このプロトコルは、ジェーンドッド、トーマスJessellとアンドリューラムズデンの研究室で開発されました。コンラートバスラー、アンCalof、トーマスエドランド、フィルハミルトン、Domna Karagogeos、アリエルルイス私Altabaと俊哉山田を含む多くの人々は、どのようにコラーゲンの脊髄の文化の外植片に決定。 Marysia Placzekとマルクテッシェ-ラヴィーンは、軸索ガイダンスの分子を識別する手段として、生体外植片からで軸索の成長を使用してのテクニックを開拓してきました。バトラーの実験室での作業は、NIH / NINDSからダイムの月とR01 NS063999からの補助金によってサポートされています。

パート1：ハンギングドロップを使用してトランスフェクトしたCOS細胞の集合体の調製<ol><li>種子のCOS - 7（COS）35ミリメートル培養皿中の細胞。彼らは、製造業者のプロトコルに従ってLipofectamine2000を用いて細胞に発現プラスミドの80％コンフルエント、トランスフェ1μgのに到達したとき。 </li><li>吊り滴を準備するには、トランスフェクション培地を吸引し、1Xリン酸緩衝溶液（PBS）を1mlでトランスフェクトしたCOS細胞をすすいでください。 15分間酵素無細胞解離培地の0.5ミリリットルを持つ細胞を扱う。反応を停止するのOpti - MEM + 1Xペニシリン/ストレプトマイシン/グルタミン（P / S / G）+ 10％ウシ胎児血清（FBS）の溶液の1mlを追加します。 </li><li>培養皿の表面からそれらを削除し、15ミリリットルコニカルチューブに移すために細胞をひいて粉にする。細胞をペレットに2000Kで2分間スピン。のOpti - MEM + 1 × P / S / G + 10％FBSの100μlの上清とペレットを再懸濁しますを削除します。 </li><li>皿の底の上に蓋と場所反転、35培養皿のふたの内側に複数の20μlのドロップを特定し、細胞集合体になるまで数時間、37℃のインキュベーターに残す。 </li></ol>パート2：背側脊髄の外植片の調製<ol><li>母親の子宮からE11のラット胚を摘出し、必要になるまでL15培地には氷上に置きます。 </li><li>シャープタングステン針で、すぐに前肢芽の下の領域から、それぞれの胚の幹から4〜6セグメントのセクションを削除します。プラスチック製のピペットを使用して、4ウェルNunc社製皿の1つのウェルに組織片を収集し、氷上に置きます。 </li><li>すべての胚のセグメントが解剖されているときは、1ミリリットルL15のソリューション+ヌンク料理のほか秒で1mgのディスパーゼに作品を転送するためにプラスチック製のピペットを使用してください。 5分よりもはや室温でインキュベートする。以上ディスパーゼで組織片をインキュベートしないでください。 </li><li>インキュベーションの間に、ペトリ皿と混在させる渦にL15の約10ミリリットルに熱不活性化正常ヤギ血清（HIGS）の0.5ミリリットルを追加します。第3のウェルNunc社製シャーレの中にこの溶液を1mlを移し、インキュベーションが終わったときに、この溶液中に組織片を転送する。氷上に置きます。注：組織片が時間氷上に&quot;残り&quot;が許可されている場合、後続の解離が容易になる。 </li></ol>パート3：プライミングコラーゲン<ol><li> 360μlコラーゲンに40μlの10倍の最小必須培地を追加して、チューブを軽く叩いたり、簡単にボルテックスで迅速かつ十分に混ぜる。 picofugeで急速にスピンダウン。溶液は黄色になります。 可能な限り氷の上に置かないでください。 </li><li> （下記の注を参照）少しオレンジコラーゲン溶液を回すのに十分な0.8M炭酸水素ナトリウム（NaHCO 3を ）追加する。再び、フリックで急速に、徹底的にミックス、そしてpicofugeで簡単にスピンダウン。解決策は、混合後のピンク色のままの場合、あなたはあまりにも多くのNaHCO 3を追加し、コラーゲンの新しいチューブを再び開始する必要があります。 </li><li>この時点で、コラーゲン溶液が準備されている。それは氷の上に液体のままですが、室温時に持って約5分（ピンクを回す）内に固化される。 </li><li> 4ウェルNunc社製ディッシュの各ウェルにおけるコラーゲンのスポット20μlの。あなたのピペットの先端を使用して、小さな&quot;パッド&quot;を形成するコラーゲンを広げ。コラーゲンは常温で設定してみましょう。 </li></ol>注：のNaHCO 3の正確な量は、コラーゲンのバッチごとに滴定する必要があります。 （11μl）、低起動し、その溶液はオレンジ色のわずかな日陰になるまで0.51μlの段階を追加。 &quot;右の&quot;量は、通常、コラーゲンのピンクを回すというのNaHCO 3の最小量よりも少ない1ULです。 NaHCO 3の量は直線的に拡大または縮小していません。 パート4：背側脊髄の外植片とコラーゲンマトリックスにおけるCOSの細胞凝集体とそれらの位置の解剖<ol><li>新鮮なシャープタングステン針と細かいピンセット（＃5または＃55）を使用して、脊髄を囲む中胚葉を削除します。 </li><li>タングステン針で床の板に平行に切断、慎重に脊髄の腹側五分の一を削除する。 </li><li>エッジはようにきれいに可能な限りカットしていることを確認し、背側脊髄外植片を二等分する。必要に応じて、エッジを再トリム。約に引っ張らガラスキャピラリーチューブを取り付けた口のピペットを使用して、別のコラーゲンのパッドにそれぞれ背側脊髄外植片を移す。直径0.5ミリメートル。 </li><li>背側脊髄外植片の外側縁とほぼ同じ幅の正方形にトランスフェクトしたCOS細胞の集合体をカット。 </li><li>口のピペットを使用して、背側脊髄外植片とコラーゲンのパッド上にこれらの四角形の1つを転送する。 </li><li>口のピペットで、口のピペットを使用して余分な液体をオフに吸引除去する。吸引以上しないでください。 </li><li>パッドの上に下塗りコラーゲンの4μlを適用する、と、タングステン針を用いて、直接組織に触れることなく、外植片を介してコラーゲンを移動する。外植片を周囲のコラーゲンでタングステン針を移動させることにより、東洋植ので、COS細胞の集合体は背側脊髄外植片の側縁部に隣接していること。 </li><li>コラーゲンが設定された後、外植片はコラーゲンで完全に覆われていることを保証するためにコラーゲンの4μlのアプリケーションを繰り返す。 </li><li>組織培養フードの中で30〜40時間のOpti - MEM + 1xP/S/Gと文化の0.5ミリリットルを追加します。 </li><li>氷の上にプレートを保つ、1xPBSで0.5ミリリットルを4％パラホルムアルデヒドで45分間外植片を修正。 </li><li> 1xPBSのブロッキング溶液+ 0.1％のTriton - X100 +1％HIGS（PBTN）の0.5ミリリットルで2回洗浄。 4のブロックは、℃で時間の少なくともカップルのために、一晩のインキュベーションは免疫組織化学によってプロセスして望ましいとあります。 </li></ol>パート5：免疫組織化学による交連軸索の可視化<ol><li> PBTNでブロッキング後、鉗子と48ウェルディッシュの場所で4ウェルプレートの外植片を切り取る。 </li><li>各ウェルに一次抗体の200μlのを追加し、4℃で一晩おきます℃、交連軸索を可視化するために、マウス抗Tag1に抗体の午前1時06ソリューションを使用してください。シグナル伝達分子がタグ付けされたエピトープされている場合、トランスフェクションが成功したかどうかを判断するために、また、テストされているシグナル伝達分子のどちらかに対する一次抗体、または関連するエピトープが含まれています。 </li><li> 4でPBTNの5 × 1時間℃で各サンプルを洗う</li><li>各ウェルに二次抗体の200μlのを追加し、4℃で一晩おきます℃、抗Tag1に抗体を一次抗体として使用された場合、FITCまたはCy3のいずれかに結合したヤギ抗マウスIgM二次抗体を使用してください。 FITCまたはCy3標識に結合され、光に敏感な二次抗体を使用している場合、この時点でからホイルで覆われた48ウェルディッシュを保つ。 </li><li>各サンプル5 4で× 1時間PBTNの各℃を洗う</li><li> 3よくうつ病のスライドに植を転送する、Vectashield媒体とカバーガラスに余分な液体、マウントを取り外します。 4℃または-20℃のいずれかで光金庫に保管</li></ol>パート6：植の代表画像成功した実験では、外植片とCOS集計は互いに隣接して保持され、コラーゲンのセットと離れ離れになるではない。軸索の最低限の繁茂があるでしょう。重要な軸索の増殖は、背側脊髄外植片は、長すぎるために培養されたことを示します。外植片からの成長はblebsは存在しません。組織は、培養液に直接接触する場合ブレブ形成が発生します。 軸索の向きを変更するシグナル伝達分子の能力は、共焦点顕微鏡を用いて評価する。軸索の再配向の程度は、COS細胞の集合体3に最も近い軸索の方向転換の平均角度を測定することによって定量することができます。図1に示すように、COS細胞はmycタグを発現BMP7（青）は、屋根板の外植片が交連軸索（図をはじくための方法に似て離れてBMP7のソース（図1B）からTag1に+交連軸索（緑）を再設定する1A）。コントロールベクターを発現COS細胞は交連軸索3,4の方向には影響を与えません。これらの外植片は、運動ニューロンと背側介在を飾る転写因子ISL1 / 2（赤）、に対する抗体で標識した。この結果は、BMPファミリーのメンバーは、屋根板chemorepellent 3の活性を仲介する最初の兆候だ。 <img src="/files/ftp_upload/1853/1853fig1tmb.jpg" border="0" /> 図1：してください<a href="https://www.jove.com/files/ftp_upload/1853/1853fig1.jpg">こちらをクリックして</a>図1の拡大バージョンを参照すること。

