Research supported by Alberta Livestock and Meat Agency, Alberta Innovates - BioSolutions, Alberta Milk, and Livestock Research Branch, Alberta Agriculture and Forestry.

この研究の動物の一部は、アルバータ州の動物実験委員会の大学によって承認され、すべての動物実験手順（プロトコル＃AUP00000131）と代謝研究アルバータ大学の単位、エドモントン、カナダ、で行われ、そして動物は応じての世話をしました。アニマルケアのガイドライン（1993年）のカナダ人評議会へ。 1.胚の生産、全RNAおよびDNアーゼ治療の単離<ol><li>動物実験プロトコルおよび胚の収集のために私たちの以前の出版物17、18に参照してください。 </li><li>プールし、液体窒素中で各乳牛から同様の段階の胚を凍結した後、RNA抽出まで-80℃に保つスナップ。 </li><li> （キットの情報は材質一覧で利用可能です）ピコ規模なサンプルからRNAを抽出することが可能になり、カラムベースのRNA抽出キットを経由して全RNAを抽出します。 注：推奨キットが含まれています：コンディショニング・バッファ、抽出BUFをFER、70％エタノール、洗浄バッファー1、洗浄バッファー2、溶出バッファー、収集管とマイクロ遠心チューブを有するRNA精製カラム。 </li><li> 10μLのExtraction Bufferは、胚を含むチューブに追加し、42℃で30分間インキュベートします。 2分間800×gで遠心分離管をマイクロ遠心チューブに細胞抽出物を収集します。 </li><li>精製カラムのフィルター膜上に250μLコンディショニング・バッファをピペット。室温で5分間コンディショニングバッファを有するRNA精製カラムをインキュベートします。 1分間16,000×gでコレクションチューブに精製カラムを遠心します。 </li><li>ピペットRNA抽出緩衝液および胚の混合物中に70％のエタノールの10μL。ピペッティングによりよく混ぜます。プレコンディショニング精製カラムにピペット混合物。 </li><li> 100×gで2分間、RNA、遠心分離精製カラムを結合して、すぐに通る流れを除去するために30秒間16,000×gで遠心分離することによって従ってください。 </li><li>ピペット10081; 8000×gで1分間の精製カラムと遠心への洗浄緩衝液1のL。 注：DNアーゼ処理は、RNA単離の後、逆転写又は増幅を行う場合にお勧めします。 </li><li>右のステップ1.8の後、ゲノムDNAを消化。 注：推奨のDNaseキットは素材リストで利用可能です。推奨されるキットが含まれています：DNアーゼIストック溶液およびバッファを。 </li><li> 35μLバッファーで5μLのDNase Iストック溶液を混合することにより、DNアーゼインキュベーション混合物を準備します。静かに転倒混和。 </li><li>精製カラムの膜に直接40μLのDNaseインキュベーション混合物をピペット。室温で15分間インキュベートします。 </li><li>精製カラム膜へのピペット40μL洗浄緩衝液1、次いで15秒間8000×gで遠心します。 </li><li> 8,000×gで1分間の精製カラムと遠心にピペットで100μLの洗浄緩衝液2。 </li><li>精製カラムと遠心機の中に別の100μLの洗浄緩衝液2をピペット16,000×gで2分間。 </li><li>新しい0.5 mlのマイクロ遠心チューブに精製カラムを転送します。直接精製カラムのメンブレン上に溶出バッファーのピペット11μL。溶出緩衝液を分配しながら、膜への溶出バッファーの最大吸収を確実にするために、穏やかに膜の表面にピペットの先端をタッチします。 </li><li>室温で1分間カラムをインキュベートします。カラムに溶出バッファーを配布するために千×gで1分間カラムを遠心してから、RNAを溶出するために16,000×gで1分間スピン。 </li><li> RNAの質と量19を評価するための製造元の指示に従ってバイオアナライザー機器を使用してください。 </li><li>使用するまで-80℃でRNAサンプルを保管してください。 </li></ol> 2.マイクロアレイ解析のためのRNA増幅とラベリング注：RNA増幅とラベリング手順で作業初めてのユーザーのために、RNA ALOにスパイクの5 ngのを使用RNA増幅およびハイブリダイゼーションの品質管理測定を監視するために、実際のaRNAサンプルを用いてngの。スパイク中のRNAミックスが所定の割合で10 in vitroで合成し、ポリアデニル化転写物を含んでいます。手順が適切に実行すると、標識された転写物は、具体的にのみ配列中の相補的なコントロールプローブにハイブリダイズしたデータは、最小限の自己または交差ハイブリダイゼーションと品質管理保証を追跡するためにスキャンすることができます。アレイおよびデータ分析をスキャンした後スパイクインコントロール強度と正規化手順についての詳細の説明は、以前の出版物20、21を参照してくださいです。以下の手順は、通常のマイクロアレイユーザに与えられます。 <ol><li> 11μL - 10の全体積で100 pgの高品質なRNAサンプル（RNA完全性番号（RIN）の値は、少なくとも&gt; 7.0）を準備します。 </li><li>増幅キットはにできることでRNAサンプルを増幅（100頁わずかとして）ピコスケールのサンプルで動作します。 注：推奨増幅キット情報は、マテリアルリストで利用可能で、任意の変更を加えることなく、製造元の指示に従います。 </li><li>増幅されたaRNAのサイズ範囲のプロファイルを評価するために、蛍光ベースの定量を使用してください。 </li><li>増幅されたaRNAの濃度を測定する（260および280 nmの吸光度に基づいて）分光光度計の測定器を使用してください。 </li><li>標識のための準備ができるまで-80℃で増幅されたaRNAを保管してください。 </li><li> ULS aRNAのラベリングキットのユーザーガイドに記載の蛍光標識のために増幅されたRNA2μgのを使用してください。 