我々はこの仕事のための資金を提供するためのオハイオ州立大学総合がんセンターに感謝しています。

1。統合された組換えの基質を有する細胞株<ol><li> HeLa細胞を安定PDR - GFP（M.ジャシーン、メモリアルスローンケタリング癌センターからプラスミド7贈り物）で形質転換し、形質転換体の選択のためのピューロマイシンで維持した。 </li><li>細胞は、10％ウシ胎児血清、2mMのグルタミン、1mMピルビン酸ナトリウム、ペニシリン/ストレプトマイシン（100 U / mL/0.1 mg / mLの）、および1.5 ug / mlとピューロマイシンを含む、DMEM / FBSで継代されています。 </li></ol>プラスミドPDR - GFPの再結合の基質、および安定な形質転換体では、GFPの2つの非アクティブ対立遺伝子（図1）が含まれています。一つの対立遺伝子が原因でI - SCEIは18 bpの制限酵素認識部位のそのコード配列に含めるに不活性である。これらの細胞におけるI - SCEIの発現は、二本鎖DNAの切断（DSB）を作成します。そこにこのDNA上の非アクティブなGFPの第二の対立遺伝子は、ですが、最初の対立遺伝子のI - SCEIのサイトと相関し、その配列の一部は、野生型である。この第二のGFPの対立遺伝子は、DSBの相同組換え修復のためのシーケンスのドナーとして機能することができ、容易にフローサイトメトリーによって検出されるアクティブなGFPの対立遺伝子、になるだろう。また、DSBは、非相同末端結合するI - SCEIの制限部位を破壊するとGFP陰性細胞という結果になる、ことによって修復することができます。セル内の相同組換えのレベルは、GFP陽性細胞の割合を計数することにより決定されます。 この細胞株、pHeLa - DR13 - 9は、ゼロバックグラウンドのGFPの信号とI - SCEI発現プラスミド、pCBASceによるトランスフェクションの際にGFP陽性細胞の比較的高い割合をすべてのクロー​​ンの中から選ばれました。 （pCBASceとそのベクトル制御、pCAGGSは、両方のメモリアルスローンケタリング癌センターのM.ジャシーンによって供給された。） HeLa細胞- DR13 - 9細胞株は、要求に応じて、著者から入手可能です。 2。内因性BRCA1を破壊し、復帰突然変異体BRCA1を追加するためにトランスフェクション。 <ol><li>第一トランスフェクション（通常は火曜日）前日は：HeLa細胞 - DR13 - 9細胞の少なくとも90％コンフルエントの10cmディッシュから始めます。 </li><li> 1 mLのトリプシン処理と9 mLのDMEM / FBSを加えて反応を停止し、ピペッティングによりよく混和する。 </li><li>トリプシン処理細胞の40ウェルあたりのDME / FBSを0.5mL 24ウェルプレートを追加。 </li><li> 1日目のトランスフェクション（水）：はるかに少ないが、最初からやり直す場合細胞は、〜40〜50％コンフルエントにする必要があります。混合によるトランスフェクション：5 pmolのsiRNAは+ 12.5μLのOpti - MEM中でプラスミドDNAを発現さ0.3 UGのBRCA1 -変異体、および12.5μLのOpti - MEMに0.5μLリポフェクタミン2000。リポフェクタミンプロトコル（Invitrogen社）によるとトランスフェクションに進んでください。 </li><li> 4-6時間後にメディアを交換してください。我々はBRCA1を除去するために使用するsiRNAは、80798（アクセッション番号AY273801）にBRCA1 3&#39; - UTR配列の80780を指定してシーケンスのGCUCCUCUCACUCUUCAGUに基づいています。 </li><li> 2日目：6ウェルプレートに24ウェルプレートからトリプシン処理により細胞を転送。 </li><li> 3日目のトランスフェクション：25 pmolのsiRNAを+プラスミドDNAを発現さ0.75 UG BRCA1変異+ 62.5μLのOpti - MEMおよび62.5μLのOpti - MEMに2.5μLリポフェクタミン2000、0.75 UGのpCBASce（またはpCAGGSベクトル制御）。 4-6時間後にメディアを交換してください。 </li></ol> 3。トランスフェクタントの解析。 <ol><li> 6日目に、各ウェルから細胞をトリプシン処理し、1 mLのDMEM / FBSで停止します。 GFPの発現のため細胞を分析する日5または7日に行われますが、我々は最高のその日の6作品を見つけることができます。 </li><li>フローサイトメトリーで1ウェルあたり10,000細胞を分析する。私たちは、オハイオ州立大学総合がんセンター分析サイトメトリーの共有リソースにベクトンディッキンソンFACSキャリバーの楽器を使用してください。シングル、生きている細胞は、前方と側方散乱パラメータを用いてゲートです。 GFP陽性細胞は、GFP蛍光検出器（FL1）を用いて検出され、その結果はヒストグラム（図2、底部）として表示されます。 </li><li>必要に応じて、左上の細胞は蛍光顕微鏡で写真撮影のために色を保ち続けることができます。 </li></ol> 4。代表的な結果： BRCA1は、相同組換え6,11によって二本鎖DNA切断の修復のための経路を調節する。相同組換えによって修復を受けているこれらの細胞は容易に蛍光顕微鏡（図2、上）によって検出されています。検出されたGFP陽性細胞（図2、右）の減少におけるBRCA1の結果の枯渇。 FACSキャリバーからの出力は、X軸とY軸（図2、下）にある細胞の数のセルあたりのGFPの強度ヒストグラムです。単一の実験からの結果の典型的なセットでは、細胞の15.5％がI - SCEIの発現と制御RNAiの枯渇（図3）の後にGFP陽性であった。比較することにより、BRCA1およびベクトルの枯渇は0.7％にGFP -変換を削減再度追加します。追加バック野生型BRCA1のまたはBRCA1（I21V）完全に復元された相同組換えの、として15から16パーセントGFP陽性細胞を取得することによって検出された。追加バックBRCA1（M1の8T）変異体（図3、レーン5）は、ベクトル制御のアドオンバックと同様の効果を持っていた。このバリアントは、内因性のタンパク質9と同様のレベルで発現しているので、我々はこのようにBRCA1（M18T）変異体は相同組換えに非機能的であると解釈。 我々は通常、I - SCEIの発現プラスミドをトランスフェクトした後、GFP陽性に変換細胞の10-20％を持っている。 BRCA1の枯渇は、再現性を約8〜10倍でGFP陽性細胞の転換を減らすことができます。 GFP陽性細胞の実際の割合は、実験内のコントロールの枯渇に対する相対的なBRCA1の枯渇、以下の実験間、GFP陽性細胞の比率に変更される可能性があるところではありますが非常に一貫しています。我々は、100％に制約のない変換速度を設定することで、結果を正規化し、BRCA1の枯渇やパーセンテージなどのアドインが背を向け、結果を表現する。このように、我々は複数の実験を組み合わせることができます、そして実験的な変化は高くありません。 これまでの堅牢な結果が得られているバックBRCA1の変異体の追加：各BRCA1の変異は、完全に相同組換えの活性を回復しているか、効果がなかったしました。これらの結果を得るのに重要な考慮事項は、当社のトランスフェクションの条件は、未処理の細胞の内因性タンパク質の濃度とほぼ等しいBRCA1タンパク質のレベルを生成することである。 BRCA1のであって欠陥のある変異体の過剰発現が高すぎるとは、いくつかの正の相同組換えの活動になることがあります。 我々が克服するために必要な問題は、トランスフェクションの毒性であった。 BRCA1自体の枯渇は細胞の成長を遅らせることができる、と皿上の細胞の数に対して相対的リポフェクタミン- 2000とプラスミドDNAのバランスは、一貫性のあるレベルに維持する必要があります。あまりにも多くのリポフェクタミンまたはプラスミドDNAは、細胞数を減らし、細胞の数は、信頼性の高いフローサイトメトリーの結果を得るには不十分になります。トランスフェクションされた細胞の数に対するプラスミドとリポフェクタミン試薬のバランスが細胞の生存を維持するために非常に重要です。 <img src="/files/ftp_upload/2468/2468fig1.jpg" alt="図1" /> 図1組換えの基質。 M.ジャシーン7,12のグループによって開発されていた戦略が描かれている。 PDR - GFPプラスミドI - SCEIの制限エンドヌクレアーゼ部位を含むそのうちの一つGFPの二つの欠陥対立遺伝子が含まれています。 HeLa細胞由来の細胞株は、このDNAのゲノム9つのサイトに統合された基板、およびGFP陽性への一つの対立遺伝子の変換における相同組換えの結果により、I - SCEIのサイトにある二本鎖DNAの休憩のアクティブな修復が含まれています。 <img src="/files/ftp_upload/2468/2468fig2.jpg" alt="図2" /> 図2。蛍光顕微鏡によるGFP陽性細胞の検出。細胞は、このプロトコルに従って、テスト、および制御枯渇細胞は、GFPの発現（左）と細胞の高い割合で検出される、相同組換えでアクティブであった。 GFP陽性細胞（右）の数の急激な減少のRNAi結果によるBRCA1の枯渇。 <img src="/files/ftp_upload/2468/2468fig3.jpg" alt="図3" /> 図3。BRCA1のミスセンス変異のアドオンバックの結果。単一の実験からの結果は、このヒストグラムに表示されます。プラスミドを発現しているトランスフェクトI - SCEIの不在（レーン1）で検出さGFP -陽性細胞は存在しない。レーン2-6の結果はすべて発現するプラスミドをI - SCEIは3日目にトランスフェクトされた細胞から採取した。無関係な遺伝子（ルシフェラーゼ）のために枯渇し、ベクトルで、再び追加された細胞で、細胞の15.5％が（レーン2）のGFP陽性であった。 BRCA1およびベクトルアドインバックの枯渇は、相同組換えでBRCA1の損失（レーン3）の効果を明らかにする。 BRCA1の枯渇とアドインバック野生型BRCA1（レーン4）、BRCA1（M18T）（レーン5）、およびBRCA1（I21V）（レーン6）のの効果が示されています。これらの結果は、単一の実験からのもので、特定の変異BRCA1のタンパク質によってサポートされている相同組換えの指標を得るために少なくとも二つ以上の繰り返し実験と組み合わせることになります。