<ol>
	<li>Altman, J., Bayer, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The development of the rat spinal cord. , (1984).</li><li>Placzek, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+recombinations+in+collagen+gels.">Tissue recombinations in collagen gels.</a> Methods in molecular biology. , 325-335 (2008).</li><li>Augsburger, A., Schuchardt, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BMPs+as+mediators+of+roof+plate+repulsion+of+commissural+neurons.">BMPs as mediators of roof plate repulsion of commissural neurons.</a> Neuron. 24, 127-141 (1999).</li><li>Butler, S. J., Dodd, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+role+for+BMP+heterodimers+in+roof+plate-mediated+repulsion+of+commissural+axons.">A role for BMP heterodimers in roof plate-mediated repulsion of commissural axons.</a> Neuron. 38, 389-401 (2003).</li><li>Kennedy, T. E., Serafini, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Netrins+are+diffusible+chemotropic+factors+for+commissural+axons+in+the+embryonic+spinal+cord.">Netrins are diffusible chemotropic factors for commissural axons in the embryonic spinal cord.</a> Cell. 78, 425-435 (1994).</li><li>Charron, F., Stein, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+morphogen+sonic+hedgehog+is+an+axonal+chemoattractant+that+collaborates+with+netrin-1+in+midline+axon+guidance.">The morphogen sonic hedgehog is an axonal chemoattractant that collaborates with netrin-1 in midline axon guidance.</a> Cell. 113, 11-23 (2003).</li><li>Lumsden, A. G., Davies, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemotropic+effect+of+specific+target+epithelium+in+the+developing+mammalian+nervous+system.">Chemotropic effect of specific target epithelium in the developing mammalian nervous system.</a> Nature. 323, 538-539 (1986).</li><li>Dodd, J., Morton, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+regulation+of+axonal+glycoprotein+expression+on+subsets+of+embryonic+spinal+neurons.">Spatial regulation of axonal glycoprotein expression on subsets of embryonic spinal neurons.</a> Neuron. 1, 105-116 (1988).</li><li>Yamauchi, K., Phan, K. D., Butler, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BMP+type+I+receptor+complexes+have+distinct+activities+mediating+cell+fate+and+axon+guidance+decisions.">BMP type I receptor complexes have distinct activities mediating cell fate and axon guidance decisions.</a> Development. 135, 1119-1128 (2008).</li></ol>

このアッセイを行うに成功したかどうかを判断する重要な要因が最初に、組織があまりにも長時間ディスパーゼで処理されるべきではないされ、そのような治療は、組織が非常に粘着性と生存率の減少となることになります。二、コラーゲンは完全にプライムし、可能な限り氷上で保存する必要があります。それが設定を開始する場合は、処理することはますます困難になる、ピンクターン?...

assaying the ability of diffusible signaling molecules to reorient embryonic spinal commissural axons