注：シアニン547及びシアニン647色素は光劣化及びシアニン647色素に敏感であるので、容易にオゾンによって分解され、標識操作は、光の最小量のオゾンのない環境下で行われます。 </li><li>オゾンのレベルは0.001 ppm以下になるまで、オゾンボックス（ 図1A）をオンにします。 </li><li&gt;はシアニン547またはシアニン647、ラベリングバッファーの2μLの2μLを追加することによって、オゾンボックス（ 図1B）内の反応セットアップを行い、その後、セーフライトの下でRNaseフリー水で20μLの最終容量に調整します。光源に暗室フィルタを追加します。 </li><li>穏やかに混合し、15分間85℃でサーモサイクラーにチューブをインキュベートします。少なくとも1分間、氷の上に置いてサンプル。 CY-色素標識増幅されたRNAをクリーンアップするために、（1.9から1.11へ）DNase処理に関連する手順を除いて、RNA抽出キットを進める前に、チューブの内容を収集するためにスピンダウン。 </li><li>測定および標識された増幅されたRNAの濃度を測定し、使用前に3日間、-80℃の最大で標識したaRNAを維持するために、分光光度計の測定器を使用してください。 </li></ol> 3.マイクロアレイハイブリダイゼーションおよび洗浄注：次の重要なステップは、二色マイクロアレイベースの遺伝子発現解析プロトコル（バージョンに応じて適応されます6.5、2010年5月）。 <ol><li>各シアニン547-から825 ngの同量のを追加し、シアニン647標識したaRNA及び25Xフラグメンテーションと10倍のブロッキングバッファーと混合します。 </li><li> 15分間60℃で混合物を加熱し、1分間氷上で冷却します。 </li><li> 2×ハイブリダイゼーション緩衝液の同量を加え、よく混ぜます。 </li><li> 13,000 rpmで遠心分離し、次いで混合物は、ハイブリダイゼーションのための準備ができています。 </li><li>アレイスライド上に混合物から100μLをロードし、ハイブリダイゼーションチャンバ内のスライドをシール。 </li><li>オーブンで10 rpmで回転させ、65℃で17時間ハイブリダイゼーションオーブンに組み立て室をロードします。 </li><li>安定化に浸漬し、30秒のための溶液を乾燥することによりオゾン保護処理とプロトコルに従って配列を洗ってください。 </li><li>無塵粒子を溶液からアレイを削除し、アレイ表面が乾燥していることを確認してください</li></ol> 4.スキャンマイクロアレイスライド<ol><li>暗い場所で配列を維持し、scannをオンにしますえー（15分の待ち時間）を使用する準備ができるまで。 </li><li>スキャナーに、左側の列のバーコードをロードし、スキャンを開始します。ソフトウェアのユーザーガイドに従ってスポット分析を行うと（GPR）ファイル形式としてデータを保存します。 </li></ol> 5.統計解析<ol><li> FlexArrayソフトウェアをダウンロードhttp://genomequebec.mcgill.ca/FlexArray/license.php「輸入生データ」をクリックし、FlexArrayにGPRファイルをロードします。 </li><li>必要に応じて指示に従うことによって正規化および統計分析を実行します。注：それは以前に22に記載されたように、本研究ではポジティブスポット選択のためのしきい値を計算します。 </li><li>すべてのハイブリダイズしたスポットやバックグラウンド信号を表示するFlexArrayのプロットビューアから手動ドロップダウンを選択します。注：マイクロアレイへのハイブリダイゼーション後のコントロールにスパイクの同様の絵は、以前の研究8で見ることができます。 </li><li>選択しますFlexArrayからのアレイの正規化プロセスの間に変位することにより、以下の配列内の黄土正規化は、それに応じてマニュアルをドロップダウン。 </li><li>エクスポート、他の用途のためのExcelファイルにFlexArrayからすべての正規化されたデータ。 </li></ol> 6.バイオインフォマティクス解析ウシ胚特異的転写産物からのプローブ配列の元の注釈は（BESTv1）マイクロアレイは8以前に記載されています。本研究では、20403ユニークな遺伝子シンボル（GS）は、（得られた<a href="http://ecsource.jove.com/files/ftp_upload/53754/Supplement_file_1.xlsx">File1のサプリメント</a> EmbryoGENEラボラトリー情報管理システム（LIMS）ELMA（http://elma.embryo-gene.ca）を介して）。 6765ユニークな遺伝子（のリストは<a href="http://ecsource.jove.com/files/ftp_upload/53754/Supplement_file_2.xlsx">File2の補足</a> ）（マイクロアレイ正規化処理後に選択し、すべての正の信号に対応したステップ50.5）ウシの7日目の胚の遺伝子発現プロファイルから。陽性シグナル閾値はA値の信号強度から透過刊行16に基づいて計算しました。 <ol><li>分類システム23、24 PANTHER（進化的関係を通じてタンパク質解析）との機能解析を実行するには、以下のリンク（http://www.pantherdb.org/about.jsp）を開きます。 </li><li>統計的な過剰発現試験を用いて、胚発生の間に特定の生物学的プロセスを特定し、参照リスト25のようにボスの牡牛座 （データベース内のすべての遺伝子）のデフォルト設定を使用するように6765胚特異的GSのリストをアップロードします。 注：これは統計的に過剰または過少発現二項検定を使用しているGOタームから進化の関連プロセスの識別を可能にします。 </li><li>ソフトウェアのデフォルトとして&lt;0.05でP値のしきい値を設定します。データがダウンロードされ、博覧会することができますさらなる選択16用のExcelファイルにRTED。 </li></ol>