<ol>
	<li>Carvalho, 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=Determination+of+cancer+risk+associated+with+germ+line+BRCA1+missense+variants+by+functional+analysis.">Determination of cancer risk associated with germ line BRCA1 missense variants by functional analysis.</a> Cancer Res. 67, 1494-1501 (2007).</li><li>Couch, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+epidemiology+of+BRCA1.">Genetic epidemiology of BRCA1.</a> Cancer Biol Ther. 3, 509-514 (2004).</li><li>Couch, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+functional+effects+of+unclassified+genetic+variants.">Assessment of functional effects of unclassified genetic variants.</a> Hum Mutat. 29, 1314-1326 (2008).</li><li>Tomlinson, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+a+breast+cancer+cell+line+derived+from+a+germ-line+BRCA1+mutation+carrier.">Characterization of a breast cancer cell line derived from a germ-line BRCA1 mutation carrier.</a> Cancer Res. 58, 3237-3242 (1998).</li><li>Ruffner, H., Joazeiro, C. A., Hemmati, D., Hunter, T., Verma, I. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer-predisposing+mutations+within+the+RING+domain+of+BRCA1:+loss+of+ubiquitin+protein+ligase+activity+and+protection+from+radiation+hypersensitivity.">Cancer-predisposing mutations within the RING domain of BRCA1: loss of ubiquitin protein ligase activity and protection from radiation hypersensitivity.</a> Proc Natl Acad Sci U S A. 98, 5134-5139 (2001).</li><li>Moynahan, M. E., Chiu, J. W., Koller, B. H., Jasin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brca1+controls+homology-directed+DNA+repair.">Brca1 controls homology-directed DNA repair.</a> Mol Cell. 4, 511-518 (1999).</li><li>Nakanishi, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+Fanconi+anemia+monoubiquitination+pathway+promotes+homologous+DNA+repair.">Human Fanconi anemia monoubiquitination pathway promotes homologous DNA repair.</a> Proc Natl Acad Sci U S A. 102, 1110-1115 (2005).</li><li>Pierce, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Double-strand+breaks+and+tumorigenesis.">Double-strand breaks and tumorigenesis.</a> Trends Cell Biol. 11, 52-59 (2001).</li><li>Ransburgh, D. J., Chiba, N., Ishioka, C., Toland, A. E., Parvin, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+breast+tumor+mutations+in+BRCA1+that+abolish+its+function+in+homologous+DNA+recombination.">Identification of breast tumor mutations in BRCA1 that abolish its function in homologous DNA recombination.</a> Cancer Res. 70, 988-995 (2010).</li><li>Easton, D. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+systematic+genetic+assessment+of+1,433+sequence+variants+of+unknown+clinical+significance+in+the+BRCA1+and+BRCA2+breast+cancer-predisposition+genes.">A systematic genetic assessment of 1,433 sequence variants of unknown clinical significance in the BRCA1 and BRCA2 breast cancer-predisposition genes.</a> Am J Hum Genet. 81, 873-883 (2007).</li><li>Snouwaert, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BRCA1+deficient+embryonic+stem+cells+display+a+decreased+homologous+recombination+frequency+and+an+increased+frequency+of+non-homologous+recombination+that+is+corrected+by+expression+of+a+brca1+transgene.">BRCA1 deficient embryonic stem cells display a decreased homologous recombination frequency and an increased frequency of non-homologous recombination that is corrected by expression of a brca1 transgene.</a> Oncogene. 18, 7900-7907 (1999).</li><li>Pierce, A. J., Hu, P., Han, M., Ellis, N., Jasin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ku+DNA+end-binding+protein+modulates+homologous+repair+of+double-strand+breaks+in+mammalian+cells.">Ku DNA end-binding protein modulates homologous repair of double-strand breaks in mammalian cells.</a> Genes Dev. 15, 3237-3242 (2001).</li><li>Spearman, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinically+applicable+models+to+characterize+BRCA1+and+BRCA2+variants+of+uncertain+significance.">Clinically applicable models to characterize BRCA1 and BRCA2 variants of uncertain significance.</a> J Clin Oncol. 26, 5393-5400 (2008).</li><li>Sweet, K., Senter, L., Pilarski, R., Wei, L., Toland, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+BRCA1+ring+finger+variants+of+uncertain+significance.">Characterization of BRCA1 ring finger variants of uncertain significance.</a> Breast Cancer Res Treat. 119, 737-743 (2009).</li></ol>