脊椎動物の脊髄1の背側交連軸索は軸索ガイダンスシグナルを識別するための貴重なモデルシステムとなっている。ここで、我々は、in vitroアッセイ、交連軸索の方向2に外因性と内因性シグナルの効果を研究するために広く用いられている&quot;方向転換アッセイ&quot;、 で説明します。このアッセイは、ジェーンドッド、トーマスJessellとアンドリューラムズデンの研究室で多数の人々によって開発されました（詳細については、確認応答を参照）、このアッセイのバージョンがでchemorepellent BMPを含む、主要な軸索ガイダンス分子の再配向の活動を示すために使用された屋根板3,4と脊髄での床の板でNetrin1 5とソニックヘッジホッグ（Shh）6 chemoattractive活動。 脊髄の背側三分の二の2-3のセグメントを構成する外植片は、胎生（E）11ラットから切除し、三次元コラーゲンゲル7で培養される。 E11背側脊髄外植片は、糖タンパク質、Tag1に8の彼らの軸索の表現によって識別することができる新たに生まれた交連ニューロンが含まれています。文化30〜40時間かけて、交連軸索の軌跡は、 生体内で見られるのと同様のタイムコースでこれらの背側外植片にrecapitulatedている。この軸索軌道は、テストの組織または背側外植片の側縁の一つと接触して候補シグナル伝達分子を発現するCOS細胞の集合のどちらかを置くことによって挑戦することができます。追加された組織の近傍に延在する交連軸索は、内因性の屋根板と異所性の側面組織からの信号の両方の影響を受けて成長します。交連軸索は、これらの状況下でreorientedされる程度を定量化することができる。このアッセイを使用して、それは両方が交連軸索3,4と同様交連軌道9を指示するためにこの信号のための必要性を新しい方向に向けるために特定の信号の十分性を調べることが可能です。

Watch this Scientific Journal Video about Assaying the Ability of Diffusible Signaling Molecules to Reorient Embryonic Spinal Commissural Axons at JoVE.com

Assaying the Ability of Diffusible Signaling Molecules to Reorient Embryonic Spinal Commissural Axons

このアッセイは、交連軸索の向きを変更、シグナル伝達分子、ここに骨形成タンパク質7（BMP7）の能力を評価する。胚の背側脊髄の外植片は、COS細胞の分泌候補増殖因子の集合体に隣接して培養する。外植片の中で成長しているReoriented交連軸索は、免疫組織化学により可視化。

胚脊髄交連軸索アンテナの向きに拡散性のシグナル伝達分子の能力を検定する

Dorsal commissural axons in the vertebrate spinal cord1 have been an invaluable model system in which to identify axon guidance signals.  Here, we ...

assaying-ability-diffusible-signaling-molecules-to-reorient-embryonic

Research

JoVE Journal

Biology

8.7K ビュー.  University of Southern California. このアッセイは、交連軸索の向きを変更、シグナル伝達分子、ここに骨形成タンパク質7（BMP7）の能力を評価する。胚の背側脊髄の外植片は、COS細胞の分泌候補増殖因子の集合体に隣接して培養する。外植片の中で成長しているReoriented交連軸索は、免疫組織化学により可視化。

ビデオ: Assaying the Ability of Diffusible Signaling Molecules to Reorient Embryonic Spinal Commissural Axons

Fabrication of a Microfluidic Device for the Compartmentalization of Neuron Soma and Axons

In this video, we demonstrate the technique of soft lithography with polydimethyl siloxane (PDMS) which we use to fabricate a microfluidic device for culturing neurons. Previously, a silicon wafer was patterned with the design for the neuron microfluidic device using SU-8 and photolithography to create a master mold, or what we simply refer to as a "master". Next, we pour the silicon polymer PDMS on top of the master which is then cured by heating the PDMS to 80&deg;C for 1 hour. The PDMS forms a negative mold of the device. The PDMS is then carefully cut and lifted away from the master. Holes are punched where the reservoirs will be and the excess PDMS trimmed away from the device. Nitrogen is used to blow away any excess debris from the device. At this point the devices are now ready for use and can either bonded to corning No. 1 cover glass with a plasma sterilizer/cleaner or can be reversibly bound to the cover glass by simply placing the device on top of the cover glass. The reversible bonding of the device to glass is covered in a separate video and requires first that the device be sterilized either with 70% ethanol or by autoclaving. Plasma treating sterilizes the devices so no further treatment is necessary. It is, however, important, when plasma-treating the devices, to add liquid to the devices within 10 minutes of the plasma treatment while the surfaces are still hydrophilic. Waiting longer than 10 minutes to add liquid to the device makes it difficult for the liquid to enter the device. The neuron devices are typically plasma-bound to cover glass and 0.5 mg/ml poly-L-lysine (PLL) in pH 8.5 borate buffer is immediately added to the device. After a minimum of 3 hours incubating with PLL, the devices are washed with dH2O water a minimum of 3 times with at least 15 minutes between each wash. Next, the water is removed and fresh media is added to the device. At this point the device is ready for use. It is important to remember at this point to never remove all the media from the device. Always leave media in the main channel.

In this video we demonstrate the technique of soft lithography with polydimethyl siloxane (PDMS) which we use to farbricate a microfluidic device for culturing neurons.