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D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PANTHER:+a+library+of+protein+families+and+subfamilies+indexed+by+function.">PANTHER: a library of protein families and subfamilies indexed by function.</a> Genome Res. 13 (9), 2129-2141 (2003).</li><li>Mi, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+PANTHER+database+of+protein+families,+subfamilies,+functions+and+pathways.">The PANTHER database of protein families, subfamilies, functions and pathways.</a> Nucleic Acids Res. 33, 284-288 (2005).</li><li>Mi, H., Muruganujan, A., Casagrande, J. T., Thomas, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large-scale+gene+function+analysis+with+the+PANTHER+classification+system.">Large-scale gene function analysis with the PANTHER classification system.</a> Nat. Protoc. 8, 1551-1566 (2013).</li><li>Ross, P. J., Wang, K., Kocabas, A., Cibelli, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Housekeeping+gene+transcript+abundance+in+bovine+fertilized+and+cloned+embryos.">Housekeeping gene transcript abundance in bovine fertilized and cloned embryos.</a> Cell Reprogram. 12 (6), 709-717 (2010).</li><li>Gilbert, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+dynamics+of+gene+products+fluctuation+during+bovine+pre-hatching+development.">The dynamics of gene products fluctuation during bovine pre-hatching development.</a> Mol. Reprod. Dev. 76, 762-772 (2009).</li><li>Vallee, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Revealing+the+bovine+embryo+transcript+profiles+during+early+in+vivo+embryonic+development.">Revealing the bovine embryo transcript profiles during early in vivo embryonic development.</a> Reproduction. 138 (1), 95-105 (2009).</li><li>Dafforn, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Linear+mRNA+amplification+from+as+little+as+5+ng+total+RNA+for+global+gene+expression+analysis.">Linear mRNA amplification from as little as 5 ng total RNA for global gene expression analysis.</a> Biotechniques. 37 (5), 854-857 (2004).</li><li>Beaujean, N., Jammes, H., Jouneau, A., Dufort, I., Rovert, C., Sirard, 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=Nuclear+Reprogramming.">Nuclear Reprogramming.</a> Studying Bovine Early Embryo Transcriptome by Microarray. , (2015).</li><li>Patterson, T. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Performance+comparison+of+one-color+and+two-color+platforms+within+the+MicroArray+Quality+Control+(MAQC)+project.">Performance comparison of one-color and two-color platforms within the MicroArray Quality Control (MAQC) project.</a> Nat. Biotechnol. 24 (9), 1140-1150 (2006).</li></ol>

The authors have nothing to disclose.