利害の衝突は宣言されません。

この方法の利点は、我々は、野生型内因的に発現BRCA1を持つ細胞を用いた相同組換えによるDNA修復を調節する上でBRCA1蛋白質の機能を勉強できるということです。我々は、相同組換えの過程を研究するのとDNA修復の機能のためにBRCA1の変異体をテストするための新規な方法を得るために、RNAiによる枯渇を利用する2つの別々の確立された方法を採用している。この方法の成功の鍵は、我々はGFPの変換の非常に高いレベルを得るようなゲノムの組換えの基質と、そのようなHeLa細胞のような容易にtransfectable細胞株を、確立することだった。我々は、検出可能なバックグラウンドのGFPシグナルを持っていたものを得るために元の変換の複数のクローンをスクリーニングしたが、プラスミドI - SCEIの発現のトランスフェクションによりGFPの変換の非常に高いレベルを持っていた。 この方法では、我々は今相同組換えにおける機能のための他の特定のBRCA1のアミノ酸残基を調査している。この作品からの生物学的洞察と一緒に、これらの結論は、臨床癌の遺伝学に適用することができます。多くの珍しい亜種が存在するので、BRCA1のミスセンス変異の割合が高い未知の臨床的意義を持っており、遺伝的分離分析3,10,13,14によって癌の関連付けを決定するために、これらの多くのための十分な情報があります。この方法を使用して、相同組換えの制御におけるBRCA1の生物学的機能上の特定の変異の影響は、それらのゲノム中にこれらの亜種を運ぶ女性たちに知らせるために使用することができる。

