著者らは、年間でプロトコルを改善し、すべてのジャーヴィスラボのメンバーに感謝したいと思います。

<ol>
	<li>Clayton, D. F., Huecas, M. E., Sinclair-Thompson, E. Y., Nastiuk, K. L., Nottebohm, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probes+for+rare+mRNAs+reveal+distributed+cell+subsets+in+canary+brain.">Probes for rare mRNAs reveal distributed cell subsets in canary brain.</a> Neuron. 1, 249-261 (1988).</li><li>Wada, K., Sakaguchi, H., Jarvis, E. D., Hagiwara, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+expression+of+glutamate+receptors+in+avian+neural+pathways+for+learned+vocalization.">Differential expression of glutamate receptors in avian neural pathways for learned vocalization.</a> J. Comp. Neurol. 476, 44-64 (2004).</li><li>Haesler, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FoxP2+expression+in+avian+vocal+learners+and+non-learners.">FoxP2 expression in avian vocal learners and non-learners.</a> J. Neurosci. 24, 3164-3175 (2004).</li><li>Jarvis, E. D., Nottebohm, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Motor-driven+gene+expression.">Motor-driven gene expression.</a> Proc. Natl. Acad. Sci. U.S.A. 94, 4097-4102 (1997).</li><li>Holzenberger, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+expression+of+insulin-like+growth+factor+II+in+the+songbird+brain.">Selective expression of insulin-like growth factor II in the songbird brain.</a> J. Neurosci. 17, 6974-6987 (1997).</li><li>Mahmood, R., Mason, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In-situ+hybridization+of+radioactive+riboprobes+to+RNA+in+tissue+sections.">In-situ hybridization of radioactive riboprobes to RNA in tissue sections.</a> Methods Mol. Biol. 461, 675-686 (2008).</li><li>Wilkinson, D. G., Nieto, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+messenger+RNA+by+in+situ+hybridization+to+tissue+sections+and+whole+mounts.">Detection of messenger RNA by in situ hybridization to tissue sections and whole mounts.</a> Methods Enzymol. 225, 361-373 (1993).</li><li>Acloque, H., Wilkinson, D. G., Nieto, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+hybridization+analysis+of+chick+embryos+in+whole-mount+and+tissue+sections.">In situ hybridization analysis of chick embryos in whole-mount and tissue sections.</a> Methods Cell Biol. 87, 169-185 (2008).</li><li>Moorman, A. F., Houweling, A. C., de Boer, P. A., Christoffels, V. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensitive+nonradioactive+detection+of+mRNA+in+tissue+sections:+novel+application+of+the+whole-mount+in+situ+hybridization+protocol.">Sensitive nonradioactive detection of mRNA in tissue sections: novel application of the whole-mount in situ hybridization protocol.</a> J. Histochem. Cytochem. 49, 1-8 (2001).</li><li>Horita, H., Wada, K., Rivas, M. V., Hara, E., Jarvis, E. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+dusp1+immediate+early+gene+is+regulated+by+natural+stimuli+predominantly+in+sensory+input+neurons.">The dusp1 immediate early gene is regulated by natural stimuli predominantly in sensory input neurons.</a> J. Comp. Neurol. 518, 2873-2901 (2010).</li><li>Braissant, O., Wahli, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simplified+in+situ+hybridization+protocol+using+non-radioactively+labelled+probes+to+detect+abundant+and+rare+mRNAs+on+tissue+sections.">A simplified in situ hybridization protocol using non-radioactively labelled probes to detect abundant and rare mRNAs on tissue sections.</a> Biochemica. 1, 10-16 (1998).</li><li>Kubikova, L., Wada, K., Jarvis, E. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dopamine+receptors+in+a+songbird+brain.">Dopamine receptors in a songbird brain.</a> J. Comp. Neurol. 518, 741-769 (2010).</li><li>Wada, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+molecular+neuroethological+approach+for+identifying+and+characterizing+a+cascade+of+behaviorally+regulated+genes.">A molecular neuroethological approach for identifying and characterizing a cascade of behaviorally regulated genes.</a> Proc. Natl. Acad. Sci. U.S.A. 103, 15212-15217 (2006).</li><li>Jarvis, E. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Avian+brains+and+a+new+understanding+of+vertebrate+brain+evolution.">Avian brains and a new understanding of vertebrate brain evolution.</a> Nat. Rev. Neurosci. 6, 151-159 (2005).</li><li>Hoke, K. L., Ryan, M. J., Wilczynski, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Social+cues+shift+functional+connectivity+in+the+hypothalamus.">Social cues shift functional connectivity in the hypothalamus.</a> Proc. Natl. Acad. Sci. U.S.A. 102, 10712-10717 (2005).</li><li>Burmeister, S. S., Jarvis, E. D., Fernald, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+behavioral+and+genomic+responses+to+social+opportunity.">Rapid behavioral and genomic responses to social opportunity.</a> PLoS. Biol. 3, e363 (2005).</li><li>Jarvis, E. D., Schwabl, H., Ribeiro, S., Mello, C. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain+gene+regulation+by+territorial+singing+behavior+in+freely+ranging+songbirds.">Brain gene regulation by territorial singing behavior in freely ranging songbirds.</a> Neuroreport. 8, 2073-2077 (1997).</li><li>Jarvis, E. D., Scharff, C., Grossman, M. R., Ramos, J. A., Nottebohm, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=For+whom+the+bird+sings:+context-dependent+gene+expression.">For whom the bird sings: context-dependent gene expression.</a> Neuron. 21, 775-788 (1998).</li><li>Feenders, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mapping+of+movement-associated+areas+in+the+avian+brain:+a+motor+theory+for+vocal+learning+origin.">Molecular mapping of movement-associated areas in the avian brain: a motor theory for vocal learning origin.</a> PLoS One. 3, e1768 (2008).</li><li>Chen, C. C., Fernald, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distributions+of+two+gonadotropin-releasing+hormone+receptor+types+in+a+cichlid+fish+suggest+functional+specialization.">Distributions of two gonadotropin-releasing hormone receptor types in a cichlid fish suggest functional specialization.</a> J. Comp. Neurol. 495, 314-323 (2006).</li></ol>

