Name: co-localization of cell lineage markers and the tomato signal
Uploaded: 2016-12-28T14:00:00.000+00:00
Duration: 10 min 56 s
Description: Watch this Scientific Journal Video about Co-localization of Cell Lineage Markers and the Tomato Signal at JoVE.com

This study was supported by NIH grant DE025014 to JQF.

すべてのプロトコルは、歯科のテキサスA＆M大学で施設内動物管理使用委員会（IACUC）によってレビューされ、承認されました。 1.動物の繁殖<ol><li>本研究では3動物モデルを使用してください。顆形成に胚の軟骨細胞の運命を調査し、最初の使用Col10a1-Creを 15匹とのRosa26 tdTomatoでそれらを横断するには（B6; 129S6- Gtの（ROSA）26Sor TM9（CAG-tdTomato）Hze / J）マウスはCol10a1-を得るために、 CREとのRosa26 tdTomatoマウス。次に、2.3Col1a1-GFPマウス 16と、これらのマウスを渡ります。 Col10a1-Creをを使用します。 ROSA26 tdTomato; 2.3Col1a1-GFPマウスは、細胞系譜トレース実験（ 図1B）を実行します。 </li><li>出生後軟骨細胞の運命を研究するために、ステップ1.1と同様の手順に従いますが、 アグリカン-CREのERT2（ のAgg-CreをERT2を使用）17,18ラインとのAgg-CreをERT2を保ちます。 ROSA26 tdTomato; 2.3Col1a1-GFPマウスは、細胞系譜トレース実験（ 図1C）を実行します。生後14日目にタモキシフェンを注入します。 </li><li>免疫蛍光と技術をトレースする細胞系譜を結合するには、 のAgg-CreをERT2マウスとのRosa26 tdTomatoマウスを渡ります。実験で両方の遺伝子を運ぶマウスを使用し、生後3日目（ 図1D）にタモキシフェンを注入。 </li></ol> 2.材料の準備<ol><li> 10mg / mlの濃度で10％エタノールおよび90％のトウモロコシ油中タモキシフェン粉末を溶解します。注射のための投与量は75ミリグラム/ kgです。 </li><li> 7.4のpH値を調整するために2 M水酸化ナトリウムを使用して、4％の濃度にPBS中パラホルムアルデヒド（PFA）粉末を溶解します。屠殺後（下顎頭と後ろ足がここで使用されている）は、組織を固定するために、4％PFAのソリューションを使用してください。理由は、そのの毒性は、手袋、フェイスマスクとフードにPFAを扱います。 </li><li> 7.4のpH値を調整するために2 M水酸化ナトリウムを使用して、10％の濃度になるように蒸留水にEDTA粉末を溶解します。下顎頭と後肢を脱灰するために10％のEDTAを使用してください。 </li><li> 15％および30％の濃度で、PBS中のスクロース粉末を溶解します。脱灰後の組織を脱水するためにショ糖溶液を使用してください。 </li><li> 2 mg / mlで、pHが5.0の濃度でPBS中のヒアルロニダーゼの粉末を溶かします。このソリューションは、免疫抗原回復のためのものです。 </li><li> Runx2のまたはDMP1免疫蛍光染色のためにPBS中の3％ウシ血清アルブミン（BSA）、20％ヤギ血清を含有するブロッキング溶液を調製し、1.5 mlのチューブを使用します。 </li><li> Runx2のかDMP1免疫蛍光染色のために、PBS中の2％ヤギ血清が含まれている一次抗体溶液を準備するために1.5 mlチューブを使用してください。 Runx2のための400および1：100 DMP1のための一次抗体の濃度が1です。 </li><li>準備するために1.5 mlチューブを使用します偽陽性の結果を回避するために、免疫蛍光染色のための対照としてのウサギIgG溶液。液をPBS中の2％ヤギ血清が含まれています。 Runx2の制御のためのウサギIgGの濃度は1：400; DMP1は、1：100。同時に実験およびコントロール染色を実行します。 </li><li> Runx2のまたはDMP1免疫蛍光染色のためにPBS中の2％ヤギ血清を含有する二次抗体溶液を調製する1.5mlチューブを使用します。 500：1の希釈で二次抗体を使用してください。 </li></ol> 3.スライドさせて共焦点顕微鏡のための準備（のみトレース細胞系統、免疫蛍光ではありませんコンビネーション） <ol><li>有利な時点でのAgg-CRE ERT2マウスではタモキシフェンを注入します。 <ol><li>まず、そのケージからマウスを削除します。その後、腹部を露出させ、マウスの背中の皮膚を取得し、それをひっくり返すために左手の親指と人差し指を使用しています。シリンジを保持するために右手を使用してください。注射のための最適なエントリー・ポイントはリットルでありますEFTや下腹部の右側に、肝臓や膀胱を回避することができます。 </li><li>マウスの後肢に注射器を平行に保ち、腹腔内に注入します。注射のための投与量は75ミリグラム/ kgです。それぞれ、18グラム - 9グラム、11 - - 13グラム、および16の2週間、3週間、および4週齢の年齢のマウスの重量は約7です。 </li></ol></li><li>キシラジン/ Ketasetの組み合わせでマウスを麻酔。 <ol><li>作業溶液を調製するには、まず、それぞれ、1mg / mlのおよび5mg / mlの濃度に蒸留水でキシラジンとKetasetを希釈します。 2比：次に、1でキシラジンとKetasetを混ぜます。注射のための投与量は30μL/ gです。 </li><li>ステップ3.1のように注入します。マウスの足首をつまんで麻酔を確認してください。それは何の反応を持っていない場合は、マウスは無意識です。 </li></ol></li><li>麻酔した後、4％PFAでマウスを灌流。 <ol><li>マウスは意識を失った後、entirelするボード上でマウスの4脚を固定しますyは腹部を露出させます。 </li><li> 70％エタノールで腹部を飽和させ、中央の線に沿って首に下腹部から切除を行います。ピンチと同時に腹膜を明らかにするために側面に皮膚を引っ張ります。縦方向の切除を行うために解剖ハサミを使用してください。 </li><li>カットオフと心臓を露出するためにフロントリブを削除します。 、22 Gシリンジで左心室から心臓に穴を開け、シリンジを保持し、同時に右心耳にスロットを切りました。 </li><li>ゆっくりと右心耳からのカットから血液をフラッシュしながら、心血管系に沿って灌流される4％PFAを、注入します。灌流のためのPFAの体積を1ml /グラムです。ハードダクト建物の排気システムにあるクラスI生物学的安全キャビネットの中で、この手順を実行します。 </li></ol></li><li>ピールマウスの皮膚を切り、4℃で一晩固定するための4％PFAの40ミリリットルが含まれている50-mlのポリプロピレン製遠心管に全身を置きます。 </li><li>慎重に身体から下顎骨と後肢を除去し、表面に筋肉や腱を除去するために、解剖ハサミと＃3、＃5鉗子を使用してください。ハードダクト建物の排気システムにあるクラスI生物学的安全キャビネットの中で、この手順を実行します。 </li><li>第三大臼歯の先端領域で、2枚に下顎をカット。同様に、脱灰を促進するために、骨髄腔を露出させるために中間シャフトに大腿骨および脛骨を切断します。顆と関節丘処理を含む部分を入れて、10％のEDTAの40ミリリットルに後ろ足と一緒に2、4℃で脱灰するために - 50-mlのポリプロピレン遠心管中で4日間。 </li><li> 50mlのポリプロピレン遠心管中で4℃で一晩顆と後肢を脱水し、15％ショ糖の50ミリリットルを使用してください。 </li><li> 50mlのポリプロピレン遠心管中で4℃で一晩顆と後肢を脱水し、30％ショ糖を使用してください。 </li><li>矢状面に沿って、で試料を埋め込みます凍結切片機で切削プレート上の10月<ol><li>水平顆またはマウント型内の後ろ足を置きます。 10月に組織を水没し、10月がフリーズするまで凍結切片機にそれを残します。 </li><li>切削プレート上で10月ブロックをマウントします。 10月は完全に凍結されていることを確実にするために切断する前に、約15分間待ちます。 </li></ol></li><li> 10-μmの切片に顆と後肢をカットします。 -20℃でスライドや店舗のセクションを収集します。 </li><li>染色の前に水を除去するために、37℃の室内でスライドをインキュベートします。 </li><li> 5分間蒸留水で2回スライドを洗浄します。 </li><li>各セクションの周りに水を拭き取り。セクションを一周し、円形にDAPIまたは非蛍光退色防止マウントソリューションを削除するには、疎水性のバリアペンを使用してください。慎重にカバースリップを置きます。 </li></ol> Runx2のとDMP1 4.免疫組織化学染色注：IgGのコン免疫組織化学染色は、偽陽性シグナルを回避するトロールが必要です。実験群および対照群についての染色を同時に行う必要があります。 <ol><li>染色の前に水を除去するために37℃のチャンバー内でスライドをインキュベートします。 </li><li>蒸留水で2回スライドを洗浄します。 </li><li>スライド上のすべてのセクションを一周する疎水性バリアペンを使用してください。この段階から、完全にセクションをカバーするために円形に調製した溶液のすべてを追加します。 </li><li> 37℃で30分間加湿チャンバー内にヒアルロニダーゼを持つセクションを扱います。 （ステップ4.5 - 4.8）溶液の体積は、セクションのサイズによって異なります。顆と長骨のために100μlのための溶液50μlを使用してください。 3回（0.1％のTween 20を含んでいるPBS）PBSTで洗浄します。 </li><li>準備し、各セクションにブロッキング溶液を塗布し、室温で1時間加湿チャンバーでそれらをインキュベートします。 </li><li>プライマリを持つセクションをインキュベート一晩4℃で抗体溶液（ウサギ抗マウスRunx2の、またはウサギ抗マウスDMP1）。 PBSで3回洗浄します。 </li><li>室温で2時間、二次抗体溶液（ヤギ抗ウサギアレクサフルオロ488）を用いて切片をインキュベートします。 PBSで3回洗浄します。 </li><li>セクションの周りに水を拭き取り、スライド上のセクションをカバーするために円形にDAPIをドロップします。慎重にカバースリップを置きます。 </li></ol> 5.共焦点顕微鏡<ol><li> 488ミクロン（緑）から561ミクロン（赤）の範囲の波長で共焦点顕微鏡を用いた蛍光細胞画像をキャプチャします。 10X、20X、および63Xレンズを使用して、19 200 Hzで、複数の積み重ねられた画像を撮影（1024×1024の寸法）。 </li></ol>