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<td>DMEM</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11960</td>
<td>&nbsp;</td>
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<td>FBS</td>
<td>&nbsp;</td>
<td>USA Scientific</td>
<td>9871-5200</td>
<td>&nbsp;</td>
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<td>siRNA</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>N/A</td>
<td>&nbsp;</td>
<tr>
<td>Lipofectamine-2000</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11668</td>
<td>&nbsp;</td>
<tr>
<td>TrypLE</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>12604</td>
<td>&nbsp;</td>
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<td>Puromycin</td>
<td>&nbsp;</td>
<td>Enzo Life Sciences</td>
<td>BML-GR312</td>
<td>&nbsp;</td>
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<td>Opti-MEM I</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>31985</td>
<td>&nbsp;</td>
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<td>Sodium Pyruvate</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11360</td>
<td>&nbsp;</td>
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<td>GlutaMAX I</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>35050</td>
<td>&nbsp;</td>
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<td>Penicillin-Streptomycin</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15140</td>
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		</div>

identifying the effects of brca1 mutations on homologous recombination using cells that express endogenous wild-type brca1

ミスセンス変異の機能解析は、内因性蛋白質の細胞内の存在によって複雑になることがあります。 BRCA1の構造機能解析は、完全長のBRCA1タンパク質とhypomorphic BRCA1タンパク質1,2,3,4,5を発現する細胞系での作業における固有の困難のための堅牢な分析の欠如によって複雑にされています。我々は、細胞内の内因性のBRCA1タンパク質が急性BRCA1 mRNAの3&#39; - UTRを標的RNAiにより枯渇し、BRCA1の変異型を発現するプラスミドをコトランスフェクトすることにより置き換えられてそれによってシステムを開発した。この手順の利点の一つは、BRCA1と同時交換の急性サイレンシングは、細胞がBRCA1の機能の損失を補償するために、時間の経過とともに発生する可能性のある二次変異または翻案することなく拡張できるようにすることです。この枯渇とアドインバックプロシージャは、容易に相同組換え活性を測定したHeLa細胞由来の細胞株で行われていた。相同組換えアッセイは、組換えの基質がゲノム（図1）6,7,8,9に統合されそれによって以前に発表された方法に基づいています。この組換えの基板は、非アクティブなGFPの対立遺伝子内部に珍しいカットI - SCEIの制限酵素部位を有し、そして下流の第二非アクティブなGFPの対立遺伝子である。相同組換えによって修復されることがあります二本鎖の休憩、のI - SCEI結果を表現し、相同組換えは、ブレークを修復しない場合は、容易にGFPタンパク質の発現のフローサイトメトリーにより採点され、アクティブなGFPの対立遺伝子を作成し、そのプラスミドのトランスフェクション。内因性のBRCA1の枯渇は、相同組換えの活性の8〜10倍減少をもたらし、そして野生型プラスミドを完全に復元された相同組換え機能のバック追加。完全長のBRCA1の特定の点変異体を共トランスフェクトしたプラスミドから発現させた場合、特定のミスセンス変異の効果は、得点することができます。例として、BRCA1（M18T）タンパク質、未知の臨床的意義10の変異体の発現は、これらの細胞で発現され、それはBRCA1依存相同組換えを復元に失敗しました。また意義が不明な別の変異体のそれとは対照的に、表現、、BRCA1（I21Vが）完全に復元されたBRCA1依存相同組換えの機能。 BRCA1のミスセンス変異の機能をテストするのこの戦略は、中心体機能（KAISら、未発表の観察）をアッセイする別の生物学的システムに適用されている。全体的に、このアプローチは、劣性分析する必要があります任意の遺伝子のミスセンス変異体の解析に適しています。

Watch this Scientific Journal Video about Identifying the Effects of BRCA1 Mutations on Homologous Recombination using Cells that Express Endogenous Wild-type BRCA1 at JoVE.com

Identifying the Effects of BRCA1 Mutations on Homologous Recombination using Cells that Express Endogenous Wild-type BRCA1

我々は、RNAiを用いて細胞から内因性のBRCA1蛋白質を破壊し、コーディングの変更が含まれているBRCA1点変異体でそれを置き換えることにより、DNA損傷の相同組換え修復のためのアッセイに基づいて組織培養でのBRCA1の変異体をテストするための方法を提供する。

内因性野生型BRCA1を発現する細胞を用いて相同組換えにBRCA1の変異の影響を識別

The functional analysis of missense mutations can be complicated by the presence in the cell of the endogenous protein. Structure-function analyses of the ...

identifying-effects-brca1-mutations-on-homologous-recombination-using

Research

JoVE Journal

Biology

14.5K ビュー.  The Ohio State University. 我々は、RNAiを用いて細胞から内因性のBRCA1蛋白質を破壊し、コーディングの変更が含まれているBRCA1点変異体でそれを置き換えることにより、DNA損傷の相同組換え修復のためのアッセイに基づいて組織培養でのBRCA1の変異体をテストするための方法を提供する。

ビデオ: Identifying the Effects of BRCA1 Mutations on Homologous Recombination using Cells that Express Endogenous Wild-type BRCA1

Studying the Effects of Matrix Stiffness on Cellular Function using Acrylamide-based Hydrogels