我々は、開示することは何もありません。

mRNA発現のin situハイブリダイゼーション放射性は広く、地域組織の組織、細胞の種類、脳機能活性2-5,10,12-14を研究するためなど、複数の目的に使用されています。後で使用するためには、そのmRNA発現、脳内の神経活動の増加、頻繁に呼び出さ活​​動依存的遺伝子または即時早期遺伝子に依存している遺伝子にあります。孵化/新生児、少年を含む、複数の年齢、複数の組織で、脳、皮膚、筋肉を含む。これらの用途で、我々の方法は、哺乳動物（例えばヒト）、魚、両生類、鳥類を含め、複数種を越えて適用されています、成人、ここで全体の胚セクションの2,3,5,15-17。我々のプロトコルの特殊な機能が含まれます：（1）は、解剖学的特異性と定量的な特異性のバランスを生成します。 X線フィルム上に遺伝子発現を定量化するために、我々は画像のデジタル写真を撮ること（例：図1Aおよび1B）、フォーを使用しtoshop（アドビシステムズ社）の関心領域内のピクセル密度を測定し、組織の外膜の上にバックグラウンドレベルを減算するヒストグラム機能はまだスライドガラス2,4に。細胞レベルでの発現を定量するために、我々は高倍率下での細胞（ 図4】40-100X）を介して銀粒子の画像を撮影する。我々は、その後スライドガラス上の細胞なしで同じような領域でのバックグラウンドのカウントを減算し、画像内の銀粒子の数をカウントするためにNIHでのWayne Rasbandによる画像Jのしきい値および測定機能を使用して、セルの数で割るセル当たりの式の値。4,18を取得する（2）が原因鉱物油浴によって作成されたタイトなカバースリップのシールに、同時に100から200スライドの処理を可能にする、相対的に高いスループットをすることができます。標準のin-situハイブリダイゼーション法では、パラフィルム、マニキュア、スライドは多くのスペースを取る他の手段で、スライドを封止するために時間がかかる、（3）これは非常にあるデキストラン硫酸とハイブリダイゼーション緩衝液2,13でデンハルト溶液による低濃度の転写産物の高感度（4）暗視野の撮像手法は、4,5解剖顕微鏡に暗視野照明の下で写真を撮るために、コントラストの高い画像が得られます。さらに、このような活動依存的遺伝子発現のように、遺伝子発現の小さな変化の高感度な検出が特定の行動を19の知覚と生成の間にアクティブに特定の脳領域を識別することができます。非放射性プロトコルへの相対的な制限が、後者は携帯電話の解像度と、細胞内のmRNAの場所に明確であることであり、エマルジョンでの作業は、操作や光に対して非常に敏感です。同じ組織10,17,20で複数の遺伝子のmRNA発現にラベルを付けるためにin situハイブリダイゼーションなどの非放射性として、他の方法と手法を組み合わせることができます。それは免疫細胞化学と組み合わせることができます特定の細胞型10との共局在mRNAにために、同じサンプルにRNAとタンパク質の発現の両方にラベルを付けます。このような二重標識実験のために必要なプロトコルの変更は、引用文献に記載されています。 要約すると、我々のアプローチは、地域組織、組織の機能活性、遺伝子機能を理解するためにタイミングと遺伝子発現の細胞の位置の理解を容易にします。