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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tamoxifen+induces+rapid,+reversible+atrophy,+and+metaplasia+in+mouse+stomach.">Tamoxifen induces rapid, reversible atrophy, and metaplasia in mouse stomach.</a> Gastroenterology. 142 (1), 21-24 (2012).</li><li>Lee, M. H., Kim, J. W., Kim, J. H., Kang, K. S., Kong, G., Lee, M. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+expression+profiling+of+murine+hepatic+steatosis+induced+by+tamoxifen.">Gene expression profiling of murine hepatic steatosis induced by tamoxifen.</a> Toxicol Lett. 199 (3), 416-424 (2010).</li><li>Nakamura, E., Nguyen, M. T., Mackem, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetics+of+tamoxifen-regulated+Cre+activity+in+mice+using+a+cartilage-specific+CreER(T)+to+assay+temporal+activity+windows+along+the+proximodistal+limb+skeleton.">Kinetics of tamoxifen-regulated Cre activity in mice using a cartilage-specific CreER(T) to assay temporal activity windows along the proximodistal limb skeleton.</a> Dev Dyn. 235 (9), 2603-2612 (2006).</li></ol>

The authors declare that they have no competing financial interests or other conflicts of interest.

技術的制限のために、 インビボでの細胞の挙動を研究することは常に困難です。しかし、細胞系譜トレース技術は、細胞生物学7-9を研究するための強力なツールであることが証明されています。本研究では、さらに、免疫蛍光法と組み合わせることにより、このプロトコールを改良します。この方法では、細胞の運命は、系統追跡の適用を拡大する、複数の関連マーカーによ?...

開発中に、軟骨内骨形成は、骨格の体積の80％以上を占めています。広く、それが肥大軟骨細胞のアポトーシスから始まると考えられています。続いて、下層の骨髄由来の細胞が侵入し、骨髄および骨膜由来の細胞1,2による新たな骨沈着に続いて血管新生を、開始します。肥大軟骨細胞（HCS）の細胞の運命は、しかし、数十年の3のための議論の課題となっています。最初に、のHCは、軟骨細胞分化経路の末端であるとみなされ、アポトーシスは、一般のHCの最終的な運命であると考えられました。今、一部の研究者は、少なくとも一部のHCが生き残ると軟骨内骨形成に寄与し得ることを示唆しています。彼らは成長板軟骨細胞は、超微細構造に基づいて、骨芽細胞に分化転換する能力、免疫組織化学染色を持っていたし、in vitroで 、これらの方法のいずれもが、ワット、46を研究することを提案したが、骨芽細胞系への軟骨細胞の寄与を実証するに決定的なERE。 細胞系譜トレース技術は、細胞の運命を研究するためのより厳密な方法を提供します。簡単に言えば、唯一の細胞の特定の型で表現されるリコンビナーゼ酵素は、レポーター遺伝子の発現を刺激します。このように、この細胞の種類とその子孫は永久に7のラベルが付いています 。 Cre-loxP系は、一般に系統追跡に使用されます。 CRE（リコンビナーゼ酵素）は、2つのloxP部位の間にSTOPシーケンスを切除し、特定の細胞株（ 図1A）でレポーターを活性化します。いくつかのケースでは、研究者は、Creは、エストロゲン受容体（CreをERT2）8の修正された形に融合させ、タモキシフェンなど、薬剤を使用してのCreを活性化するために有利な時点を選択することができます。蛍光レポーターは、彼らが劇的に複雑さを軽減するための実験をトレースする系統における標準となっていますそして、8,9をトレース細胞の運命の精度と効率を向上させます。それはそれは簡単に7（ 図1A）を可視化すること、明るい蛍光タンパク質と最強のエピ蛍光を有しているためtdTomatoは、蛍光レポーターの中で最良の選択となってきています。 トレースシステムのRosa26 tdTomato系統を使用することにより、当社グループおよび他の研究者は、HCSは、開発10-14の間に骨細胞へのそれらの表現型を変更することができることを示しています。これを達成するために、我々は、Rosa26遺伝子tdtomato（全ての細胞中に遍在式）/のCre（軟骨細胞に特異的な）マウスとの組み合わせを追跡する二組の開発：2.3Col1a1-GFP（骨芽細胞に特異的）および免疫蛍光（骨細胞に特異的）。データは、両方の方法は、 インビボでの細胞の運命を研究するための実行可能な方法であることを示しています。