Tissue stiffness is an important determinant of cellular function, and changes in tissue stiffness are commonly associated with fibrosis, cancer and cardiovascular disease1-11. Traditional cell biological approaches to studying cellular function involve culturing cells on a rigid substratum (plastic dishes or glass coverslips) which cannot account for the effect of an elastic ECM or the variations in ECM stiffness between tissues. To model in vivo tissue compliance conditions in vitro, we and others use ECM-coated hydrogels. In our laboratory, the hydrogels are based on polyacrylamide which can mimic the range of tissue compliances seen biologically12. "Reactive" cover slips are generated by incubation with NaOH followed by addition of 3-APTMS. Glutaraldehyde is used to cross-link the 3-APTMS and the polyacrylamide gel. A solution of acrylamide (AC), bis-acrylamide (Bis-AC) and ammonium persulfate is used for the polymerization of the hydrogel. N-hydroxysuccinimide (NHS) is incorporated into the AC solution to crosslink ECM protein to the hydrogel. Following polymerization of the hydrogel, the gel surface is coated with an ECM protein of choice such as fibronectin, vitronectin, collagen, etc.
The stiffness of a hydrogel can be determined by rheology or atomic force microscopy (AFM) and adjusted by varying the percentage of AC and/or bis-AC in the solution12. In this manner, substratum stiffness can be matched to the stiffness of biological tissues which can also be quantified using rheology or AFM. Cells can then be seeded on these hydrogels and cultured based upon the experimental conditions required. Imaging of the cells and their recovery for molecular analysis is straightforward. For this article, we define soft substrata as those having elastic moduli (E) &lt;3000 Pascal and stiff substrata/tissues as those with E &gt;20,000 Pascal.

The effect of substrata stiffness on cellular function can be modeled in vitro using polyacrylamide hydrogels of varying compliances.

アクリルアミド系ハイドロゲルを用いた細胞機能のマトリックスの剛性の影響を研究

Tissue stiffness is an important determinant of cellular function, and changes in tissue stiffness are commonly associated with fibrosis, cancer and ...

生物学

Analyzing the Function of Small GTPases by Microinjection of Plasmids into Polarized Epithelial Cells

Epithelial cells polarize their plasma membrane into biochemically and functionally distinct apical and basolateral domains where the apical domain faces the 'free' surfaces and the basolateral membrane is in contact with the substrate and neighboring cells. Both membrane domains are separated by tight junctions, which form a diffusion barrier. Apical-basolateral polarization can be recapitulated successfully in culture when epithelial cells such as Madin-Darby Canine Kidney (MDCK) cells are seeded at high density on polycarbonate filters and cultured for several days 1 2. Establishment and maintenance of cell polarity is regulated by an array of small GTPases of the Ras superfamily such as RalA, Cdc42, Rab8, Rab10 and Rab13 3 4 5 6 7. Like all GTPases these proteins cycle between an inactive GDP-bound state and an active GTP-bound state. Specific mutations in the nucleotide binding regions interfere with this cycling 8. For example, Rab13T22N is permanently locked in the GDP-form and thus dubbed 'dominant negative', whereas Rab13Q67L can no longer hydrolyze GTP and is thus locked in a 'dominant active' state 7. To analyze their function in cells both dominant negative and dominant active alleles of GTPases are typically expressed at high levels to interfere with the function of the endogenous proteins 9. An elegant way to achieve high levels of overexpression in a short amount of time is to introduce the plasmids encoding the relevant proteins directly into the nuclei of polarized cells grown on filter supports using microinjection technique. This is often combined with the co-injection of reporter plasmids that encode plasma membrane receptors that are specifically sorted to the apical or basolateral domain. A cargo frequently used to analyze cargo sorting to the basolateral domain is a temperature sensitive allele of the vesicular stomatitis virus glycoprotein (VSVGts045) 10. This protein cannot fold properly at 39&deg;C and will thus be retained in the endoplasmic reticulum (ER) while the regulatory protein of interest is assembled in the cytosol. A shift to 31&deg;C will then allow VSVGts045 to fold properly, leave the ER and travel to the plasma membrane 11. This chase is typically performed in the presence of cycloheximide to prevent further protein synthesis leading to cleaner results. Here we describe in detail the procedure of microinjecting plasmids into polarized cells and subsequent incubations including temperature shifts that allow a comprehensive analysis of regulatory proteins involved in basolateral sorting.

This article details the procedures involved in overexpression and analysis of small GTPases in polarized epithelial cells using microinjection technique.

偏上皮細胞へのプラスミドのマイクロインジェクションによる低分子量GTPaseの機能を解析する

Epithelial cells polarize their plasma membrane into biochemically and functionally distinct apical and basolateral domains where the apical domain faces ...

A PCR-based Genotyping Method to Distinguish Between Wild-type and Ornamental Varieties of Imperata cylindrica