<table border="1"><tbody><tr><td> 試薬の名前 </td><td> 会社 </td><td> カタログ番号 </td><td> コメント </td></tr><tr><td>無水酢酸</td><td> VWR </td><td> MK242002 </td><td></td></tr><tr><td>クロロホルム</td><td> VWR </td><td> BDH1109 </td><td></td></tr><tr><td>クレシルバイオレット酢酸</td><td>シグマ</td><td> C5042 </td><td></td></tr><tr><td>クライオスタット</td><td>サーモフィッシャーサイエンティフィック</td><td>マイクロモルHM550 </td><td></td></tr><tr><td>脱イオン化ホルムアミド</td><td>シグマ</td><td> F9037 </td><td></td></tr><tr><td> DTT </td><td>プロメガ</td><td> P1171 </td><td> 100mMの</td></tr><tr><td> EDTA </td><td>シグマ</td><td> ED </td><td></td></tr><tr><td>埋め込み型</td><td> VWR </td><td> 15160-215 </td><td></td></tr><tr><td>フィッシャーブランドSuperfrostプラススラ​​イド</td><td>フィッシャー·サイエンティフィック</td><td> 22-034-979 </TD&gt; <td></td></tr><tr><td>ホルムアルデヒド</td><td> VWR </td><td> BDH0506-4LP </td><td></td></tr><tr><td>ホルムアミド</td><td>シグマ</td><td> F7508 </td><td></td></tr><tr><td> GENECLEANキット</td><td> Q-バイオ遺伝子</td><td> 1001-200 </td><td></td></tr><tr><td>コダックバイオマックスMRフィルム</td><td>シグマ</td><td> Z350370 </td><td></td></tr><tr><td>コダックNTBエマルジョン</td><td>ケアストリームヘルス社</td><td> 8895666 </td><td></td></tr><tr><td>コダックプロフェッショナル開発者D19 </td><td>コダック</td><td> 1462593 </td><td></td></tr><tr><td>コダックプロフェッショナルフィクサー</td><td>コダック</td><td> 1971746 </td><td></td></tr><tr><td> β-メルカプトエタノール</td><td>カルビオケム</td><td> 444203 </td><td></td></tr><tr><td>鉱油</td><td> VWR </td><td> IC15169491 </td><td></td></tr><tr><td> NaOHで</td><td> VWR </td><td> SX0600-1 </td><td></td></tr><tr><td>パラホルムアルデヒド</td><td>シグマ</td><td> 76240 </td><td></td></tr><tr><td>ポリ</td><td>インビトロジェン</td><td> POLYA.GF </td><td></td></tr><tr><td> RATP </td><td>プロメガ</td><td> P1132 </td><td> 10mMの</td></tr><tr><td> rCTP </td><td>プロメガ</td><td> P1142 </td><td> 10mMの</td></tr><tr><td> rGTP </td><td>プロメガ</td><td> P1152 </td><td> 10mMの</td></tr><tr><td> RNasin </td><td>プロメガ</td><td> N2111 </td><td> 40Units/μl </td></tr><tr><td> S 35 UTP </td><td>パーキンエルマー</td><td> NEG039C001MC </td><td></td></tr><tr><td>安全解決ソリューション</td><td>安全性は、研究プロダクツ国際を解決する</td><td> 111177 </td><td></td></tr><tr><td>酢酸ナトリウム</td><td>シグマ</td><td> S7899 </td><td> 3M </td></tr><tr><td>リン酸ナトリウム二塩基</td><td>シグマ</td><td> S3264 </td><td></td></tr><tr><td>ナトリウム一塩基性リン</td><td>シグマ</td><td> S3139 </td><td></td></tr><tr><td> SP6RNAポリメラーゼ</td><td>プロメガ</td><td> P1085 </td><td></td></tr><tr><td>メタルラックを染色</td><td>電子顕微鏡学</td><td> 70312から54 </td><td></td></tr><tr><td> T7 RNAポリメラーゼ</td><td>プロメガ</td><td> P2075 </td><td></td></tr><tr><td>組織テック10月</td><td>桜</td><td> 4583 </td><td></td></tr><tr><td> 5倍の転写バッファー</td><td>プロメガ</td><td> P1181 </td><td></td></tr><tr><td>トリエタノールアミン</td><td> VWR </td><td> IC15216391 </td><td></td></tr><tr><td>トリス-HCl（1M、pH8.0）に</td><td> VWR </td><td> 101449-446 </td><td></td></tr><tr><td> tRNAの</td><td>ロシュ社</td><td> 10109509001 </td><td></td></tr></tbody></table>ソリューション： <ol><li>リボプローブを作るための試薬：1.5μlのDNAテンプレート（0.2から0.3μg/μLの）、5最適化された転写緩衝液2μl、100mMのDTT 1μlの（Promegaからポリメラーゼが付属しており、ミックスよく室温）、RNasin（40ユニット/μl）、AGCリボヌクレオチド混合液を、S 35 UTPの3.5μlを、1μlのRNAポリメラーゼ1.5μlを0.3μL。ヌクレアーゼ·フリー水10μlに起動します。 </li><li> AGCリボヌクレオチド混合液：ミックス一緒に10mMのATP、GTPとCTPの等量。 </li><li>フリーS 35 UTPを削除するエタノール沈殿用酢酸ナトリウム溶液：40μlのRNaseおよびDNaseフリー水、3M酢酸ナトリウム5μlの、100％エタノール125μlの。 </li><li>ハイブリダイゼーション緩衝液（10ミリリットル株）：100％脱イオンホルムアミド5mlの、5M NaClを600μlの、1Mトリス塩酸液（pH = 8.0）、0.5M EDTA 240液（pH = 8.0）、100×デンハルトの溶液100μl、 1M DTT 100μlの、20mg/ml tRNAの250μlの20 mg / mlのポリと、デキストラン硫酸ナトリウム1gを125μlの。 10ミリリットルの合計体積をもたらすためにDEPC処理水を追加します。激しく振とうして、すべてのデキストラン硫酸ナトリウムが溶解するまで55℃でインキュベートする。にハイブリダイゼーションバッファーを格納する〜6ヶ月に適しています-20°C、。 </li><li> 4％パラホルムアルデヒド緩衝溶液：ホルムアルデヒド用に指定され、50〜ヒート°Cのホットプレート上で撹拌しながらフラスコ内で760ミリリットルの蒸留水にホルムアルデヒドの40グラムを追加します。パラホルムアルデヒドを溶解するのを助けるために10NのNaOH 320μlを添加します。 （〜10分）に溶解した後、10倍のPBS 100ミリリットルを追加し、1リットルになるまで蒸留水を使用してボリュームを起動します。ホルムアルデヒドが溶解するまで溶液を攪拌し、加熱する。 pHは7.4でなければなりません。 </li><li>アセチル化バッファー：トリエタノールアミン13.6ミリリットルを加え、蒸留水1リットル中の無水酢酸の2.52ミリリットル。 </li><li> 20×SSPE溶液：3MのNaCl、200mMののNaH 2 PO 4-H 2 O、および蒸留水で200 mMのEDTA。 10N NaOHでpH7.4に解決策を調整します。 </li><li> 10×PBS緩衝液：蒸留水のKH 2 PO 4のNaClの80グラム、KClの2.0グラム、14.4グラムのNa 2 HPO 4、2.4グラム。 pHを7.0にソリューションを調整し、1 Liteに総量をもたらすR。 </li><li>第二洗浄液：0.1％β-メルカプトエタノールおよび2x SSPEで50％のホルムアミド</li><li>クレシルバイオレット酢酸溶液：水道水の3％クレシルバイオレット酢酸（蒸留水が良い染色を防ぎます）、室温でフラスコ内を撹拌しながら一晩溶解した。 1ミリメートルワットマン濾紙とブフナー漏斗を介して真空吸引してフィルタリングします。 </li></ol>