<table><tbody><tr><td>Tamoxifen&amp;nbsp;</td><td>Sigma</td><td>T5648</td><td>activate the Cre event&amp;nbsp;</td></tr><tr><td>Paraformaldehyde</td><td>Sigma&amp;nbsp;</td><td>P6148</td><td>fix the sample</td></tr><tr><td>Ethylenediaminetetraacetic acid</td><td>Alfa Aesar</td><td>A10713</td><td>decalcify the hard tissue</td></tr><tr><td>Sucrose&amp;nbsp;</td><td>Sigma</td><td>S0389</td><td>dehydrate the tissue</td></tr><tr><td>Hyaluronidase from bovine testes&amp;nbsp;</td><td>Sigma</td><td>H4272</td><td>retrieve antigen for immunochemical staining</td></tr><tr><td>Bovine serum albumin</td><td>Sigma&amp;nbsp;</td><td>A3059</td><td>blocking solution&amp;nbsp;</td></tr><tr><td>primary antibody for Runx2&amp;nbsp;</td><td>Cell Signal</td><td>D1L7F</td><td>primary antibody for immunochemical staining</td></tr><tr><td>primary antibody for DMP1</td><td>provided by Dr. Chunlin Qin</td><td></td><td>primary antibody for immunochemical staining</td></tr><tr><td>anti-rabbit IgG</td><td>Sigma</td><td>18140</td><td>control for immunochemical staining</td></tr><tr><td>secondary antibody&amp;nbsp;</td><td>Invitrogen</td><td>A11008</td><td>second antibody for immunochemical staining</td></tr><tr><td>OCT</td><td>Tissue-Tek</td><td>4583</td><td>embed the sample for frozen section</td></tr><tr><td>Tween 20</td><td>Fisher Scientific</td><td>BP337</td><td>PBST</td></tr><tr><td>non-fluorescing antifade mountant</td><td>Life technologies</td><td>P36934</td><td>mounting slides</td></tr><tr><td>DAPI</td><td>Life technologies</td><td>P36931</td><td>nuclear staining</td></tr><tr><td>Hydrophobic Barrier Pen</td><td>Vector Laboratories</td><td></td><td>circle the section on the slide for for immunochemical staining&amp;nbsp;</td></tr><tr><td>Xylazine</td><td>AnaSed</td><td></td><td>anesthetization&amp;nbsp;</td></tr><tr><td>Ketaset&amp;nbsp;</td><td>Zoetis</td><td></td><td>anesthetization&amp;nbsp;</td></tr><tr><td>cryosection machine</td><td>Leica</td><td>CM1860 UV</td><td></td></tr><tr><td>confocal microscope</td><td>Leica</td><td>DM6000 CFS&amp;nbsp;</td><td></td></tr></tbody></table>

co-localization of cell lineage markers and the tomato signal

細胞系統追跡システムは、発生生物学研究に主に使用されてきました。 Creリコンビナーゼの使用は、特定の細胞株およびすべての子孫におけるレポーターの活性化を可能にします。ここでは、軟骨細胞は、直接のCre、Col10a1-のCreおよびアグリカン-CreをERT2（ のAgg-CreをERT2）の2種類を使用して、長い骨と下顎頭の開発中に骨芽細胞および骨細胞に変身することを実証するための技術をトレースする細胞系譜を使用し、Rosa26遺伝子と交配tdTomato。 Col10とアグリカンの両方が軟骨細胞のためのマーカーをよく認識しています。 これに基づき、我々は、蛍光免疫組織化学-する特定の細胞マーカーの発現を分析することによって、細胞の運命を定義すると併せてトレース新しい方法細胞系統を開発しました。 Runx2の（早期骨形成細胞のマーカー）および象牙質マトリックスprotein1（DMP1;後期骨形成細胞のマーカー）しました。軟骨細胞由来の骨細胞およびそれらの分化状態を識別するために使用されます。この組み合わせは、細胞系統のトレースの適用を広げるだけでなく、化合物をマウスの生成を簡素化するだけでなく。さらに重要なことは、親細胞の子孫の数、位置、および分化の状態だけではトレース細胞系統よりも多くの情報を提供し、同時に表示されます。結論として、細胞系譜トレース技術および免疫蛍光の同時適用は、in vivoで細胞生物学を研究するための強力なツールです。

軟骨細胞は直接下顎頭と長骨における骨細胞（骨芽細胞および骨細胞）に変換します。 アグリカン 、軟骨形成のための重要な遺伝子は、主に早期に成熟した軟骨細胞18で表されます。その結果、 のAgg-CreをERT2年齢の2週間でのタモキシフェンの注射; ROSA26 tdTomatoマウス...

Watch this Scientific Journal Video about Co-localization of Cell Lineage Markers and the Tomato Signal at JoVE.com

Co-localization of Cell Lineage Markers and the Tomato Signal

2.3Col1a1-GFP（骨芽細胞に特異的）で1と（骨細胞に特異的）免疫蛍光との1：私たちは（特に軟骨細胞で発現さ）/のCreマウス（遍在すべての細胞で発現）Rosa26遺伝子tdtomatoのトレースの組み合わせの2セットを開発しました。データは、骨細胞への軟骨細胞の直接形質転換を実証します。

細胞系譜マーカーの共局在とトマト信号

The cell lineage tracing system has been used predominantly in developmental biology studies. The use of Cre recombinase allows for the activation of the ...

co-localization-of-cell-lineage-markers-and-the-tomato-signal

Research

JoVE Journal

Developmental Biology

12.1K ビュー.  Texas A&M University College of Dentistry.  2.3Col1a1-GFP（骨芽細胞に特異的）で1と（骨細胞に特異的）免疫蛍光との1：私たちは（特に軟骨細胞で発現さ）/のCreマウス（遍在すべての細胞で発現）Rosa26遺伝子tdtomatoのトレースの組み合わせの2セットを開発しました。データは、骨細胞への軟骨細胞の直接形質転換を実証します。 

ビデオ: Co-localization of Cell Lineage Markers and the Tomato Signal

A Method for Lineage Tracing of Corneal Cells Using Multi-color Fluorescent Reporter Mice

Lineage tracing experiments define the origin, fate and behavior of cells in a specific tissue or organism. This technique has been successfully applied for many decades, revealing seminal findings in developmental biology. More recently, it was adopted by stem cell biologists to identify and track different stem cell populations with minimal experimental intervention. The recent developments in mouse genetics, the availability of a large number of mouse strains, and the advancements in fluorescent microscopy allow the straightforward design of powerful lineage tracing systems for various tissues with basic expertise, using commercially available tools. We have recently taken advantage of this powerful methodology to explore the origin and fate of stem cells at the ocular surface using R26R-Confetti mouse. This model offers a multi-color genetic system, for the expression of 4 fluorescent genes in a random manner. Here we describe the principles of this methodology and provide an adaptable protocol for designing lineage tracing experiments; specifically for the corneal epithelium as well as for other tissues.

In this article we describe the principles for designing and performing a multi-color lineage tracing experiment using R26R-Confetti mice. We provide a specific protocol for tracking corneal epithelial cells which can be modified for other tissues of interest.

マルチカラー蛍光レポーターマウスを用いた角膜細胞の系譜トレースする方法

Lineage tracing experiments define the origin, fate and behavior of cells in a specific tissue or organism. This technique has been successfully applied ...

発生生物学

Cell Lineage Analyses and Gene Function Studies Using Twin-spot MARCM

Mosaic analysis with a repressible cell marker (MARCM) is a positive mosaic labeling system that has been widely applied in Drosophila neurobiological studies to depict intricate morphologies and to manipulate the function of genes in subsets of neurons within otherwise unmarked and unperturbed organisms. Genetic mosaics generated in the MARCM system are mediated through site-specific recombination between homologous chromosomes within dividing precursor cells to produce both marked (MARCM clones) and unmarked daughter cells during mitosis. An extension of the MARCM method, called twin-spot MARCM (tsMARCM), labels both of the twin cells derived from a common progenitor with two distinct colors. This technique was developed to enable the retrieval of useful information from both hemi-lineages. By comprehensively analyzing different pairs of tsMARCM clones, the tsMARCM system permits high-resolution neural lineage mapping to reveal the exact birth-order of the labeled neurons produced from common progenitor cells. Furthermore, the tsMARCM system also extends gene function studies by permitting the phenotypic analysis of identical neurons of different animals. Here, we describe how to apply the tsMARCM system to facilitate studies of neural development in Drosophila.

Here, we present a protocol for a mosaic labeling technique that permits the visualization of neurons derived from a common progenitor cell in two distinct colors. This facilitates neural lineage analysis with the capability of birth-dating individual neurons and studying gene function in the same neurons of different individuals.

ツインスポットMARCMを使用して細胞系譜の解析と遺伝子機能研究

Mosaic analysis with a repressible cell marker (MARCM) is a positive mosaic labeling system that has been widely applied in Drosophila neurobiological ...

Confocal Live Imaging of Shoot Apical Meristems from Different Plant Species

The shoot apical meristem (SAM) functions as a conserved stem cell reservoir and it generates almost all aboveground tissues during the postembryonic development. The activity and morphology of SAMs determine important agronomic traits, such as shoot architecture, size and number of reproductive organs, and most importantly, grain yield. Here, we provide a detailed protocol for analyzing both the surface morphology and the internal cellular structure of the living SAMs from different species through laser scanning confocal microscope. The whole procedure from the sample preparation to the acquisition of high resolution three-dimensional (3D) images can be accomplished within as short as 20 minutes. We demonstrate that this protocol is highly efficient for studying not only the inflorescence SAMs of the model species but also the vegetative meristems from different crops, providing a simple but powerful tool to study the organization and development of meristems across different plant species.

This protocol presents how to live image and analyze the shoot apical meristems from different plant species using laser scanning confocal microscopy.

別の植物種から茎頂の共焦点のライブ イメージング

The shoot apical meristem (SAM) functions as a conserved stem cell reservoir and it generates almost all aboveground tissues during the postembryonic ...

Visualization of IL-22-expressing Lymphocytes Using Reporter Mice

Reporter mice have been widely used to observe the localization of expression of targeted genes. This protocol focuses on a strategy to establish a new transgenic reporter mouse model. We chose to visualize interleukin (IL) 22 gene expression because this cytokine has important activities in the intestine, where it contributes to repair tissues damaged by inflammation. Reporter systems offer considerable advantages over other methods of identifying products in vivo. In the case of IL-22, other studies had first isolated cells from tissues and then re-stimulated the cells in vitro. IL-22, which is normally secreted, was trapped inside cells using a drug, and intracellular staining was used to visualize it. This method identifies cells capable of producing IL-22, but it does not determine whether they were doing so in vivo. The reporter design includes inserting a gene for a fluorescent protein (tdTomato) into the IL-22 gene in such a way that the fluorescent protein cannot be secreted and therefore remains trapped inside the producing cells in vivo. Fluorescent producers can then be visualized in tissue sections or by ex vivo analysis through flow cytometry. The actual construction process for the reporter included recombineering a bacterial artificial chromosome that contained the IL-22 gene. This engineered chromosome was then introduced into the mouse genome. Homeostatic IL-22 reporter expression was observed in different mouse tissues, including the spleen, thymus, lymph nodes, Peyer&#39;s patch, and intestine, by flow cytometry analysis. Colitis was induced by T-cell (CD4+CD45RBhigh) transfer, and reporter expression was visualized. Positive T cells were first present in the mesenteric lymph nodes, and then they accumulated inside the lamina propria of the distal small intestine and colon tissues. The strategy using BACs gave good-fidelity reporter expression compared to IL-22 expression, and it is simpler than knock-in procedures.

We describe here a transgenic reporter mouse model to visualize the IL-22-producing cells inside different mouse tissues. This method can be used to track the location of other cytokines or secretary proteins in the mouse.

レポーターマウスを用いたIL-22を発現するリンパ球の可視化

Reporter mice have been widely used to observe the localization of expression of targeted genes. This protocol focuses on a strategy to establish a new ...

免疫・感染学

Identification of Plasmodesmal Localization Sequences in Proteins In Planta

Plasmodesmata (Pd) are cell-to-cell connections that function as gateways through which small and large molecules are transported between plant cells. Whereas Pd transport of small molecules, such as ions and water, is presumed to occur passively, cell-to-cell transport of biological macromolecules, such proteins, most likely occurs via an active mechanism that involves specific targeting signals on the transported molecule. The scarcity of identified plasmodesmata (Pd) localization signals (PLSs) has severely restricted the understanding of protein-sorting pathways involved in plant cell-to-cell macromolecular transport and communication. From a wealth of plant endogenous and viral proteins known to traffic through Pd, only three PLSs have been reported to date, all of them from endogenous plant proteins. Thus, it is important to develop a reliable and systematic experimental strategy to identify a functional PLS sequence, that is both necessary and sufficient for Pd targeting, directly in the living plant cells. Here, we describe one such strategy using as a paradigm the cell-to-cell movement protein (MP) of the Tobacco mosaic virus (TMV). These experiments, that identified and characterized the first plant viral PLS, can be adapted for discovery of PLS sequences in most Pd-targeted proteins.

Identification of Plasmodesmal Localization Sequences in Proteins In Planta

Plant intercellular connections, the plasmodesmata (Pd), play central roles in plant physiology and plant-virus interactions. Critical to Pd transport are sorting signals that direct proteins to Pd. However, our knowledge about these sequences is still in its infancy. We describe a strategy to identify Pd localization signals in Pd-targeted proteins.

タンパク質Planta Plasmodesmal ローカリゼーション シーケンスの同定

Plasmodesmata (Pd) are cell-to-cell connections that function as gateways through which small and large molecules are transported between plant cells. ...

In Situ Hybridization for the Precise Localization of Transcripts in Plants

With the advances in genomics research of the past decade, plant biology has seen numerous studies presenting large-scale quantitative analyses of gene expression. Microarray and next generation sequencing approaches are being used to investigate developmental, physiological and stress response processes, dissect epigenetic and small RNA pathways, and build large gene regulatory networks1-3. While these techniques facilitate the simultaneous analysis of large gene sets, they typically provide a very limited spatiotemporal resolution of gene expression changes. This limitation can be partially overcome by using either profiling method in conjunction with lasermicrodissection or fluorescence-activated cell sorting4-7. However, to fully understand the biological role of a gene, knowledge of its spatiotemporal pattern of expression at a cellular resolution is essential. Particularly, when studying development or the effects of environmental stimuli and mutants can the detailed analysis of a gene's expression pattern become essential. For instance, subtle quantitative differences in the expression levels of key regulatory genes can lead to dramatic phenotypes when associated with the loss or gain of expression in specific cell types. 
Several methods are routinely used for the detailed examination of gene expression patterns. One is through analysis of transgenic reporter lines. Such analysis can, however, become time-consuming when analyzing multiple genes or working in plants recalcitrant to transformation. Moreover, an independent validation to ensure that the transgene expression pattern mimics that of the endogenous gene is typically required. Immunohistochemical protein localization or mRNA in situ hybridization present relatively fast alternatives for the direct visualization of gene expression within cells and tissues. The latter has the distinct advantage that it can be readily used on any gene of interest. In situ hybridization allows detection of target mRNAs in cells by hybridization with a labeled anti-sense RNA probe obtained by in vitro transcription of the gene of interest. 
Here we outline a protocol for the in situ localization of gene expression in plants that is highly sensitivity and specific. It is optimized for use with paraformaldehyde fixed, paraffin-embedded sections, which give excellent preservation of histology, and DIG-labeled probes that are visualized by immuno-detection and alkaline-phosphatase colorimetric reaction. This protocol has been successfully applied to a number of tissues from a wide range of plant species, and can be used to analyze expression of mRNAs as well as small RNAs8-14.

In Situ Hybridization for the Precise Localization of Transcripts in Plants

The in situ hybridization protocol described here allows a direct localization of mRNA and small RNA expression at the cellular level with high sensitivity and specificity. The procedure is optimized for paraffin-embedded plant tissue sections, is applicable to a wide range of plants and tissues, and can be completed within ten days.

植物の転写産物の正確な位置のためのin situハイブリダイゼーション

With the advances in genomics research of the past decade, plant biology has seen numerous studies presenting large-scale quantitative analyses of gene ...