Wild-type I. cylindrica (cogongrass) is one of the top ten worst invasive plants in the world, negatively impacting agricultural and natural resources in 73 different countries throughout Africa, Asia, Europe, New Zealand, Oceania and the Americas1-2. Cogongrass forms rapidly-spreading, monodominant stands that displace a large variety of native plant species and in turn threaten the native animals that depend on the displaced native plant species for forage and shelter. To add to the problem, an ornamental variety [I. cylindrica var. koenigii (Retzius)] is widely marketed under the names of Imperata cylindrica 'Rubra', Red Baron, and Japanese blood grass (JBG). This variety is putatively sterile and noninvasive and is considered a desirable ornamental for its red-colored leaves. However, under the correct conditions, JBG can produce viable seed (Carol Holko, 2009 personal communication) and can revert to a green invasive form that is often indistinguishable from cogongrass as it takes on the distinguishing characteristics of the wild-type invasive variety4 (Figure 1). This makes identification using morphology a difficult task even for well-trained plant taxonomists. Reversion of JBG to an aggressive green phenotype is also not a rare occurrence. Using sequence comparisons of coding and variable regions in both nuclear and chloroplast DNA, we have confirmed that JBG has reverted to the green invasive within the states of Maryland, South Carolina, and Missouri. JBG has been sold and planted in just about every state in the continental U.S. where there is not an active cogongrass infestation. The extent of the revert problem in not well understood because reverted plants are undocumented and often destroyed.
Application of this molecular protocol provides a method to identify JBG reverts and can help keep these varieties from co-occurring and possibly hybridizing. Cogongrass is an obligate outcrosser and, when crossed with a different genotype, can produce viable wind-dispersed seeds that spread cogongrass over wide distances5-7. JBG has a slightly different genotype than cogongrass and may be able to form viable hybrids with cogongrass. To add to the problem, JBG is more cold and shade tolerant than cogongrass8-10, and gene flow between these two varieties is likely to generate hybrids that are more aggressive, shade tolerant, and cold hardy than wild-type cogongrass. While wild-type cogongrass currently infests over 490 million hectares worldwide, in the Southeast U.S. it infests over 500,000 hectares and is capable of occupying most of the U.S. as it rapidly spreads northward due to its broad niche and geographic potential3,7,11. The potential of a genetic crossing is a serious concern for the USDA-APHIS Federal Noxious Week Program. Currently, the USDA-APHIS prohibits JBG in states where there are major cogongrass infestations (e.g., Florida, Alabama, Mississippi). However, preventing the two varieties from combining can prove more difficult as cogongrass and JBG expand their distributions. Furthermore, the distribution of the JBG revert is currently unknown and without the ability to identify these varieties through morphology, some cogongrass infestations may be the result of JBG reverts. Unfortunately, current molecular methods of identification typically rely on AFLP (Amplified Fragment Length Polymorphisms) and DNA sequencing, both of which are time consuming and costly. Here, we present the first cost-effective and reliable PCR-based molecular genotyping method to accurately distinguish between cogongrass and JBG revert.

A PCR-based Genotyping Method to Distinguish Between Wild-type and Ornamental Varieties of Imperata cylindrica

We provide a cost-effective and rapid molecular genotyping protocol that employs variety-specific PCR primers that target DNA sequence differences within the chloroplast trnL-F spacer region to differentiate between varieties of Imperata cylindrica (cogongrass) that cannot be distinguished by morphology alone. These varieties include the federally listed noxious weed, cogongrass and closely-related, wide-spread ornamental variety, I. cylindrica var. koenigii (Japanese blood grass).

の野生型と観賞用品種を区別するPCRベースのジェノタイピング法チガヤ</em

Wild-type I. cylindrica (cogongrass) is one of the top ten worst invasive plants in the world, negatively impacting agricultural and natural resources in ...

Recombineering Homologous Recombination Constructs in Drosophila

The continued development of techniques for fast, large-scale manipulation of endogenous gene loci will broaden the use of Drosophila melanogaster as a genetic model organism for human-disease related research. Recent years have seen technical advancements like homologous recombination and recombineering. However, generating unequivocal null mutations or tagging endogenous proteins remains a substantial effort for most genes. Here, we describe and demonstrate techniques for using recombineering-based cloning methods to generate vectors that can be used to target and manipulate endogenous loci in vivo. Specifically, we have established a combination of three technologies: (1) BAC transgenesis/recombineering, (2) ends-out homologous recombination and (3) Gateway technology to provide a robust, efficient and flexible method for manipulating endogenous genomic loci. In this protocol, we provide step-by-step details about how to (1) design individual vectors, (2) how to clone large fragments of genomic DNA into the homologous recombination vector using gap repair, and (3) how to replace or tag genes of interest within these vectors using a second round of recombineering. Finally, we will also provide a protocol for how to mobilize these cassettes in vivo to generate a knockout, or a tagged gene via knock-in. These methods can easily be adopted for multiple targets in parallel and provide a means for manipulating the Drosophila genome in a timely and efficient manner.

Recombineering Homologous Recombination Constructs in Drosophila

Homologous recombination techniques greatly advance Drosophila genetics by enabling the creation of molecularly precise mutations. The recent adoption of recombineering allows one to manipulate large pieces of DNA and transform them into Drosophila6. The methods presented here combine these techniques to rapidly generate large homologous recombination vectors.

における相同組換えコンストラクトを遺伝子改変<em&gt;ショウジョウバエ</em

The continued development of techniques for fast, large-scale manipulation of endogenous gene loci will broaden the use of Drosophila melanogaster as a ...

Measuring the Effects of Bacteria on C. Elegans Behavior Using an Egg Retention Assay

C. elegans egg-laying behavior is affected by environmental cues such as osmolarity1 and vibration2. In the total absence of food C. elegans also cease egg-laying and retain fertilized eggs in their uterus3. However, the effect of different sources of food, especially pathogenic bacteria and particularly Enterococcus faecalis, on egg-laying
	behavior is not well characterized. The egg-in-worm (EIW) assay is a useful tool to quantify the effects of different types of bacteria, in this case E. faecalis, on egg- laying behavior.
	