ニューロン相馬と軸索のコンパートメントのためのマイクロ流体デバイスの作製

In this video, we demonstrate the technique of soft lithography with polydimethyl siloxane (PDMS) which we use to fabricate a microfluidic device for ...

生物学

Spinal Cord Electrophysiology

The neonatal mouse spinal cord is a model for studying the development of neural circuitries and locomotor movement. We demonstrate the spinal cord dissection and preparation of recording bath artificial cerebrospinal fluid used for locomotor studies. Once dissected, the spinal cord ventral nerve roots can be attached to a recording electrode to record the electrophysiologic signals of the central pattern generating circuitry within the lumbar cord.

A demonstration of the isolation of neonatal mouse spinal cord for electrophysiologic studies.

脊髄電気生理学

The neonatal mouse spinal cord is a model for studying the development of neural circuitries and locomotor movement. We demonstrate the spinal cord ...

Mouse Embryonic Lung Culture, A System to Evaluate the Molecular Mechanisms of Branching

Lung primordial specification as well as branching morphogenesis, and the formation of various pulmonary cell lineages requires a specific interaction of the lung endoderm with its surrounding mesenchyme and mesothelium. Lung mesenchyme has been shown to be the source of inductive signals for lung branching morphogenesis. Epithelial-mesenchymal-mesothelial interactions are also critical to embryonic lung morphogenesis. Early embryonic lung organ culture is a very useful system to study epithelial-mesenchymal interactions. Both epithelial and mesenchymal morphogenesis proceeds under specific conditions that can be readily manipulated in this system (in the absence of maternal influence and blood flow). More importantly this technique can be readily done in a serumless, chemically defined culture media. Gain and loss of function can be achieved using expressed proteins, recombinant viral vectors and/or analysis of transgenic mouse strains, antisense RNA, as well as RNA interference gene knockdown.

Early embryonic lung organ culture is a very useful system to study epithelial-mesenchymal interactions. Both epithelial and mesenchymal morphogenesis proceeds under specific conditions that can be readily manipulated in this system.

マウス胎児肺の文化、分岐の分子機構を評価するシステム

Lung primordial specification as well as branching morphogenesis, and the formation of various pulmonary cell lineages requires a specific interaction of ...

Adenovirus-mediated Genetic Removal of Signaling Molecules in Cultured Primary Mouse Embryonic Fibroblasts

The ability to genetically remove specific components of various cell signalling cascades has been an integral tool in modern signal transduction analysis. One particular method to achieve this conditional deletion is via the use of the Cre-loxP system. This method involves flanking the gene of interest with loxP sites, which are specific recognition sequences for the Cre recombinase protein. Exposure of the so-called floxed (flanked by loxP site) DNA to this enzyme results in a Cre-mediated recombination event at the loxP sites, and subsequent excision of the intervening gene3. Several different methods exist to administer Cre recombinase to the site of interest. In this video, we demonstrate the use of an adenovirus containing the Cre recombinase gene to infect primary mouse embryonic fibroblasts (MEFs) obtained from embryos containing a floxed Rac1 allele1. Our rationale for selecting Rac1 MEFs for our experiments is that clear morphological changes can be seen upon deletion of Rac1, due to alterations in the actin cytoskeleton2,5. 72 hours following viral transduction and Cre expression, cells were stained using the actin dye phalloidin and imaged using confocal laser scanning microscopy. It was observed that MEFs which had been exposed to the adeno-Cre virus appeared contracted and elongated in morphology compared to uninfected cells, consistent with previous reports2,5. The adenovirus method of Cre recombinase delivery is advantageous as the adeno-Cre virus is easily available, and gene deletion via Cre in nearly 100% of the cells can be achieved with optimized adenoviral infection.

In this video we use an adenovirus carrying the Cre recombinase gene to infect primary mouse embryonic fibroblasts carrying a floxed Rac1 allele.

培養一次マウス胚線維芽細胞におけるシグナル伝達分子のアデノウイルス媒介遺伝子除去

The ability to genetically remove specific components of various cell signalling cascades has been an integral tool in modern signal transduction ...