radioactive in situ hybridization for detecting diverse gene expression patterns in tissue

遺伝子は、遺伝子の機能の理解に貢献するで表現されているタイミング、レベル、細胞内局在、細胞の種類を知っている。これらの各機能は、細胞内のmRNAのin situハイブリダイゼーションを行うことができます。ここでは、クレイトンら （1988）1特に大人の脊椎動物の脳2-5、長年にわたって私たちのラボで正常に働いているから修正in situハイブリダイゼーション法で放射性を提示する。標的配列への長い相補的RNA（cRNA）をプローブは、低濃度の転写産物6,7の検出を可能にします。 cRNAプローブに放射性ヌクレオチドの取り込みのいずれかの組織の上にコーティングされた光に敏感なX線フィルム又はエマルジョンにより、低濃度の転写産物と定量分析の更なる検出感度を可能にします。これらの検出方法は、標的遺伝子の発現の長期的な記録を提供しています。非放射性プローブ​​をメタに比べてそのようなDIG標識としてODS、放射性プローブ​​のハイブリダイゼーション法は、低濃度の転写産物を検出するためにHRP-抗体および/またはTSAキットを使用して複数の増幅工程を必要としません。したがって、このメソッドは、定量分析のための信号強度と標的mRNAの量の間の線形関係を提供しています。それは同時に100から200までのスライドを処理することができます。それは胚の発達段階の異なるにも適しています。胚組織は、それが困難な組織切片を持つ細胞集団間の境界を確認しながら、細胞間の少ない凝集して、成体組織よりも脆弱であるため、ほとんどの遺伝子発現の発達研究では、全体の胚および一部の非放射性のアプローチ8,9を使用しています 。対照的に、感度のより広い範囲のために私たちの放射性アプローチは、それが簡単に集団間の境界を見て作り、組織領域間の遺伝子発現の解像度で高コントラストを得ることができる。この方法を使用して、研究者は明らかにすることができ新たに同定された遺伝子の可能性の意義、さらに興味のある遺伝子の機能を予測する。

Watch this Scientific Journal Video about Radioactive in situ Hybridization for Detecting Diverse Gene Expression Patterns in Tissue at JoVE.com

Radioactive in situ Hybridization for Detecting Diverse Gene Expression Patterns in Tissue

このプロトコルが正常に定量的な脊椎動物種間で複数の組織の種類のレベルとmRNA発現の空間パターンを検出するために使用されています。メソッドは、低濃度の転写物を検出すると同時にスライドの何百もの処理を可能にすることができます。我々は、例として、鳥類の胚の脳形成の発現プロファイリングを使用して、このプロトコルを提示します。

放射性その場で</em組織における多様な遺伝子発現パターンを検出するための>ハイブリダイゼーション

Knowing the timing, level, cellular localization, and cell type that a gene is expressed in contributes to our understanding of the function of the gene. ...

radioactive-situ-hybridization-for-detecting-diverse-gene-expression

Research

JoVE Journal

Biology

Radioactive in situ Hybridization for Detecting Diverse Gene Expression Patterns in Tissue

15.5K ビュー.  Duke University. このプロトコルが正常に定量的な脊椎動物種間で複数の組織の種類のレベルとmRNA発現の空間パターンを検出するために使用されています。メソッドは、低濃度の転写物を検出すると同時にスライドの何百もの処理を可能にすることができます。我々は、例として、鳥類の胚の脳形成の発現プロファイリングを使用して、このプロトコルを提示します。

ビデオ: Radioactive in situ Hybridization for Detecting Diverse Gene Expression Patterns in Tissue

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell  electroporation system and Gene Pulser  electroporation buffer were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

エレクトロポレーションのためのマウス骨髄からの一次造血細胞培養の準備

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

生物学

Non-radioactive in situ Hybridization Protocol Applicable for Norway Spruce and a Range of Plant Species

The high-throughput expression analysis technologies available today give scientists an overflow of expression profiles but their resolution in terms of tissue specific expression is limited because of problems in dissecting individual tissues. Expression data needs to be confirmed and complemented with expression patterns using e.g. in situ hybridization, a technique used to localize cell specific mRNA expression. The in situ hybridization method is laborious, time-consuming and often requires extensive optimization depending on species and tissue. In situ experiments are relatively more difficult to perform in woody species such as the conifer Norway spruce (Picea abies). Here we present a modified DIG in situ hybridization protocol, which is fast and applicable on a wide range of plant species including P. abies. With just a few adjustments, including altered RNase treatment and proteinase K concentration, we could use the protocol to study tissue specific expression of homologous genes in male reproductive organs of one gymnosperm and two angiosperm species; P. abies, Arabidopsis thaliana and Brassica napus. The protocol worked equally well for the species and genes studied. AtAP3 and BnAP3 were observed in second and third whorl floral organs in A. thaliana and B. napus and DAL13 in microsporophylls of male cones from P. abies. For P. abies the proteinase K concentration, used to permeablize the tissues, had to be increased to 3 g/ml instead of 1 g/ml, possibly due to more compact tissues and higher levels of phenolics and polysaccharides. For all species the RNase treatment was removed due to reduced signal strength without a corresponding increase in specificity. By comparing tissue specific expression patterns of homologous genes from both flowering plants and a coniferous tree we demonstrate that the DIG in situ protocol presented here, with only minute adjustments, can be applied to a wide range of plant species. Hence, the protocol avoids both extensive species specific optimization and the laborious use of radioactively labeled probes in favor of DIG labeled probes. We have chosen to illustrate the technically demanding steps of the protocol in our film.
Anna Karlgren and Jenny Carlsson contributed equally to this study.
Corresponding authors: Anna Karlgren at Anna.Karlgren@ebc.uu.se and Jens F. Sundstr&#246;m at Jens.Sundstrom@vbsg.slu.se

Non-radioactive in situ Hybridization Protocol Applicable for Norway Spruce and a Range of Plant Species

We describe a modified DIG in situ hybridization protocol, which is fast and applicable on a wide range of plant species including Norway spruce. With just a few adjustments, including altered RNase treatment and proteinase K concentration, the protocol may be used in studies of different tissues and species.

非放射性その場で</emノルウェースプルースと植物種の範囲に適用>ハイブリダイゼーションプロトコール

The high-throughput expression analysis technologies available today give scientists an overflow of expression profiles but their resolution in terms of ...

Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell electroporation system and Gene Pulser electroporation buffer (Bio-Rad) were specifically developed to easily transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells. We will demonstrate how to perform a simple experiment to quickly identify the best electroporation conditions. We will demonstrate how to run several samples through a range of electroporation conditions so that an experiment can be conducted at the same time as optimization is performed. We will also show how optimal conditions identified using 96-well electroporation plates can be used with standard electroporation cuvettes, facilitating the switch from electroporation plates to electroporation cuvettes while maintaining the same electroporation efficiency. In the video, we will also discuss some of the key factors that can lead to the success or failure of electroporation experiments.

This procedure shows how to use the Gene Pulser MXcell electroporation system to rapidly and easily identify the best electroporation conditions for mouse embryonic fibroblasts (MEFs) or other primary cells. Considerations for troubleshooting are also discussed in the associated video.

高効率で初代培養細胞をトランスフェクトするためにジーンパルサーMXcellのエレクトロポレーションシステムの使用

Using an Automated Cell Counter to Simplify Gene Expression Studies: siRNA Knockdown of IL-4 Dependent Gene Expression in Namalwa Cells

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only to study the effects of downregulating single genes, but also to interrogate signaling pathways and other complex interaction networks.  These pathway analyses require both the use of relevant cellular models and methods that cause less perturbation to the cellular physiology.  Electroporation is increasingly being used as an effective way to introduce siRNA and other nucleic acids into difficult to transfect cell lines and primary cells without altering the signaling pathway under investigation. There are multiple critical steps to a successful siRNA experiment, and there are ways to simplify the work while improving the data quality at several experimental stages.  To help you get started with your siRNA mediated gene knockdown project, we will demonstrate how to perform a pathway study complete from collecting and counting the cells prior to electroporation through post transfection real-time PCR gene expression analysis.  The following study investigates the role of the transcriptional activator STAT6 in IL-4 dependent gene expression of CCL17 in a Burkitt lymphoma cell line (Namalwa).  The techniques demonstrated are useful for a wide range of siRNA-based experiments on both adherent and suspension cells.  We will also show how to streamline cell counting with the TC10 automated cell counter, how to electroporate multiple samples simultaneously using the MXcell electroporation system, and how to simultaneously assess RNA quality and quantity with the Experion automated electrophoresis system.

This procedure describes a quick and easy workflow to introduce siRNA into difficult to transfect cell lines and follow gene expression by real-time PCR. Use of an automated cell counter, multi-well electroporation plate, and automated electrophoresis station provide quick and reliable results without the need for expensive robotic handling.

ナマルバ細胞におけるIL - 4依存性遺伝子発現のsiRNAのノックダウン：遺伝子発現研究を簡素化する自動化セルカウンターの使い方

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only ...

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.
Telomerase is a specialized reverse transcriptase1 that adds short DNA repeats, called telomeres, to the 3' end of linear chromosomes2 that serve to protect them from end-to-end fusion and degradation. Following DNA replication, a short segment is lost at the end of the chromosome3 and without telomerase, cells continue dividing until eventually reaching their Hayflick Limit4. Additionally, telomerase is dormant in most somatic cells5 in adults, but is active in cancer cells6 where it promotes cell immortality7.
The minimal telomerase enzyme consists of two core components: the protein subunit (TERT), which comprises the catalytic subunit of the enzyme and an integral RNA component (TER), which contains the template TERT uses to synthesize telomeres8,9. Prior to 2008, only structures for individual telomerase domains had been solved10,11. A major breakthrough in this field came from the determination of the crystal structure of the active12, catalytic subunit of T. castaneum telomerase, TcTERT1.
Here, we present methods for producing large quantities of the active, soluble TcTERT for structural and biochemical studies, and the reconstitution of the telomerase RNP complex in vitro for telomerase activity assays. An overview of the experimental methods used is shown in Figure 1.

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.

アクティブT.の試験管内再構成でcastaneumのテロメラーゼ

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more ...

A High Throughput in situ Hybridization Method to Characterize mRNA Expression Patterns in the Fetal Mouse Lower Urogenital Tract

Development of the lower urogenital tract (LUT) is an intricate process. This complexity is evidenced during formation of the prostate from the fetal male urethra, which relies on androgenic signals and epithelial-mesenchymal interactions1,2. Understanding the molecular mechanisms responsible for prostate development may reveal growth mechanisms that are inappropriately reawakened later in life to give rise to prostate diseases such as benign prostatic hyperplasia and prostate cancer.
The developing LUT is anatomically complex. By the time prostatic budding begins on 16.5 days post conception (dpc), numerous cell types are present. Vasculature, nerves and smooth muscle reside within the mesenchymal stroma3. This stroma surrounds a multilayered epithelium and gives rise to the fetal prostate through androgen receptor-dependent paracrine signals4. The identity of the stromal androgen receptor-responsive genes required for prostate development and the mechanism by which prostate ductal epithelium forms in response to these genes is not fully understood. The ability to precisely identify cell types and localize expression of specific factors within them is imperative to further understand prostate development. In situ hybridization (ISH) allows for localization of mRNAs within a tissue. Thus, this method can be used to identify pattern and timing of expression of signaling molecules and their receptors, thereby elucidating potential prostate developmental regulators.
Here, we describe a high throughput ISH technique to identify mRNA expression patterns in the fetal mouse LUT using vibrating microtome-cut sections. This method offers several advantages over other ISH protocols. Performing ISH on thin sections adhered to a slide is technically difficult; cryosections frequently have poor structural quality while both cryosections and paraffin sections often result in weak signal resolution. Performing ISH on whole mount tissues can result in probe trapping. In contrast, our high throughput technique utilizes thick-cut sections that reveal detailed tissue architecture. Modified microfuge tubes allow easy handling of sections during the ISH procedure. A maximum of 4 mRNA transcripts can be screened from a single 17.5dpc LUT with up to 24 mRNA transcripts detected in a single run, thereby reducing cost and maximizing efficiency. This method allows multiple treatment groups to be processed identically and as a single unit, thereby removing any bias for interpreting data. Most pertinently for prostate researchers, this method provides a spatial and temporal location of low and high abundance mRNA transcripts in the fetal mouse urethra that gives rise to the prostate ductal network.

A High Throughput in situ Hybridization Method to Characterize mRNA Expression Patterns in the Fetal Mouse Lower Urogenital Tract

Here, we describe an efficient high throughput in situ hybridization (ISH) method for visualizing patterns of mRNA expression in developing fetal mouse prostate tissue sections. The method can be easily adapted to visualize mRNA expression patterns in other mouse tissues or in tissues from other species.

ハイスループットその場で</em胎児マウス下部尿生殖路のmRNAの発現パターンを特徴づける>ハイブリダイゼーション法

Development of the lower urogenital tract (LUT) is an intricate process. This complexity is evidenced during formation of the prostate from the fetal male ...

Analysis of Single-cell Gene Transcription by RNA Fluorescent In Situ Hybridization (FISH)

Adhesion of Plasmodium falciparum infected erythrocytes (IE) to human endothelial receptors during malaria infections is mediated by expression of PfEMP1 protein variants encoded by the var genes.
The haploid P. falciparum genome harbors approximately 60 different var genes of which only one has been believed to be transcribed per cell at a time during the blood stage of the infection. How such mutually exclusive regulation of var gene transcription is achieved is unclear, as is the identification of individual var genes or sub-groups of var genes associated with different receptors and the consequence of differential binding on the clinical outcome of P. falciparum infections. Recently, the mutually exclusive transcription paradigm has been called into doubt by transcription assays based on individual P. falciparum transcript identification in single infected erythrocytic cells using RNA fluorescent in situ hybridization (FISH) analysis of var gene transcription by the parasite in individual nuclei of P. falciparum IE1.
Here, we present a detailed protocol for carrying out the RNA-FISH methodology for analysis of var gene transcription in single-nuclei of P. falciparum infected human erythrocytes. The method is based on the use of digoxigenin- and biotin- labeled antisense RNA probes using the TSA Plus Fluorescence Palette System2 (Perkin Elmer), microscopic analyses and freshly selected P. falciparum IE. The in situ hybridization method can be used to monitor transcription and regulation of a variety of genes expressed during the different stages of the P. falciparum life cycle and is adaptable to other malaria parasite species and other organisms and cell types.

