real-time Imaging of Single Engineered RNA Transcripts in Living Cells Using Ratiometric Bimolecular Beacons

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the development of diverse techniques to visualize individual RNA transcripts in single living cells. One promising technique that has recently been described utilizes an oligonucleotide-based optical probe, ratiometric bimolecular beacon (RBMB), to detect RNA transcripts that were engineered to contain at least four tandem repeats of the RBMB target sequence in the 3&#8217;-untranslated region. RBMBs are specifically designed to emit a bright fluorescent signal upon hybridization to complementary RNA, but otherwise remain quenched. The use of a synthetic probe in this approach allows photostable, red-shifted, and highly emissive organic dyes to be used for imaging. Binding of multiple RBMBs to the engineered RNA transcripts results in discrete fluorescence spots when viewed under a wide-field fluorescent microscope. Consequently, the movement of individual RNA transcripts can be readily visualized in real-time by taking a time series of fluorescent images. Here we describe the preparation and purification of RBMBs, delivery into cells by microporation and live-cell imaging of single RNA transcripts.

Ratiometric bimolecular beacons (RBMBs) can be used to image single engineered RNA transcripts in living cells. Here, we describe the preparation and purification of RBMBs, delivery of RBMBs into cells by microporation and fluorescent imaging of single RNA transcripts in real-time.

レシオメトリック二分子ビーコンを使用して、生細胞における単エンジニアのRNA転写物のリアルタイムイメージング

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the ...

The c-FOS Protein Immunohistological Detection: A Useful Tool As a Marker of Central Pathways Involved in Specific Physiological Responses In Vivo and Ex Vivo

Many studies seek to identify and map the brain regions involved in specific physiological regulations. The proto-oncogene c-fos, an immediate early gene, is expressed in neurons in response to various stimuli. The protein product can be readily detected with immunohistochemical techniques leading to the use of c-FOS detection to map groups of neurons that display changes in their activity. In this article, we focused on the identification of brainstem neuronal populations involved in the ventilatory adaptation to hypoxia or hypercapnia. Two approaches were described to identify involved neuronal populations in vivo in animals and ex vivo in deafferented brainstem preparations. In vivo, animals were exposed to hypercapnic or hypoxic gas mixtures. Ex vivo, deafferented preparations were superfused with hypoxic or hypercapnic artificial cerebrospinal fluid. In both cases, either control in vivo animals or ex vivo preparations were maintained under normoxic and normocapnic conditions. The comparison of these two approaches allows the determination of the origin of the neuronal activation i.e., peripheral and/or central. In vivo and ex vivo, brainstems were collected, fixed, and sliced into sections. Once sections were prepared, immunohistochemical detection of the c-FOS protein was made in order to identify the brainstem groups of cells activated by hypoxic or hypercapnic stimulations. Labeled cells were counted in brainstem respiratory structures. In comparison to the control condition, hypoxia or hypercapnia increased the number of c-FOS labeled cells in several specific brainstem sites that are thus constitutive of the neuronal pathways involved in the adaptation of the central respiratory drive.

The c-FOS Protein Immunohistological Detection: A Useful Tool As a Marker of Central Pathways Involved in Specific Physiological Responses In Vivo and Ex Vivo

Here, we present a protocol based on c-FOS protein immunohistological detection, a classical technique used for the identification of neuronal populations involved in specific physiological responses in vivo and ex vivo.

C-FOSタンパク質免疫組織学的検出：特定の生理反応に関与する中央経路のマーカーとして有用ツール<em&gt;インビボ</em&gt;と<em&gt; ex vivoで</em

Many studies seek to identify and map the brain regions involved in specific physiological regulations. The proto-oncogene c-fos, an immediate early gene, ...

Filtration Isolation of Nucleic Acids:  A Simple and Rapid DNA Extraction Method

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad to extract cellular DNA from whole blood in less than 2 min. The blood specimen is treated with detergent, mixed briefly and applied by pipet to the separation membrane. The lysate wicks into the blotting pad due to capillary action, capturing the genomic DNA on the surface of the separation membrane. The extracted DNA is retained on the membrane during a simple wash step wherein PCR inhibitors are wicked into the absorbent blotting pad. The membrane containing the entrapped DNA is then added to the PCR reaction without further purification. This simple method does not require laboratory equipment and can be easily implemented with inexpensive laboratory supplies. Here we describe a protocol for highly sensitive detection and quantitation of HIV-1 proviral DNA from 100 &#181;l whole blood as a model for early infant diagnosis of HIV that could readily be adapted to other genetic targets.

We describe here a simple and rapid paper-based DNA extraction method of HIV proviral DNA from whole blood detected by quantitative PCR. This protocol can be extended for use in detecting other genetic markers or using alternative amplification methods.

核酸の濾過単離：簡易迅速DNA抽出方法

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad ...