この文書に含まれている映像のために細胞培養を提供するための博士テリーSchochetに、細胞培養をめっきし、維持するためにケビン宮代に感謝。さらに、aRNAの手順で入力するためのケビン宮代、ピーターバックリー、およびティーナPertizにありがとう。この作業のための資金は国立老化研究所、精神保健及びペンシルベニア州の連邦からの人事ファクトファインダーファンドの国立研究所からだった。

<ol>
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E., Andrews, C., Miyashiro, K., McIntosh, T., Becker, K., Barrett, T., Hinkle, D., Dent, G., Marciano, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=mRNA+expression+analysis+of+tissue+sections+and+single+cells.">mRNA expression analysis of tissue sections and single cells.</a> The Journal of Neuroscience. 21, 8310-8314 (2001).</li><li>Van Gelder, R. N., von Zastrow, M. E., Yool, A., Dement, W. C., Barchas, J. D., Eberwine, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amplified+RNA+synthesized+from+limited+quantities+of+heterogeneous+cDNA.">Amplified RNA synthesized from limited quantities of heterogeneous cDNA.</a> Proc. Natl. Acad. Sci. USA. 87, 1663-1667 (1990).</li><li>Miyashiro, K., Dichter, M., Eberwine, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+nature+and+differential+distribution+of+mRNAs+in+hippocampal+neuritis:+Implications+for+neuronal+functioning.">On the nature and differential distribution of mRNAs in hippocampal neuritis: Implications for neuronal functioning.</a> Proc. Natl. Acad. Sci. USA. 91, 10800-10804 (1994).</li><li>Sul, J., Wo, C. K., Zeng, F., Jochems, J., Lee, M. T., Kim, T. K., Peritz, T., Buckley, P., Cappelleri, D. J., Maronski, M., Kim, M., Kumar, V., Meaney, D., Kim, J., Eberwine, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptional+transfer+produces+a+predictable+cellular+phenotype.">Transcriptional transfer produces a predictable cellular phenotype.</a> Proc. Natl. Acad. Sci. USA. 106, 7624-7629 (2009).</li><li>Sow, F. B., Gallup, J. M., Sacco, R. E., Ackerman, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser+capture+microdissection+revisited+as+a+tool+for+transcriptomic+analysis:+Application+of+an+excel-based+qPCR+preparation+software+(PREXCEL-Q).">Laser capture microdissection revisited as a tool for transcriptomic analysis: Application of an excel-based qPCR preparation software (PREXCEL-Q).</a> Int. J. Biomed. Sci. 5, (2010).</li><li>Vandewoestyne, M., Deforce, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser+capture+microdissection+in+forensic+research:+a+review.">Laser capture microdissection in forensic research: a review.</a> Int J Legal Med. 124, (2010).</li><li>Eberwine, J., Bartfai, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+cell+transcriptomics+of+hypothalamic+warm+sensitive+neurons+that+control+core+body+temperature+and+fever+response+Signaling+asymmetry+and+an+extension+of+chemical+neuroanatomy.">Single cell transcriptomics of hypothalamic warm sensitive neurons that control core body temperature and fever response Signaling asymmetry and an extension of chemical neuroanatomy.</a> Pharmacol. Ther. , (2010).</li></ol>

博士はEberwineは博士Eberwineが、株主であるLBS技術にライセンスされているaRNAの特許の発明者です。

注意事項とトラブルシューティング<ul><li>あなたは前と後に標的細胞が単離されたことを示す画像と、残りの半影はそのまま残されたものになるとよいでしょう。 </li><li>あなたが収穫されるためあまり解決策は、ピペットを入力している場合は、3ウェイルアーロックフィッティングとチューブに正圧を保持するシリンジ用プランジャーを使用することができます。任意のボリュームは、処理の種類によって異なりますが、最大5μlのは、ほとんどのアプリケーションで問題ないはずです。 </li></ul>分子生物学的手法の一般的なヒント<ul><li>渦すべての株式のチューブを、反応に添加する前に、それらをスピンダウン。 NEVER渦酵素。 </li><li>徹底的にバッファが適切な濃度であることを保証するために酵素を加える前に反応を混ぜる。 </li><li>可能であればstratacoolerを使用して-20℃で酵素の在庫を保管してください。そうでない場合は、、右使用前に取り外して氷上に保持し、-20℃にすぐに戻ります</li><li>常にピペッティングエラーを減らすために複数の反応を準備する時、マスターミックスのこと。サンプル数に基づいて必要な量を計算すると10-20％を追加。 </li><li>店舗RNA -80 ° Cを遅らせる劣化へ。 RNAは酸性条件下で発生するRNAのa​​purinationを遅らせるためにバッファに格納されている最も安定しています。 </li></ul> aRNAの手順の一般的な概要図4Aは、aRNAの手順の最初のラウンドを示しています。第一鎖の反応では、T7 - oligo（dT）プライマーのポリ- T部分は、ポリA尾部に結合することによってmRNA種（長い白い長方形）のために選択されます。いくつかのマイクロRNAは、ポリアデニル化されると、このプロシージャによって取得されます。さらに重要なことは、しかし、細胞の中で最も豊富なRNAは、リボソームRNAは、しません。このオリゴは、テンプレートとしてmRNAを用いたcDNA（長い灰色の長方形）の相補鎖を合成する逆転写酵素のためのプライマーとして機能します。 T7 -オリゴ（dT）プライマーのT7の部分は、開始mRNAに配列をアンチセンスでフレーム内のT7 RNAポリメラーゼプロモーターが組み込まれています。これは、in vitroでの転写反応の後半で使用されています。 次に、前の手順で作成した発現/ DNAハイブリッド中のmRNAは、ラギング鎖のDNA合成で作成された岡崎フラグメントと同様のRNA&quot;プライマー&quot;を（小さな白い長方形）を作成RNアーゼHによって部分的に加水分解される。 DNAポリメラーゼは、私がテンプレートとしてmRNAに相補的なDNAを使用してプライムDNA合成にRNA断片を使用しています。それは次のRNA断片に到達すると、その5&#39;から3&#39;ヌクレアーゼ活性は、リボヌクレオチドを​​除去し、デオキシリボヌクレオチドに置き換えます。 DNAリガーゼは、リーディング鎖の交換が​​完了していない任意の鎖を連結するために追加されます。 RNA断片は、DNAポリメラーゼIが作成するための最初のプライマーとして役立ったところ、T4 DNAポリメラーゼは、エリアを埋めるために追加されて平滑末端に二重次にIVT反応を実行する前に精製される二本鎖cDNAを。 IVT反応では、T7 RNAポリメラーゼは、鋳型としてセンス鎖を使って二重鎖cDNAと合成するアンチセンスRNA分子（長い黒い長方形）に組み込まれたT7プロモータに結合する。これは、アンチセンスRNA分子の数千が各二本鎖cDNA分子（図4A）から生成される増幅のステップとして機能します。 図4Bに示すように第二ラウンドは、、出発RNAはアンチセンス（黒く塗りつぶした矩形）であるとT7 -オリゴの標的にされたポリアデニル化した尾部を欠いているので、最初のラウンドのそれとは若干異なる逆転写反応から始まり、最初のラウンドで（dT）プライマー。したがって、この反応は、ランダムプライマー（小さなグレーの長方形）と続いて変性RNAを発揮する。第二鎖の反応は、前述の逆転写反応で作成したセンスRNAの3&#39;末端にポリA配列に結合するT7 -オリゴ（dT）プライマーでプライミングされています。別のIVT反応は、最初のラウンドと同様に実行されます。この第二ラウンドは、通常、単一の細胞から増幅3回を達成するには、少なくとも一回繰り返されます。 アプリケーション我々はこの記事で紹介されていることの技術は多数のアプリケーションに変換することができます。