Analysis of Single-cell Gene Transcription by RNA Fluorescent In Situ Hybridization FISH

Fluorescent in situ hybridization (FISH) to identify mRNA transcripts in individual cells allows analysis of polygenic activity such as the simultaneous transcription of more than one member of the var multigene family in Plasmodium falciparum infected erythrocytes 1. The technique is adaptable and can be used on different types of genes, cells and organisms.

RNAの蛍光による単一細胞遺伝子転写の解析<em&gt; in situで</em&gt;ハイブリダイゼーション（FISH）

Adhesion of Plasmodium falciparum infected erythrocytes (IE) to human endothelial receptors during malaria infections is mediated by expression of PfEMP1 ...

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for using FISH to quantify the number of mRNAs in single yeast cells. Cells can be grown in any condition of interest and then fixed and made permeable. Subsequently, multiple single-stranded deoxyoligonucleotides conjugated to fluorescent dyes are used to label and visualize mRNAs. Diffraction-limited fluorescence from single mRNA molecules is quantified using a spot-detection algorithm to identify and count the number of mRNAs per cell. While the more standard quantification methods of northern blots, RT-PCR and gene expression microarrays provide information on average mRNAs in the bulk population, FISH facilitates both the counting and localization of these mRNAs in single cells at single-molecule resolution.

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

This protocol describes an experimental procedure for performing Fluorescence in situ Hybridization (FISH) for counting mRNAs in single cells at single-molecule resolution.

蛍光を用いたmRNA分子の可視化と分析<em&gt;その場で</emで&gt;ハイブリダイゼーション<em&gt;サッカロミセス·セレビシエ（Saccharomyces cerevisiae）</em

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for ...

High Resolution Whole Mount In Situ Hybridization within Zebrafish Embryos to Study Gene Expression and Function

This article focuses on whole-mount in situ hybridization (WISH) of zebrafish embryos. The WISH technology facilitates the assessment of gene expression both in terms of tissue distribution and developmental stage. Protocols are described for the use of WISH of zebrafish embryos using antisense RNA probes labeled with digoxigenin. Probes are generated by incorporating digoxigenin-linked nucleotides through in vitro transcription of gene templates that have been cloned and linearized. The chorions of embryos harvested at defined developmental stages are removed before incubation with specific probes. Following a washing procedure to remove excess probe, embryos are incubated with anti-digoxigenin antibody conjugated with alkaline phosphatase. By employing a chromogenic substrate for alkaline phosphatase, specific gene expression can be assessed. Depending on the level of gene expression the entire procedure can be completed within 2-3 days.

High Resolution Whole Mount In Situ Hybridization within Zebrafish Embryos to Study Gene Expression and Function

The zebrafish, a small tropical fish, has become a popular model for studying gene function during vertebrate development and disease. The temporal and spatial expression of target genes can be determined by in situ hybridization. Our improved protocol allows for the detection of low abundant transcripts with low non-specific background signal.

高解像度全マウント<em&gt;その場で</em遺伝子発現と機能を研究するためにゼブラフィッシュ胚内&gt;ハイブリダイゼーション

This article focuses on whole-mount in situ hybridization (WISH) of zebrafish embryos. The WISH technology facilitates the assessment of gene expression ...

MicroRNA In situ Hybridization for Formalin Fixed Kidney Tissues

In this article we describe a method for colorimetric detection of miRNA in the kidney through in situ hybridization with digoxigenin tagged microRNA probes. This protocol, originally developed by Kloosterman and colleagues for broad use with Exiqon miRNA probes1, has been modified to overcome challenges inherent in miRNA analysis in kidney tissues. These include issues such as structure identification and hard to remove residual probe and antibody. Use of relatively thin, 5 mm thick, tissue sections allowed for clear visualization of kidney structures, while a strong probe signal was retained in cells. Additionally, probe concentration and incubation conditions were optimized to facilitate visualization of microRNA expression with low background and nonspecific signal. Here, the optimized protocol is described, covering the initial tissue collection and preparation through the mounting of slides at the end of the procedure. The basic components of this protocol can be altered for application to other tissues and cell culture models.

MicroRNA In situ Hybridization for Formalin Fixed Kidney Tissues

This article describes an in situ hybridization protocol optimized for colormetric detection of microRNA expression in formalin fixed kidney sections.

マイクロRNA<em&gt;その場で</em&gt;ホルマリン固定腎臓組織のためのハイブリダイゼーション

In this article we describe a method for colorimetric detection of miRNA in the kidney through in situ hybridization with digoxigenin tagged microRNA ...

放射性その場でハイブリダイゼーション