生物学

Cell Specific Analysis of Arabidopsis Leaves Using Fluorescence Activated Cell Sorting

After initiation of the leaf primordium, biomass accumulation is controlled mainly by cell proliferation and expansion in the leaves1. However, the Arabidopsis leaf is a complex organ made up of many different cell types and several structures. At the same time, the growing leaf contains cells at different stages of development, with the cells furthest from the petiole being the first to stop expanding and undergo senescence1. Different cells within the leaf are therefore dividing, elongating or differentiating; active, stressed or dead; and/or responding to stimuli in sub-sets of their cellular type at any one time. This makes genomic study of the leaf challenging: for example when analyzing expression data from whole leaves, signals from genetic networks operating in distinct cellular response zones or cell types will be confounded, resulting in an inaccurate profile being generated.
To address this, several methods have been described which enable studies of cell specific gene expression. These include laser-capture microdissection (LCM)2 or GFP expressing plants used for protoplast generation and subsequent fluorescence activated cell sorting (FACS)3,4, the recently described INTACT system for nuclear precipitation5 and immunoprecipitation of polysomes6.
FACS has been successfully used for a number of studies, including showing that the cell identity and distance from the root tip had a significant effect on the expression profiles of a large number of genes3,7. FACS of GFP lines have also been used to demonstrate cell-specific transcriptional regulation during root nitrogen responses and lateral root development8, salt stress9 auxin distribution in the root10 and to create a gene expression map of the Arabidopsis shoot apical meristem11. Although FACS has previously been used to sort Arabidopsis leaf derived protoplasts based on autofluorescence12,13, so far the use of FACS on Arabidopsis lines expressing GFP in the leaves has been very limited4. In the following protocol we describe a method for obtaining Arabidopsis leaf protoplasts that are compatible with FACS while minimizing the impact of the protoplast generation regime. We demonstrate the method using the KC464 Arabidopsis line, which express GFP in the adaxial epidermis14, the KC274 line, which express GFP in the vascular tissue14 and the TP382 Arabidopsis line, which express a double GFP construct linked to a nuclear localization signal in the guard cells (data not shown; Figure 2). We are currently using this method to study both cell-type specific expression during development and stress, as well as heterogeneous cell populations at various stages of senescence.

Cell Specific Analysis of Arabidopsis Leaves Using Fluorescence Activated Cell Sorting

A method for producing Arabidopsis leaf protoplasts that are compatible with fluorescence activated cell sorting (FACS), allowing for studies of specific cell populations. This method is compatible with any Arabidopsis line that expresses GFP in a subset of cells.

の細胞特異的な分析<em&gt;シロイヌナズナ</em&gt;蛍光活性化細胞選別を使用した葉

After initiation of the leaf primordium, biomass accumulation is controlled mainly by cell proliferation and expansion in the leaves1. However, the ...

Lineage Tracing of Inducible Fluorescently-Labeled Stem Cells in the Adult Mouse Brain

A telomerase reverse transcriptase (Tert) lineage-tracing mouse line was developed to investigate the behavior and fate of adult tissue stem cells, by crossing the &#39;Tet-On&#39; system oTet-Cre mouse with a novel reverse tetracycline transactivator (rtTA) transgene linked to the Tert promoter, which we have demonstrated marks a novel population of adult brain stem cells. Here, administration of the tetracycline derivative doxycycline to mTert-rtTA::oTet-Cre mice will indelibly mark a population of cells that express a 4.4 kb fragment of the promoter region of the gene Tert. When combined the Rosa-mTmG reporter, mTert-rtTA::oTet-Cre::Rosa-mTmG mice will express membrane tdTomato (mTomato) until doxycycline treatment induces the replacement of mTomato expression with membrane EGFP (mGFP) in cells that also express Tert. Therefore, when these triple-transgenic lineage tracing mice receive doxycycline (the &#34;pulse&#34; period during which TERT expressing cells are marked), these cells will become indelibly marked mGFP+ cells, which can be tracked for any desirable amount of time after doxycycline removal (the &#34;chase&#34; period), even if Tert expression is subsequently lost. Brains are then perfusion-fixed and processed for immunofluorescence and other downstream applications in order to interpret changes to stem cell activation, proliferation, lineage commitment, migration to various brain niches, and differentiation to mature cell types. Using this system, any rtTA mouse can be mated to oTet-Cre and a Rosa reporter to conduct doxycycline-inducible &#34;pulse-chase&#34; lineage&#160;tracing experiments using markers of stem cells.

The ability to permanently mark stem cells and their progeny with a fluorophore using an inducible transgenic lineage tracing mouse line allows for spatial and temporal analysis of activation, proliferation, migration, and/or differentiation in vivo. Lineage tracing can reveal novel information about lineage commitment, response to intervention(s), and multipotency.

成体マウス脳における誘導性蛍光標識幹細胞の系統追跡

A telomerase reverse transcriptase (Tert) lineage-tracing mouse line was developed to investigate the behavior and fate of adult tissue stem cells, by ...