	EIW assays involve counting the number of eggs retained in the uterus of C. elegans4. The EIW assay involves bleaching staged, gravid adult C. elegans to remove the cuticle and separate the retained eggs from the animal. Prior to bleaching, worms are exposed to bacteria (or any type of environmental cue) for a fixed period of time. After bleaching, one is very easily able to count the number of eggs retained inside the uterus of the worms. In this assay, a quantifiable increase in egg retention after E. faecalis exposure can be easily measured. The EIW assay is a behavioral assay that may be used to screen for potentially pathogenic bacteria or the presence of environmental toxins. In addition, the EIW assay may be a tool to screen for drugs that affect neurotransmitter signaling since egg-laying behavior is modulated by neurotransmitters such as serotonin and acetylcholine5-9.

Measuring the Effects of Bacteria on C. Elegans Behavior Using an Egg Retention Assay

An egg-in-worm (EIW) assay is a useful method to quantify egg-laying behavior. Alterations in egg laying can be a behavioral response of the model organism Caenorhabditis elegans to potentially harmful environmental substances such as those produced by pathogenic bacteria.

上の細菌の影響を測定する<em&gt; C.エレガンス</em卵保持アッセイを用いて&gt;行動

C. elegans egg-laying behavior is  affected by environmental cues such as osmolarity1 and  vibration2. In the total absence of food C. elegans also cease ...

gDNA Enrichment by a Transposase-based Technology for NGS Analysis of the Whole Sequence of BRCA1, BRCA2, and 9 Genes Involved in DNA Damage Repair

The widespread use of Next Generation Sequencing has opened up new avenues for cancer research and diagnosis. NGS will bring huge amounts of new data on cancer, and especially cancer genetics. Current knowledge and future discoveries will make it necessary to study a huge number of genes that could be involved in a genetic predisposition to cancer. In this regard, we developed a Nextera design to study 11 complete genes involved in DNA damage repair. This protocol was developed to safely study 11 genes (ATM, BARD1, BRCA1, BRCA2, BRIP1, CHEK2, PALB2, RAD50, RAD51C, RAD80, and TP53) from promoter to 3&#39;-UTR in 24 patients simultaneously. This protocol, based on transposase technology and gDNA enrichment, gives a great advantage in terms of time for the genetic diagnosis thanks to sample multiplexing. This protocol can be safely used with blood gDNA.

gDNA Enrichment by a Transposase-based Technology for NGS Analysis of the Whole Sequence of BRCA1, BRCA2, and 9 Genes Involved in DNA Damage Repair

gDNA enrichment for NGS sequencing is an easy and powerful tool for the study of constitutional mutations. In this article, we present the procedure to analyse simply the complete sequence of 11 genes involved in DNA damage repair.

の全配列のNGS解析のためのトランスポゼースベースの技術によるgDNAのエンリッチメント<em&gt; BRCA1</em&gt;<em&gt; BRCA2</em&gt;、およびDNA損傷修復に関与する遺伝子9

The widespread use of Next Generation Sequencing has opened up new avenues for cancer research and diagnosis. NGS will bring huge amounts of new data on ...

Identifying Protein-protein Interaction Sites Using Peptide Arrays

Protein-protein interactions mediate most of the processes in the living cell and control homeostasis of the organism. Impaired protein interactions may result in disease, making protein interactions important drug targets. It is thus highly important to understand these interactions at the molecular level. Protein interactions are studied using a variety of techniques ranging from cellular and biochemical assays to quantitative biophysical assays, and these may be performed either with full-length proteins, with protein domains or with peptides. Peptides serve as excellent tools to study protein interactions since peptides can be easily synthesized and allow the focusing on specific interaction sites. Peptide arrays enable the identification of the interaction sites between two proteins as well as screening for peptides that bind the target protein for therapeutic purposes. They also allow high throughput SAR studies. For identification of binding sites, a typical peptide array usually contains partly overlapping 10-20 residues peptides derived from the full sequences of one or more partner proteins of the desired target protein. Screening the array for binding the target protein reveals the binding peptides, corresponding to the binding sites in the partner proteins, in an easy and fast method using only small amount of protein.
In this article we describe a protocol for screening peptide arrays for mapping the interaction sites between a target protein and its partners. The peptide array is designed based on the sequences of the partner proteins taking into account their secondary structures. The arrays used in this protocol were Celluspots arrays prepared by INTAVIS Bioanalytical Instruments. The array is blocked to prevent unspecific binding and then incubated with the studied protein. Detection using an antibody reveals the binding peptides corresponding to the specific interaction sites between the proteins.

Peptide array screening is a high throughput assay for identifying protein-protein interaction sites. This allows mapping multiple interactions of a target protein and can serve as a method for identifying sites for inhibitors that target a protein. Here we describe a protocol for screening and analyzing peptide arrays.

ペプチド配列の使用タンパク質間相互作用部位を同定

Protein-protein interactions mediate most of the processes in the living cell and control homeostasis of the organism. Impaired protein interactions may ...

Determination of Sialic Acids in Liver and Milk Samples of Wild-type and CMAH Knock-out Mice.

CMAH (cytidine monophosphate-N-acetylneuraminic acid hydroxylase) is responsible for the oxidation of cytidine monophosphate-N-acetylneuraminic acids in mammals. However, humans cannot oxidize cytidine monophosphate-N-acetylneuraminic acid to cytidine monophosphate-N-glycolylneuraminic acid due to a primary exon deletion of the CMAH gene. To understand the effects and implications of the lack of CMAH activity in more detail, a Cmah knock-out model in mice is of keen interest in basic and applied research. The analysis method to determine the phenotype of this mouse model is herein described in detail, and is based on the detection of both N-acetylneuraminic acid and N-glycolylenuraminic acid in the liver and milk of wild-type and Cmah knock-out mice. Endogenous sialic acids are released and derivatized with o-phenylenediamine to generate fluorogenic derivatives, which can be subsequently analyzed by HPLC. The presented protocol can be also applied for the analysis of milk and tissue samples from various other origins, and may be of use to investigate the nutritional and health effects of N-glycolylneuraminic acid.

Determination of Sialic Acids in Liver and Milk Samples of Wild-type and CMAH Knock-out Mice.

We describe a HPLC-based method for the determination of N-acetylneuraminic acid and N-glycolylenuraminic acid in mouse liver and milk.

野生型および肝臓の肝臓試料および牛乳試料中のシアル酸の定量<em&gt; CMAH</em&gt;ノックアウトマウス。

CMAH (cytidine monophosphate-N-acetylneuraminic acid hydroxylase) is responsible for the oxidation of cytidine monophosphate-N-acetylneuraminic acids in ...

A Method to Assess Bacteriocin Effects on the Gut Microbiota of Mice

Very intriguing questions arise with our advancing knowledge on gut microbiota composition and the relationship with health, particularly relating to the factors that contribute to maintaining the population balance. However, there are limited available methodologies to evaluate these factors. Bacteriocins are antimicrobial peptides produced by many bacteria that may confer a competitive advantage for food acquisition and/or niche establishment. Many probiotic lactic acid bacteria (LAB) strains have great potential to promote human and animal health by preventing the growth of pathogens. They can also be used for immuno-modulation, as they produce bacteriocins. However, the antagonistic activity of bacteriocins is normally determined by laboratory bioassays under well-defined but over-simplified conditions compared to the complex gut environment in humans and animals, where bacteria face multifactorial influences from the host and hundreds of microbial species sharing the same niche. This work describes a complete and efficient procedure to assess the effect of a variety of bacteriocins with different target specificities in a murine system. Changes in the microbiota composition during the bacteriocin treatment are monitored using compositional 16S rDNA sequencing. Our approach uses both the bacteriocin producers and their isogenic non-bacteriocin-producing mutants, the latter giving the ability to distinguish bacteriocin-related from non-bacteriocin-related modifications of the microbiota. The fecal DNA extraction and 16S rDNA sequencing methods are consistent and, together with the bioinformatics, constitute a powerful procedure to find faint changes in the bacterial profiles and to establish correlations, in terms of cholesterol and triglyceride concentration, between bacterial populations and health markers. Our protocol is generic and can thus be used to study other compounds or nutrients with the potential to alter the host microbiota composition, either when studying toxicity or beneficial effects.

Bacteriocins are believed to play a key role in defining microbial diversity in different ecological niches. Here, we describe an efficient procedure to assess how bacteriocins affect gut microbiota composition in an animal model.

マウスの腸内細菌叢に及ぼすバクテリオシンの影響を評価する方法

Very intriguing questions arise with our advancing knowledge on gut microbiota composition and the relationship with health, particularly relating to the ...

Investigating the Detrimental Effects of Low Pressure Plasma Sterilization on the Survival of Bacillus subtilis Spores Using Live Cell Microscopy

Plasma sterilization is a promising alternative to conventional sterilization methods for industrial, clinical, and spaceflight purposes. Low pressure plasma (LPP) discharges contain a broad spectrum of active species, which lead to rapid microbial inactivation. To study the efficiency and mechanisms of sterilization by LPP, we use spores of the test organism Bacillus subtilis because of their extraordinary resistance against conventional sterilization procedures. We describe the production of B. subtilis spore monolayers, the sterilization process by low pressure plasma in a double inductively coupled plasma reactor, the characterization of spore morphology using scanning electron microscopy (SEM), and the analysis of germination and outgrowth of spores by live cell microscopy. A major target of plasma species is genomic material (DNA) and repair of plasma-induced DNA lesions upon spore revival is crucial for survival of the organism. Here, we study the germination capacity of spores and the role of DNA repair during spore germination and outgrowth after treatment with LPP by tracking fluorescently-labelled DNA repair proteins (RecA) with time-resolved confocal fluorescence microscopy. Treated and untreated spore monolayers are activated for germination and visualized with an inverted confocal live cell microscope over time to follow the reaction of individual spores. Our observations reveal that the fraction of germinating and outgrowing spores is dependent on the duration of LPP-treatment reaching a minimum after 120 s. RecA-YFP (yellow fluorescence protein) fluorescence was detected only in few spores and developed in all outgrowing cells with a slight elevation in LPP-treated spores. Moreover, some of the vegetative bacteria derived from LPP-treated spores showed an increase in cytoplasm and tended to lyse. The described methods for analysis of individual spores could be exemplary for the study of other aspects of spore germination and outgrowth.

Investigating the Detrimental Effects of Low Pressure Plasma Sterilization on the Survival of Bacillus subtilis Spores Using Live Cell Microscopy

This protocol illustrates the important consecutive steps required to assess the relevance of monitoring vitality parameter and DNA repair processes in reviving Bacillus subtilis spores after treatment with low pressure plasma by tracking fluorescence-labelled DNA repair proteins via time-resolved confocal microscopy and scanning electron microscopy.

有害な効果の低圧プラズマ滅菌を使用して住んでいる携帯顕微鏡枯草菌胞子の生存に調査

Plasma sterilization is a promising alternative to conventional sterilization methods for industrial, clinical, and spaceflight purposes. Low pressure ...