単一細胞の単離のプロトコールは、急性スライスで使用するために変更することができます。14技術的に困難なものの、同じ原則が、この代替の準備に適用されます。ピペットのサイズが若干調整されている場合はさらに、細胞の生理機能の録音は、収穫が生理的な出力の背後にある分子メカニズムの十分に制御された調査を許可する前に行うことができます。別の若干の修正は15が 、このアプリケーションでは。細胞体からプロセスを分離することになってピペットで細胞体を収集し、新鮮なピペットで戻って、100から300に識別dendritを収集するESまたはコレクションチューブあたり軸索。。16 一度細胞が収穫されて、mRNAの存在量と組成の比較は、同一の細胞集団内でも異なるとの間で行うことができます。第三ラウンドにビオチン- UTPを組み込んだaRNAのは、これらの相対的なmRNAの存在量を決定するためにマイクロアレイ解析が可能になります。オリジナルのmRNAの人口の組成物はまた、次世代シーケンシングを使用してaRNAの手順の後に決定することができます。増幅されたaRNAを、前者のように後者の細胞型の表現型の変化を誘導するために、ひとつの細胞型からのmRNAの完全なセットが異なる細胞型にトランスフェクトされた細胞の表現型の変換の研究を確認するためにも使用できます。手順ラボが開発し、TIPeRとして知られている。17これらの研究は、病気の状態や細胞の表現型とそのような研究を研究するために特に有用であるが、ラボで現在進行中です。 RT - PCRや定量PCRは、細胞特異的遺伝子の発現を確認するために増幅された材料で行うことができます。さらに、トランスフェクションまたは形質導入の効率性の評価は、単一細胞レベルで行うことができます。 利点と制限事項抽象的に記載されているように、分析のための単一細胞の単離は、異種細胞集団の解析で見られる平均化の効果がなくなります。これらの平均化の効果は、豊富な転写を過剰表現して平均化し、多くの発現量の低い転写産物の検出を防止することにより、単一細胞内のmRNAの存在量を偽る。フローサイトメトリーは、個々のセルを並べ替えるために使用されますが、この方法は、細胞特異的マーカーと高価な機器の知識が必要です。18レーザーキャプチャーマイクロダイセクションはどちらUVやIRレーザーキャプチャーマイクロダイセクションシステムと単一のセル、さらには細胞内取り込みを可能にするだけの目的の細胞を必要とすることができます非常に薄い切片の表面に位置する。19はフローサイトメトリーと同様に、レーザーキャプチャーマイクロダイセクションはまた、高価な機器が必要です。 上記の電極ベースのコレクションの技術の主な利点の1つは、その貴重な電気生理学的データが同じセルで実行される機能性とトランスクリプトーム解析を可能にする、収穫の前に目的の細胞から得ることができるからである。私たちの技術の20の欠点があるそれは、マニピュレーターを使用して経験を必要としないこと。マイクロマニピュレータに精通して研究者らは、このテクニックは非常に直感的にわかりますが、そのような経験を持つ個人が必要な細かい動きに慣れる必要があります。 任意の増幅技術に内在するには、サイズと塩基組成に基づいて特定の転写産物の選択的増幅です。4,6,7ポリメラーゼ連鎖反応（PCR）などの逆転写ポリメラーゼ連鎖反応（RT - PCR）、cDNAの迅速増幅のようなベースのテクニックは線形増幅のaRNAの増幅法の結果、一方の端（RACE）の転写産物の指数関数的増幅の結果、。このように、aRNAの手順の主要な利点の一つは、より指数関数的に増幅するのではなく、唯一の直線的に増幅過程で発生したエラーやバイアスを増幅することによってmRNAの転写産物の相対的存在量を維持する能力にあるエラーやバイアスは言った。 マイクロアレイ解析と相まってaRNAの手順は、処理または未処理同様のまたは別の形態の単セル間のmRNAの存在量の比較ができます。さらに、増幅された材料は、次世代シーケンシングのために提出することができます。5,8がしかし、、手続きのためのランダムプライマーを使用することとは対照的に、オリゴ- dTプライミング手順はの偏見の増幅上記以来、このような分析には注意が必要mRNAおよび各ラウンドで、その後の増幅された物質のわずかな短縮で、一般的に、結果の3&#39;末端。ケアは、いくつかの偽の不在コールが若干短縮増幅された物質から発生することができるように標準的な方法とマイクロアレイの結果を分析することに注意してください。さらに、シーケンシングの結果は確かに全5提供する間、&quot;オリジナルのmRNAのほとんどのシーケンスを、5&#39;いくつかのmRNAのシーケンスが見逃される可能性があります。これらの理由により、増幅のラウンド数を制限する必要があります。

A correction was made to <a href="http://http://www.jove.com/video/2634/transcriptome-analysis-of-single-cells">Transcriptome Analysis of Single Cells</a>. There were errors in the volumes used in the aRNA Amplification section.

Erratum: Transcriptome Analysis of Single Cells

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Spiegelgas coverslips</td>
<td>&nbsp;</td>
<td>Carolina Biological</td>
<td>63-3029</td>
<td>&nbsp;</td>
<tr>
<td>Nunc 35x10 mm culture dishes</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>12-565-90</td>
<td>&nbsp;</td>
<tr>
<td>Water for cell culture</td>
<td>&nbsp;</td>
<td>Lonza</td>
<td>17-724Q</td>
<td>For making solutions used for cell culture and rinsing coverslips</td>
</tr>
<tr>
<td>Poly-D-Lysine MW70-150K</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>6407</td>
<td>&nbsp;</td>
<tr>
<td>Laminin, ultrapure</td>
<td>&nbsp;</td>
<td>BD Bioscience</td>
<td>354239</td>
<td>&nbsp;</td>
<tr>
<td>Boric acid</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>B0252</td>
<td>&nbsp;</td>
<tr>
<td>MEM with Earle&#x2019;s salts and glutamax</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>41090-101</td>
<td>For plating cells</td>
</tr>
<tr>
<td>D-glucose</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>G8769</td>
<td>&nbsp;</td>
<tr>
<td>Penicillin-streptomycin</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15140-122</td>
<td>&nbsp;</td>
<tr>
<td>Horse serum</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>16050</td>
<td>&nbsp;</td>
<tr>
<td>Cytosine beta-D-arabinofuranoside</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>C1768</td>
<td>&nbsp;</td>
<tr>
<td>MEM with Earle&#x2019;s salts and L-glutamine</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11095-098</td>
<td>For growing cells</td>
</tr>
<tr>
<td>Sodium pyruvate</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P2256</td>
<td>&nbsp;</td>
<tr>
<td>B27 serum-free supplement</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>17504-044</td>
<td>&nbsp;</td>
<tr>
<td>Borosilicate glass capillary tubes (1.5mm O.D, 100mm length)</td>
<td>&nbsp;</td>
<td>Kimble Chase</td>
<td>34500 99</td>
<td>Any borosilicate glass pipette will work as long as the proper bore size is attained. </td>
</tr>
<tr>
<td>HBSS</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>14175</td>
<td>Any solution will work as long as the components won&#x2019;t interfere with any future processing (i.e. no Ca2+ or Mg2+)</td>
</tr>
<tr>
<td>1ml syringe</td>
<td>&nbsp;</td>
<td>BD</td>
<td>309628</td>
<td>&nbsp;</td>
<tr>
<td>Needle</td>
<td>&nbsp;</td>
<td>BD</td>
<td>&nbsp;</td>
<td>Gauge depends on the diameter of the pipettes</td>
</tr>
<tr>
<td>dNTP mix</td>
<td>&nbsp;</td>
<td>Amersham</td>
<td>28-4065-51</td>
<td>&nbsp;</td>
<tr>
<td>dt-T7 oligo</td>
<td>&nbsp;</td>
<td>Custom</td>
<td>Midland Certified</td>
<td>&nbsp;</td>
<tr>
<td>Second strand buffer (5x)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>Y01129</td>
<td>&nbsp;</td>
<tr>
<td>DTT</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Supplied with second strand buffer</td>
</tr>
<tr>
<td>E.coli DNA Ligase</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>100002324</td>
<td>&nbsp;</td>
<tr>
<td>DNA polymerase I</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>100004926</td>
<td>&nbsp;</td>
<tr>
<td>Rnase H</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>18021-071</td>
<td>&nbsp;</td>
<tr>
<td>Megascript T7 kit</td>
<td>&nbsp;</td>
<td>Ambion</td>
<td>AM1334</td>
<td>&nbsp;</td>
<tr>
<td>Random primers</td>
<td>&nbsp;</td>
<td>BMB</td>
<td>11034731001</td>
<td>&nbsp;</td>
<tr>
<td>Superscript III Reverse Transcriptase</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>56575</td>
<td>in kit (18080-044) comes with first strand buffer (Y02321) and DTT</td>
</tr>
<tr>
<td>MEGAclear Kit</td>
<td>&nbsp;</td>
<td>Ambion</td>
<td>1908</td>
<td>&nbsp;</td>
<tr>
<td>MinElute Reaction Cleanup Kit</td>
<td>&nbsp;</td>
<td>Qiagen</td>
<td>28206</td>
<td>&nbsp;</td>
<tr>
<td>T4 DNA polymerase</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>100004994</td>
<td>&nbsp;</td>
<tr>
<td>Rnasin</td>
<td>&nbsp;</td>
<td>Promega</td>
<td>N251B</td>
<td>&nbsp;</td>
<tr>
<td>Illumina TotalPrep RNA Amplification Kit</td>
<td>&nbsp;</td>
<td>Ambion</td>
<td>AMIL1791</td>
<td>&nbsp;</td>
<tr>
<td>Flaming/Brown micropipette puller</td>
<td>&nbsp;</td>
<td>Sutter Instruments</td>
<td>P-87</td>
<td>Sutter has many other models, many are discussed in the cookbook</td>
</tr>
<tr>
<td>Micromanipulator</td>
<td>&nbsp;</td>
<td>Olympus</td>
<td>&nbsp;</td>
<td>Many other micromanipulators will work such as the newer Eppendorf models</td>
</tr>
<tr>
<td>Pipette Holder</td>
<td>&nbsp;</td>
<td>Warner Instruments</td>
<td>MP-S15A</td>
<td>Will vary with micromanipulator and pipette O.D.</td>
</tr>
<tr>
<td>Bioanalyzer RNA 6000 Nano Kit</td>
<td>&nbsp;</td>
<td>Agilent</td>
<td>5067-1511</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

transcriptome analysis of single cells

多くの遺伝子発現解析の技術は文化の組織ホモジネートまたは細胞からの細胞の不均一な集団から分離された材料に依存している。1,2,3の脳の場合には、このような海馬などの領域はそれぞれに、異なる細胞型の複雑な配置が含まれています異なるmRNAプロファイル。単一の細胞を回収する能力は、細胞集団間および内の分子の違いに綿密な調査で、より可能になります。我々は、さらなる処理のために細胞を採取するための簡便かつ迅速な方法を説明します。多くの場合、電気生理学で使用されているピペットは（誤嚥を使用して）目的の細胞を分離し、便利な分子生物学的手法の任意の数のさらなる処理のためのエッペンドルフチュー​​ブに、それを堆積させるために利用されています。我々のプロトコルは、細胞培養または急性スライスからさらには個々の細胞からの樹状突起の収穫に変更することができます。 我々はまた、単一のセルのアイソレーションの主要な下流のアプリケーションとしてaRNAの増幅法を説明します。このメソッドは、逆転写反応またはリアルタイムポリメラーゼ連鎖反応（PCR）などの他の遺伝子発現解析の手法に代わるものとして私たちの研究室で以前に開発されました。4,5,6,7,8この手法ではのリニア増幅を提供するポリアデニル化RNAは、材料の唯一のフェムトグラムから始まり、アンチセンスRNAのマイクログラム量で生じる。直線的に増幅された材料は、単離細胞のトランスクリプトームの成分の相対量のPCRの指数増幅よりも正確な推定を提供します。基本的な手順は、増幅の二回で構成されています。簡単に言うと、T7 RNAポリメラーゼプロモーターのサイトは、mRNA転写物から作成された二本鎖cDNAに組み込まれています。 in vitro転写一晩（IVT）反応は、T7 RNAポリメラーゼは、二本鎖cDNAから多くのアンチセンス転写産物を生成する実行されます。第二ラウンドでは、このプロセスを繰り返しますが、出発原料からいくつかの技術的な違いがアンチセンスRNAである。それは、増幅の3つのラウンドで、その結果、第二ラウンドを繰り返すことが標準です。多くの場合、in vitro転写反応の第三ラウンドは、生成されたアンチセンスRNAをハイブリダイズし、マイクロアレイで検出することができるようにビオチン化ヌクレオシド三リン酸を使用して実行されます。7,8

Watch this Scientific Journal Video about Transcriptome Analysis of Single Cells at JoVE.com

Transcriptome Analysis of Single Cells

この記事では、ラット初代ニューロン培養とaRNAの増幅を使用して、その後のトランスクリプトーム解析から単一細胞の収穫のための簡単​​な方法を説明します。このアプローチは、任意の細胞型への一般化です。

単一細胞のトランスクリプトーム解析

Many gene expression analysis techniques rely on material isolated from heterogeneous populations of cells from tissue homogenates or cells in ...

Research

JoVE Journal

Neuroscience

29.7K ビュー.  University of Pennsylvania. この記事では、ラット初代ニューロン培養とaRNAの増幅を使用して、その後のトランスクリプトーム解析から単一細胞の収穫のための簡単​​な方法を説明します。このアプローチは、任意の細胞型への一般化です。

ビデオ: Transcriptome Analysis of Single Cells

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding fundamental biological mechanisms. Striking progress has been particularly evident in Drosophila, whose extensive toolkit of mutants and transgenic lines provides a convenient model to study evolutionarily-conserved developmental and cell biological mechanisms. We are interested in understanding the mechanisms that control cell fate specification in the adult peripheral nervous system (PNS) in Drosophila. Bristles that cover the head, thorax, abdomen, legs and wings of the adult fly are individual mechanosensory organs, and have been studied as a model system for understanding mechanisms of Notch-dependent cell fate decisions. Sensory organ precursor (SOP) cells of the microchaetes (or small bristles), are distributed throughout the epithelium of the pupal thorax, and are specified during the first 12 hours after the onset of pupariation. After specification, the SOP cells begin to divide, segregating the cell fate determinant Numb to one daughter cell during mitosis. Numb functions as a cell-autonomous inhibitor of the Notch signaling pathway.
Here, we show a method to follow protein dynamics in SOP cell and its progeny within the intact pupal thorax using a combination of tissue-specific Gal4 drivers and GFP-tagged fusion proteins 1,2.This technique has the advantage over fixed tissue or cultured explants because it allows us to follow the entire development of an organ from specification of the neural precursor to growth and terminal differentiation of the organ. We can therefore directly correlate changes in cell behavior to changes in terminal differentiation. Moreover, we can combine the live imaging technique with mosaic analysis with a repressible cell marker (MARCM) system to assess the dynamics of tagged proteins in mitotic SOPs under mutant or wildtype conditions. Using this technique, we and others have revealed novel insights into regulation of asymmetric cell division and the control of Notch signaling activation in SOP cells (examples include references 1-6,7 ,8).

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

In this video, we describe a method for live cell imaging of asymmetrically dividing sensory organ progenitor cells and epidermal cells in intact Drosophila pupae

無傷の感覚器官の前駆細胞の生細胞イメージングショウジョウバエ蛹

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding ...

神経科学

Sequential Photo-bleaching to Delineate Single Schwann Cells at the Neuromuscular Junction

Sequential photo-bleaching provides a non-invasive way to label individual SCs at the NMJ. The NMJ is the largest synapse of the mammalian nervous system and has served as guiding model to study synaptic structure and function. In mouse NMJs motor axon terminals form pretzel-like contact sites with muscle fibers. The motor axon and its terminal are sheathed by SCs. Over the past decades, several transgenic mice have been generated to visualize motor neurons and SCs, for example Thy1-XFP1 and Plp-GFP mice2, respectively.
Along motor axons, myelinating axonal SCs are arranged in non-overlapping internodes, separated by nodes of Ranvier, to enable saltatory action potential propagation. In contrast, terminal SCs at the synapse are specialized glial cells, which monitor and promote neurotransmission, digest debris and guide regenerating axons. NMJs are tightly covered by up to half a dozen non-myelinating terminal SCs - these, however, cannot be individually resolved by light microscopy, as they are in direct membrane contact3.
Several approaches exist to individually visualize terminal SCs. None of these are flawless, though. For instance, dye filling, where single cells are impaled with a dye-filled microelectrode, requires destroying a labelled cell before filling a second one. This is not compatible with subsequent time-lapse recordings3. Multi-spectral "Brainbow" labeling of SCs has been achieved by using combinatorial expression of fluorescent proteins4. However, this technique requires combining several transgenes and is limited by the expression pattern of the promoters used. In the future, expression of "photo-switchable" proteins in SCs might be yet another alternative5. Here we present sequential photo-bleaching, where single cells are bleached, and their image obtained by subtraction. We believe that this approach - due to its ease and versatility - represents a lasting addition to the neuroscientist's technology palette, especially as it can be used in vivo and transferred to others cell types, anatomical sites or species6.
In the following protocol, we detail the application of sequential bleaching and subsequent confocal time-lapse microscopy to terminal SCs in triangularis sterni muscle explants. This thin, superficial and easily dissected nerve-muscle preparation7,8 has proven useful for studies of NMJ development, physiology and pathology9. Finally, we explain how the triangularis sterni muscle is prepared after fixation to perform correlated high-resolution confocal imaging, immunohistochemistry or ultrastructural examinations.

Visualizing individual cells in densely packed tissues, such as terminal Schwann cells (SCs) at neuromuscular junctions (NMJs), is challenging. "Sequential photo-bleaching" allows delineating single terminal SCs, for instance in the triangularis sterni muscle explant, a convenient nerve-muscle preparation, where sequential bleaching can be combined with time-lapse imaging and post-hoc immunostainings.

シーケンシャル神経筋接合部におけるシングルシュワン細胞の輪郭を描くのにフォトブリーチング

Sequential photo-bleaching provides a non-invasive way to label individual SCs at the NMJ. The NMJ is the largest synapse of the mammalian nervous system ...

Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

In this article we describe how to individually label neurons in the embryonic CNS of Drosophila melanogaster by juxtacellular injection of the lipophilic fluorescent membrane marker DiI. This method allows the visualization of neuronal cell morphology in great detail. It is possible to label any cell in the CNS: cell bodies of target neurons are visualized under DIC optics or by expression of a fluorescent genetic marker such as GFP. After labeling, the DiI can be transformed into a permanent brown stain by photoconversion to allow visualization of cell morphology with transmitted light and DIC optics. Alternatively, the DiI-labeled cells can be observed directly with confocal microscopy, enabling genetically introduced fluorescent reporter proteins to be colocalised. The technique can be used in any animal, irrespective of genotype, making it possible to analyze mutant phenotypes at single cell resolution.

Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

We present a technique for labeling single neurons in the central nervous system (CNS) of Drosophila embryos, which allows the analysis of neuronal morphology by either transmitted light or confocal microscopy.

の中枢神経系における単一細胞のラベリング<em&gt;キイロショウジョウバエ</em

In this  article we describe how to individually label neurons in the embryonic CNS of Drosophila melanogaster by juxtacellular injection of the ...

Laser Capture Microdissection of Enriched Populations of Neurons or Single Neurons for Gene Expression Analysis After Traumatic Brain Injury

Long-term cognitive disability after TBI is associated with injury-induced neurodegeneration in the hippocampus-a region in the medial temporal lobe that is critical for learning, memory and executive function.1,2 Hence our studies focus on gene expression analysis of specific neuronal populations in distinct subregions of the hippocampus. The technique of laser capture microdissection (LCM), introduced in 1996 by Emmert-Buck, et al.,3 has allowed for significant advances in gene expression analysis of single cells and enriched populations of cells from heterogeneous tissues such as the mammalian brain that contains thousands of functional cell types.4 We use LCM and a well established rat model of traumatic brain injury (TBI) to investigate the molecular mechanisms that underlie the pathogenesis of TBI. Following fluid-percussion TBI, brains are removed at pre-determined times post-injury, immediately frozen on dry ice, and prepared for sectioning in a cryostat. The rat brains can be embedded in OCT and sectioned immediately, or stored several months at -80 &deg;C before sectioning for laser capture microdissection. Additionally, we use LCM to study the effects of TBI on circadian rhythms. For this, we capture neurons from the suprachiasmatic nuclei that contain the master clock of the mammalian brain. Here, we demonstrate the use of LCM to obtain single identified neurons (injured and degenerating, Fluoro-Jade-positive, or uninjured, Fluoro-Jade-negative) and enriched populations of hippocampal neurons for subsequent gene expression analysis by real time PCR and/or whole-genome microarrays. These LCM-enabled studies have revealed that the selective vulnerability of anatomically distinct regions of the rat hippocampus are reflected in the different gene expression profiles of different populations of neurons obtained by LCM from these distinct regions. The results from our single-cell studies, where we compare the transcriptional profiles of dying and adjacent surviving hippocampal neurons, suggest the existence of a cell survival rheostat that regulates cell death and survival after TBI.

We describe how to use laser capture microdissection (LCM) to obtain enriched populations of hippocampal neurons or single neurons from frozen sections of the injured rat brain for subsequent gene expression analysis using quantitative real time PCR and/or whole-genome microarrays.

外傷性脳損傷後の神経細胞や遺伝子発現解析のための単一ニューロンの濃縮された集団のレーザーキャプチャーマイクロダイセクション

Long-term cognitive disability after TBI is associated with injury-induced neurodegeneration in the hippocampus-a region in the medial temporal lobe that ...

Peering at Brain Polysomes with Atomic Force Microscopy

The translational machinery, i.e., the polysome or polyribosome, is one of the biggest and most complex cytoplasmic machineries in cells. Polysomes, formed by ribosomes, mRNAs, several proteins and non-coding RNAs, represent integrated platforms where translational controls take place. However, while the ribosome has been widely studied, the organization of polysomes is still lacking comprehensive understanding. Thus much effort is required in order to elucidate polysome organization and any novel mechanism of translational control that may be embedded. Atomic force microscopy (AFM) is a type of scanning probe microscopy that allows the acquisition of 3D images at nanoscale resolution. Compared to electron microscopy (EM) techniques, one of the main advantages of AFM is that it can acquire thousands of images both in air and in solution, enabling the sample to be maintained under near physiological conditions without any need for staining and fixing procedures. Here, a detailed protocol for the accurate purification of polysomes from mouse brain and their deposition on mica substrates is described. This protocol enables polysome imaging in air and liquid with AFM and their reconstruction as three-dimensional objects. Complementary to cryo-electron microscopy (cryo-EM), the proposed method can be conveniently used for systematically analyzing polysomes and studying their organization.

While ribosome structure has been extensively characterized, the organization of polysomes is still understudied. To overcome this lack of knowledge, we present here a detailed preparation protocol for accurate imaging of mammalian polysomes by atomic force microscopy (AFM) in air and liquid.

原子間力顕微鏡で脳ポリソームでピアリング

The translational machinery, i.e., the polysome or polyribosome, is one of the biggest and most complex cytoplasmic machineries in cells. Polysomes, ...

Single-cell RNA-Seq of Defined Subsets of Retinal Ganglion Cells

The discovery of cell type-specific markers can provide insight into cellular function and the origins of cellular heterogeneity. With a recent push for the improved understanding of neuronal diversity, it is important to identify genes whose expression defines various subpopulations of cells. The retina serves as an excellent model for the study of central nervous system diversity, as it is composed of multiple major cell types. The study of each major class of cells has yielded genetic markers that facilitate the identification of these populations. However, multiple subtypes of cells exist within each of these major retinal cell classes, and few of these subtypes have known genetic markers, although many have been characterized by morphology or function. A knowledge of genetic markers for individual retinal subtypes would allow for the study and mapping of brain targets related to specific visual functions and may also lend insight into the gene networks that maintain cellular diversity. Current avenues used to identify the genetic markers of subtypes possess drawbacks, such as the classification of cell types following sequencing. This presents a challenge for data analysis and requires rigorous validation methods to ensure that clusters contain cells of the same function. We propose a technique for identifying the morphology and functionality of a cell prior to isolation and sequencing, which will allow for the easier identification of subtype-specific markers. This technique may be extended to non-neuronal cell types, as well as to rare populations of cells with minor variations. This protocol yields excellent-quality data, as many of the libraries have provided read depths greater than 20 million reads for single cells. This methodology overcomes many of the hurdles presented by Single-cell RNA-Seq and may be suitable for researchers aiming to profile cell types in a straightforward and highly efficient manner.

Here, we present a combinatorial approach for classifying neuronal cell types prior to isolation and for the subsequent characterization of single-cell transcriptomes. This protocol optimizes the preparation of samples for successful RNA Sequencing (RNA-Seq) and describes a methodology designed specifically for the enhanced understanding of cellular diversity.

網膜神経節細胞の定義されたサブセットの単一細胞RNA-Seq

The discovery of cell type-specific markers can provide insight into cellular function and the origins of cellular heterogeneity. With a recent push for ...

Analysis of Immune Cells in Single Sciatic Nerves and Dorsal Root Ganglion from a Single Mouse Using Flow Cytometry

Nerve-resident immune cells in the peripheral nervous system (PNS) are essential to maintaining neuronal integrity in a healthy nerve. The immune cells of the PNS are affected by injury and disease, affecting the nerve function and the capacity for regeneration. Neuronal immune cells are commonly analyzed by immunofluorescence (IF). While IF is essential for determining the location of the immune cells in the nerve, IF is only semi-quantitative and the method is limited to the number of markers that can be analyzed simultaneously and the degree of surface expression. In this study, flow cytometry was used for quantitative analysis of leukocyte infiltration into sciatic nerves or dorsal root ganglions (DRGs) of individual mice. Single cell analysis was performed using DAPI and several proteins were analyzed simultaneously for either surface or intracellular expression. Both sciatic nerves from one mouse that were treated according to this protocol generated &#8805; 30,000 single nucleated events. The proportion of leukocytes in the sciatic nerves, determined by expression of CD45, was approximately 5% of total cell content in the sciatic nerve and approximately 5-10% in the DRG. Although this protocol focuses primarily on the immune cell population within the PNS, the flexibility of flow cytometry to measure a number of markers simultaneously means that the other cells populations present within the nerve, such as Schwann cells, pericytes, fibroblasts, and endothelial cells, can also be analyzed using this method. This method therefore provides a new means for studying systemic effects on the PNS, such as neurotoxicology and genetic models of neuropathy or in chronic diseases, such as diabetes.

Quantitative analysis of cell content within the murine sciatic nerve is difficult due to the scarcity of the tissue. This protocol describes a method for tissue digestion and preparation that provides sufficient cells for flow cytometry analysis of immune cell populations from nerves of individual mice.