神経科学

Imaging Spatial Reorganization of a MAPK Signaling Pathway Using the Tobacco Transient Expression System

Visualization of dynamic signaling events in live cells has been a challenge. We expanded an established transient expression system, the biomolecular fluorescent complementation (BiFC) assay in tobacco epidermal cells, from testing protein-protein interaction to monitoring spatial distribution of signal transduction in plant cells. In this protocol, we used the BiFC assay to show that the interaction and the signaling between the Arabidopsis MAPKKK YODA and MAPK6 occur at the plasma membrane. When the scaffold protein BASL was co-expressed, the YODA-MAPK interaction redistributed and spatially co-polarized with CFP-BASL. This modified tobacco expression system allows for quick examination of signaling localization and dynamic changes (less than 4 days) and can accommodate multiple of fluorescent protein colors (at least three). We also presented detailed methods to quantify protein distribution (asymmetric spatial localization, or &#34;polarization&#34;) in tobacco cells. This advanced tobacco expression system has a potential to be used widely for quick testing of dynamic signaling events in live plant cells.

At the subcellular level, signaling events are dynamically modulated by developmental and environmental cues. Here we describe a protocol that employs the tobacco transient expression system to monitor dynamic protein-protein interaction and to disclose spatial organization of signal transduction in plant cells.

タバコ一過性発現系を使用したMAPKシグナル伝達経路の空間再編のイメージング

Visualization of dynamic signaling events in live cells has been a challenge. We expanded an established transient expression system, the biomolecular ...

Lineage Tracing and Clonal Analysis in Developing Cerebral Cortex Using Mosaic Analysis with Double Markers (MADM)

Beginning from a limited pool of progenitors, the mammalian cerebral cortex forms highly organized functional neural circuits. However, the underlying cellular and molecular mechanisms regulating lineage transitions of neural stem cells (NSCs) and eventual production of neurons and glia in the developing neuroepithelium remains unclear. Methods to trace NSC division patterns and map the lineage of clonally related cells have advanced dramatically. However, many contemporary lineage tracing techniques suffer from the lack of cellular resolution of progeny cell fate, which is essential for deciphering progenitor cell division patterns. Presented is a protocol using mosaic analysis with double markers (MADM) to perform in vivo clonal analysis. MADM concomitantly manipulates individual progenitor cells and visualizes precise division patterns and lineage progression at unprecedented single cell resolution. MADM-based interchromosomal recombination events during the G2-X phase of mitosis, together with temporally inducible CreERT2, provide exact information on the birth dates of clones and their division patterns. Thus, MADM lineage tracing provides unprecedented qualitative and quantitative optical readouts of the proliferation mode of stem cell progenitors at the single cell level. MADM also allows for examination of the mechanisms and functional requirements of candidate genes in NSC lineage progression. This method is unique in that comparative analysis of control and mutant subclones can be performed in the same tissue environment in vivo. Here, the protocol is described in detail, and experimental paradigms to employ MADM for clonal analysis and lineage tracing in the developing cerebral cortex are demonstrated. Importantly, this protocol can be adapted to perform MADM clonal analysis in any murine stem cell niche, as long as the CreERT2 driver is present.

Lineage Tracing and Clonal Analysis in Developing Cerebral Cortex Using Mosaic Analysis with Double Markers MADM

A protocol to perform lineage tracing and functional genetic analysis of candidate genes at a single cell level using mosaic analysis with double markers (MADM) is presented. MADM clonal analysis provides a quantitative framework to measure the proliferative behavior, cellular output, and lineage relationship of individual progenitors and their daughter cells.

二重マーカーを用いたモザイク解析を用いた大脳皮質の開発における系統トレースとクローン解析(MADM)

Beginning from a limited pool of progenitors, the mammalian cerebral cortex forms highly organized functional neural circuits. However, the underlying ...

細胞系譜マーカーの共局在とトマト信号

要約

さらに動画を探す

この動画の章

マルチカラー蛍光レポーターマウスを用いた角膜細胞の系譜トレースする方法

ツインスポットMARCMを使用して細胞系譜の解析と遺伝子機能研究

別の植物種から茎頂の共焦点のライブイメージング

レポーターマウスを用いたIL-22を発現するリンパ球の可視化

タンパク質Planta Plasmodesmal ローカリゼーションシーケンスの同定

植物の転写産物の正確な位置のためのin situハイブリダイゼーション

の細胞特異的な分析

成体マウス脳における誘導性蛍光標識幹細胞の系統追跡

タバコ一過性発現系を使用したMAPKシグナル伝達経路の空間再編のイメージング

二重マーカーを用いたモザイク解析を用いた大脳皮質の開発における系統トレースとクローン解析(MADM)

細胞系譜マーカーの共局在とトマト信号

要約

さらに動画を探す

この動画の章

マルチカラー蛍光レポーターマウスを用いた角膜細胞の系譜トレースする方法

ツインスポットMARCMを使用して細胞系譜の解析と遺伝子機能研究

別の植物種から茎頂の共焦点のライブ イメージング

レポーターマウスを用いたIL-22を発現するリンパ球の可視化

タンパク質Planta Plasmodesmal ローカリゼーション シーケンスの同定

植物の転写産物の正確な位置のためのin situハイブリダイゼーション

の細胞特異的な分析

成体マウス脳における誘導性蛍光標識幹細胞の系統追跡

タバコ一過性発現系を使用したMAPKシグナル伝達経路の空間再編のイメージング

二重マーカーを用いたモザイク解析を用いた大脳皮質の開発における系統トレースとクローン解析(MADM)

別の植物種から茎頂の共焦点のライブイメージング

タンパク質Planta Plasmodesmal ローカリゼーションシーケンスの同定