Photobleaching Assays (FRAP &amp; FLIP) to Measure Chromatin Protein Dynamics in Living Embryonic Stem Cells

Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Loss In Photobleaching (FLIP) enable the study of protein dynamics in living cells with good spatial and temporal resolution. Here we describe how to perform FRAP and FLIP assays of chromatin proteins, including H1 and HP1, in mouse embryonic stem (ES) cells. In a FRAP experiment, cells are transfected, either transiently or stably, with a protein of interest fused with the green fluorescent protein (GFP) or derivatives thereof (YFP, CFP, Cherry, etc.). In the transfected, fluorescing cells, an intense focused laser beam bleaches a relatively small region of interest (ROI). The laser wavelength is selected according to the fluorescent protein used for fusion. The laser light irreversibly bleaches the fluorescent signal of molecules in the ROI and, immediately following bleaching, the recovery of the fluorescent signal in the bleached area - mediated by the replacement of the bleached molecules with the unbleached molecules - is monitored using time lapse imaging. The generated fluorescence recovery curves provide information on the protein's mobility. If the fluorescent molecules are immobile, no fluorescence recovery will be observed. In a complementary approach, Fluorescence Loss in Photobleaching (FLIP), the laser beam bleaches the same spot repeatedly and the signal intensity is measured elsewhere in the fluorescing cell. FLIP experiments therefore measure signal decay rather than fluorescence recovery and are useful to determine protein mobility as well as protein shuttling between cellular compartments. Transient binding is a common property of chromatin-associated proteins. Although the major fraction of each chromatin protein is bound to chromatin at any given moment at steady state, the binding is transient and most chromatin proteins have a high turnover on chromatin, with a residence time in the order of seconds. These properties are crucial for generating high plasticity in genome expression1. Photobleaching experiments are therefore particularly useful to determine chromatin plasticity using GFP-fusion versions of chromatin structural proteins, especially in ES cells, where the dynamic exchange of chromatin proteins (including heterochromatin protein 1 (HP1), linker histone H1 and core histones) is higher than in differentiated cells2,3.

Photobleaching Assays FRAP &amp; FLIP to Measure Chromatin Protein Dynamics in Living Embryonic Stem Cells

We describe photobleaching methods including Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Loss In Photobleaching (FLIP) to monitor chromatin protein dynamics in embryonic stem (ES) cells. Chromatin protein dynamics, which is considered to be one of the means to study chromatin plasticity, is enhanced in pluripotent cells.

胚性幹細胞を生体内でクロマチン蛋白質のダイナミクスを測定するアッセイ（FRAP＆FLIP）を光退色

Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Loss In Photobleaching (FLIP) enable the study of protein dynamics in living cells with ...

Assaying the Kinase Activity of LRRK2 in vitro

Leucine Rich Repeat Kinase 2 (LRRK2) is a 2527 amino acid member of the ROCO family of proteins, possessing a complex, multidomain structure including a GTPase domain (termed ROC, for Ras of Complex proteins) and a kinase domain1. The discovery in 2004 of mutations in LRRK2 that cause Parkinson's disease (PD) resulted in LRRK2 being the focus of a huge volume of research into its normal function and how the protein goes awry in the disease state2,3. Initial investigations into the function of LRRK2 focused on its enzymatic activities4-6. Although a clear picture has yet to emerge of a consistent alteration in these due to mutations, data from a number of groups has highlighted the importance of the kinase activity of LRRK2 in cell death linked to mutations7,8. Recent publications have reported inhibitors targeting the kinase activity of LRRK2, providing a key experimental tool9-11. In light of these data, it is likely that the enzymatic properties of LRRK2 afford us an important window into the biology of this protein, although whether they are potential drug targets for Parkinson's is open to debate.
A number of different approaches have been used to assay the kinase activity of LRRK2. Initially, assays were carried out using epitope tagged protein overexpressed in mammalian cell lines and immunoprecipitated, with the assays carried out using this protein immobilised on agarose beads4,5,7. Subsequently, purified recombinant fragments of LRRK2 in solution have also been used, for example a GST tagged fragment purified from insect cells containing residues 970 to 2527 of LRRK212. Recently, Dani&euml;ls et al. reported the isolation of full length LRRK2 in solution from human embryonic kidney cells, however this protein is not widely available13. In contrast, the GST fusion truncated form of LRRK2 is commercially available (from Invitrogen, see table 1 for details), and provides a convenient tool for demonstrating an assay for LRRK2 kinase activity. Several different outputs for LRRK2 kinase activity have been reported. Autophosphorylation of LRRK2 itself, phosphorylation of Myelin Basic Protein (MBP) as a generic kinase substrate and phosphorylation of an artificial substrate - dubbed LRRKtide, based upon phosphorylation of threonine 558 in Moesin - have all been used, as have a series of putative physiological substrates including &alpha;-synuclein, Moesin and 4-EBP14-17. The status of these proteins as substrates for LRRK2 remains unclear, and as such the protocol described below will focus on using MBP as a generic substrate, noting the utility of this system to assay LRRK2 kinase activity directed against a range of potential substrates.

Assaying the Kinase Activity of LRRK2 in vitro

Leucine Rich Repeat Kinase 2 is a large multidomain kinase, mutations in which are the most common genetic cause of Parkinson's disease. Analysis of the kinase activity of this protein has proven to be a crucial tool in understanding the biology and dysfunction of this protein. In this paper, in vitro assaying of the kinase activity of LRRK2 and a selection of its mutants is described, providing an experimental system to examine phosphorylation of putative substrates and potential dysfunction of LRRK2 in disease.

LRRK2のキナーゼ活性を測定する in vitroで</em

Leucine Rich Repeat Kinase 2 (LRRK2) is a 2527 amino acid member of the ROCO family of proteins, possessing a complex, multidomain structure including a ...

A System for ex vivo Culturing of Embryonic Pancreas

The pancreas controls vital functions of our body, including the production of digestive enzymes and regulation of blood sugar levels1. Although in the past decade many studies have contributed to a solid foundation for understanding pancreatic organogenesis, important gaps persist in our knowledge of early pancreas formation2. A complete understanding of these early events will provide insight into the development of this organ, but also into incurable diseases that target the pancreas, such as diabetes or pancreatic cancer. Finally, this information will generate a blueprint for developing cell-replacement therapies in the context of diabetes.
 During embryogenesis, the pancreas originates from distinct embryonic outgrowths of the dorsal and ventral foregut endoderm at embryonic day (E) 9.5 in the mouse embryo3,4. Both outgrowths evaginate into the surrounding mesenchyme as solid epithelial buds, which undergo proliferation, branching and differentiation to generate a fully mature organ2,5,6. Recent evidences have suggested that growth and differentiation of pancreatic cell lineages, including the insulin-producing &beta;-cells, depends on proper tissue-architecture, epithelial remodeling and cell positioning within the branching pancreatic epithelium7,8. However, how branching morphogenesis occurs and is coordinated with proliferation and differentiation in the pancreas is largely unknown. This is in part due to the fact that current knowledge about these developmental processes has relied almost exclusively on analysis of fixed specimens, while morphogenetic events are highly dynamic.
 Here, we report a method for dissecting and culturing mouse embryonic pancreatic buds ex vivo on glass bottom dishes, which allow direct visualization of the developing pancreas (Figure 1). This culture system is ideally devised for confocal laser scanning microscopy and, in particular, live-cell imaging. Pancreatic explants can be prepared not only from wild-type mouse embryos, but also from genetically engineered mouse strains (e.g. transgenic or knockout), allowing real-time studies of mutant phenotypes. Moreover, this ex vivo culture system is valuable to study the effects of chemical compounds on pancreatic development, enabling to obtain quantitative data about proliferation and growth, elongation, branching, tubulogenesis and differentiation. In conclusion, the development of an ex vivo pancreatic explant culture method combined with high-resolution imaging provides a strong platform for observing morphogenetic and differentiation events as they occur within the developing mouse embryo.

A System for ex vivo Culturing of Embryonic Pancreas

Here, we describe a method for isolation, culture and manipulation of mouse embryonic pancreas. This represents an excellent ex vivo system for studying various aspects of pancreatic development, including morphogenesis, differentiation and growth. Pancreatic bud explants can be cultured for several days and used in a range of different applications, including whole-mount immunofluorescence and live imaging.

のためのシステム<em&gt; ex vivoで</em胚膵臓&gt;培養

The pancreas controls vital functions of our body,  including the production of digestive enzymes and regulation of blood sugar  levels1. Although in the ...

Blastomere Explants to Test for Cell Fate Commitment During Embryonic Development

Fate maps, constructed from lineage tracing all of the cells of an embryo, reveal which tissues descend from each cell of the embryo. Although fate maps are very useful for identifying the precursors of an organ and for elucidating the developmental path by which the descendant cells populate that organ in the normal embryo, they do not illustrate the full developmental potential of a precursor cell or identify the mechanisms by which its fate is determined. To test for cell fate commitment, one compares a cell's normal repertoire of descendants in the intact embryo (the fate map) with those expressed after an experimental manipulation. Is the cell's fate fixed (committed) regardless of the surrounding cellular environment, or is it influenced by external factors provided by its neighbors? Using the comprehensive fate maps of the Xenopus embryo, we describe how to identify, isolate and culture single cleavage stage precursors, called blastomeres. This approach allows one to assess whether these early cells are committed to the fate they acquire in their normal environment in the intact embryo, require interactions with their neighboring cells, or can be influenced to express alternate fates if exposed to other types of signals.