単一の坐骨神経とフローサイトメトリーを使用して単一のマウスの後根神経節における免疫系細胞の解析

Nerve-resident immune cells in the peripheral nervous system (PNS) are essential to maintaining neuronal integrity in a healthy nerve. The immune cells of ...

Isolation of Adult Spinal Cord Nuclei for Massively Parallel Single-nucleus RNA Sequencing

Probing an individual cell&#39;s gene expression enables the identification of cell type and cell state. Single-cell RNA sequencing has emerged as a powerful tool for studying transcriptional profiles of cells, particularly in heterogeneous tissues such as the central nervous system. However, dissociation methods required for single cell sequencing can lead to experimental changes in the gene expression and cell death. Furthermore, these methods are generally restricted to fresh tissue, thus limiting studies on archival and bio-bank material. Single nucleus RNA sequencing (snRNA-Seq) is an appealing alternative for transcriptional studies, given that it accurately identifies cell types, permits the study of tissue that is frozen or difficult to dissociate, and reduces dissociation-induced transcription. Here, we present a high-throughput protocol for rapid isolation of nuclei for downstream snRNA-Seq. This method enables isolation of nuclei from fresh or frozen spinal cord samples and can be combined with two massively parallel droplet encapsulation platforms.

Here, we present a protocol to rapidly isolate high-quality nuclei from the fresh or frozen tissue for downstream massively parallel RNA sequencing. We include detergent-mechanical and hypotonic-mechanical tissue disruption and cell lysis options, both of which can be used for isolation of nuclei.

並列の単核 RNA 配列のため大人の脊髄核の分離

Probing an individual cell&#39;s gene expression enables the identification of cell type and cell state. Single-cell RNA sequencing has emerged as a ...

Analysis of Spliceosomal snRNA Localization in Human Hela Cells Using Microinjection

Biogenesis of spliceosomal snRNAs is a complex process involving both nuclear and cytoplasmic phases and the last step occurs in a nuclear compartment called the Cajal body. However, sequences that direct snRNA localization into this subnuclear structure have not been known until recently. To determine sequences important for accumulation of snRNAs in Cajal bodies, we employed microinjection of fluorescently labelled snRNAs followed by their localization inside cells. First, we prepared snRNA deletion mutants, synthesized DNA templates for in vitro transcription and transcribed snRNAs in the presence of UTP coupled with Alexa488. Labelled snRNAs were mixed with 70 kDa-Dextran conjugated with TRITC, and microinjected to the nucleus or the cytoplasm of human HeLa cells. Cells were incubated for 1 h and fixed and the Cajal body marker coilin was visualized by indirect immunofluorescence, while snRNAs and dextran, which serves as a marker of nuclear or cytoplasmic injection, were observed directly using a fluorescence microscope. This method allows for efficient and rapid testing of how various sequences influence RNA localization inside cells. Here, we show the importance of the Sm-binding sequence for efficient localization of snRNAs into the Cajal body.

Biogenesis of spliceosomal snRNAs is a complex process involving various cellular compartments. Here, we employed microinjection of fluorescently labelled snRNAs in order to monitor their transport inside the cell.

マイクロインジェクションを用いたヒトヘラ細胞におけるスプリセオソームsnRNA局在化の解析

Biogenesis of spliceosomal snRNAs is a complex process involving both nuclear and cytoplasmic phases and the last step occurs in a nuclear compartment ...

A Scalable Model to Study the Effects of Blunt-Force Injury in Adult Zebrafish

Blunt-force traumatic brain injuries (TBI) are the most common form of head trauma, which spans a range of severities and results in complex and heterogenous secondary effects. While there is no mechanism to replace or regenerate the lost neurons following a TBI in humans, zebrafish possess the ability to regenerate neurons throughout their body, including the brain. To examine the breadth of pathologies exhibited in zebrafish following a blunt-force TBI and to study the mechanisms underlying the subsequent neuronal regenerative response, we modified the commonly used rodent Marmarou weight drop for the use in adult zebrafish. Our simple blunt-force TBI model is scalable, inducing a mild, moderate, or severe TBI, and recapitulates many of the phenotypes observed following human TBI, such as contact- and post-traumatic seizures, edema, subdural and intracerebral hematomas, and cognitive impairments, each displayed in an injury severity-dependent manner. TBI sequelae, which begin to appear within minutes of the injury, subside and return to near undamaged control levels within 7 days post-injury. The regenerative process begins as early as 48 hours post-injury (hpi), with the peak cell proliferation observed by 60 hpi. Thus, our zebrafish blunt-force TBI model produces characteristic primary and secondary injury TBI pathologies similar to human TBI, which allows for investigating disease onset and progression, along with the mechanisms of neuronal regeneration that is unique to zebrafish.

We modified the Marmarou weight drop model for adult zebrafish to examine a breadth of pathologies following blunt-force traumatic brain injury (TBI) and the mechanisms underlying subsequent neuronal regeneration. This blunt-force TBI model is scalable, induces a mild, moderate, or severe TBI, and recapitulates injury heterogeneity observed in human TBI.