The fate of an individual embryonic cell can be influenced by inherited molecules and/or by signals from neighboring cells. Utilizing fate maps of the cleavage stage Xenopus embryo, single blastomeres can be identified for culture in isolation to assess the contributions of inherited molecules versus cell-cell interactions.

胚発生時に細胞運命のコミットメントをテストするために割球植

Fate maps, constructed from lineage tracing all of the cells of an embryo, reveal  which tissues descend from each cell of the embryo.  Although fate maps ...

Mechanical Stimulation-induced Calcium Wave Propagation in Cell Monolayers: The Example of Bovine Corneal Endothelial Cells

Intercellular communication is essential for the coordination of physiological processes between cells in a variety of organs and tissues, including the brain, liver, retina, cochlea and vasculature. In experimental settings, intercellular Ca2+-waves can be elicited by applying a mechanical stimulus to a single cell. This leads to the release of the intracellular signaling molecules IP3 and Ca2+ that initiate the propagation of the Ca2+-wave concentrically from the mechanically stimulated cell to the neighboring cells. The main molecular pathways that control intercellular Ca2+-wave propagation are provided by gap junction channels through the direct transfer of IP3 and by hemichannels through the release of ATP. Identification and characterization of the properties and regulation of different connexin and pannexin isoforms as gap junction channels and hemichannels are allowed by the quantification of the spread of the intercellular Ca2+-wave, siRNA, and the use of inhibitors of gap junction channels and hemichannels. Here, we describe a method to measure intercellular Ca2+-wave in monolayers of primary corneal endothelial cells loaded with Fluo4-AM in response to a controlled and localized mechanical stimulus provoked by an acute, short-lasting deformation of the cell as a result of touching the cell membrane with a micromanipulator-controlled glass micropipette with a tip diameter of less than 1 &mu;m. We also describe the isolation of primary bovine corneal endothelial cells and its use as model system to assess Cx43-hemichannel activity as the driven force for intercellular Ca2+-waves through the release of ATP. Finally, we discuss the use, advantages, limitations and alternatives of this method in the context of gap junction channel and hemichannel research.

Intercellular Ca2+-waves are driven by gap junction channels and hemichannels. Here, we describe a method to measure intercellular Ca2+-waves in cell monolayers in response to a local single-cell mechanical stimulus and its application to investigate the properties and regulation of gap junction channels and hemichannels.

細胞単層に機械的刺激によって誘発されるカルシウム波の伝播：ウシ角膜内皮細胞の例

Intercellular communication is essential for the coordination of physiological processes between cells in a variety of organs and tissues, including the ...

Genome-wide Mapping of Drug-DNA Interactions in Cells with COSMIC (Crosslinking of Small Molecules to Isolate Chromatin)

The genome is the target of some of the most effective chemotherapeutics, but most of these drugs lack DNA sequence specificity, which leads to dose-limiting toxicity and many adverse side effects. Targeting the genome with sequence-specific small molecules may enable molecules with increased therapeutic index and fewer off-target effects. N-methylpyrrole/N-methylimidazole polyamides are molecules that can be rationally designed to target specific DNA sequences with exquisite precision. And unlike most natural transcription factors, polyamides can bind to methylated and chromatinized DNA without a loss in affinity. The sequence specificity of polyamides has been extensively studied in vitro with cognate site identification (CSI) and with traditional biochemical and biophysical approaches, but the study of polyamide binding to genomic targets in cells remains elusive. Here we report a method, the crosslinking of small molecules to isolate chromatin (COSMIC), that identifies polyamide binding sites across the genome. COSMIC is similar to chromatin immunoprecipitation (ChIP), but differs in two important ways: (1) a photocrosslinker is employed to enable selective, temporally-controlled capture of polyamide binding events, and (2) the biotin affinity handle is used to purify polyamide&#8211;DNA conjugates under semi-denaturing conditions to decrease DNA that is non-covalently bound. COSMIC is a general strategy that can be used to reveal the genome-wide binding events of polyamides and other genome-targeting chemotherapeutic agents.

Genome-wide Mapping of Drug-DNA Interactions in Cells with COSMIC Crosslinking of Small Molecules to Isolate Chromatin

Identifying the direct targets of genome-targeting molecules remains a major challenge. To understand how DNA-binding molecules engage the genome, we developed a method that relies on crosslinking of small molecules to isolate chromatin (COSMIC).