7日目のウシの胚を用いてマイクロアレイ解析を行うための第一の問題は、遺伝子発現を研究するため、高品質のRNAの十分な量を取得していません。伝統的なフェノール/クロロホルムRNA抽出およびエタノール沈殿法は、低収量およびRNA増幅反応を阻害する可能性の残りのフェノールが得られ、7日目の胚にはお勧めできません。代わりに、標準的な列ベースの方法は、全RNAを単離し、その後、...

高生産乳牛における初期胚損失は、酪農業1、2における主な課題の一つです。牛は、それらの同様の発達過程3、4に対するヒト着床前胚発生を研究するための興味深いモデルとなっています。しかしながら、多くの研究は、ウシ初期胚発生に関与する遺伝子についてのより良い理解が必要です。 1995年5に開発された最初のマイクロアレイ技術から二十年後に、より洗練されたプローブの製造技術の開発は、異なるマイクロアレイプラットフォーム6内との間に印刷エラーとアレイチップのばらつきが減少しました。改善されたマイクロアレイ技術は、初期胚Qに、臨床研究7で、より最近では、この技術の普及応用をもたらしましたualityアセスメント8。 マイクロアレイ技術のために必要な大量の材料は、マイクロアレイ技術は、最初に、このような初期胚発生などの研究分野の番号を入力することができなかった主な理由です。より最近では、RNA増幅法は、直線的にサブナノグラム出発RNA材料9からマイクログラムレベルまでRNAを増幅するために改良されてきました。市場で入手可能ないくつかの市販のRNA増幅キットがあります。しかし、より人気のよく発達したキットが11の方法を駆動リボ単一プライマー等温増幅10およびT7プロモーターに関連しています。最も人気のあるアンチセンスRNA増幅は、5 &#39;末端12にT7プロモーターに連結するオリゴdTプライマーを用いてインビトロ転写を使用します。この技術は、線形増幅F後代表アンチセンス転写物の大部分を維持することができまたはアレイハイブリダイゼーション13。この方法は、ウシの胚8から抽出した全RNAのピコグラムレベルを増幅するように適合されています。 ユニバーサルリンケージシステム（ULS）は、DNAを直接組み込むか、グアニン14のN7位の配位結合を形成することにより、白金リンク蛍光色素シアニン547またはシアニン647のいずれかでRNAを増幅した標識法です。この方法は、アミノアリルに比べて変更することなく、より安定した増幅されたaRNAを生成するために、胚の研究に適合させた酵素法15によって生成されたaRNAを変更しました。単一の色素と2つの色素の両方の標識方法は、マイクロアレイにユニバーサルリンケージシステムを使用して適応されています。大規模なマイクロアレイの比較研究は、6 1次元と2色配列のプラットフォーム間でのデータ品質の良好な相関があるということでした。 最近では、両方のT7プロモータードライブnは、アンチセンスRNA増幅およびULS標識方法は、マイクロアレイハイブリダイゼーション8、16のためのaRNA材料標識高品質の十分な量を生成するために、より信頼性の高いプロトコルを提供するために開発されてきました。そこで、本研究では、一例として、7日目ウシ胚を用いた2色マイクロアレイに関わるデータ解析にRNA抽出からの重要なステップのいくつかを実証するためのプロトコルを提供します。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>PicoPure RNA Isolation Kit</td>
			<td>Applied Biosystems</td>
			<td>KIT0204</td>
			<td></td>
		</tr>
		<tr>
			<td>RNase-Free DNase Set (50)</td>
			<td>Qiagen</td>
			<td>79254</td>
			<td></td>
		</tr>
		<tr>
			<td>Agilent RNA 6000 Pico Kit</td>
			<td>Agilent Technologies</td>
			<td>5067-1513</td>
			<td></td>
		</tr>
		<tr>
			<td>Arcturus RiboAmp HS PLUS Kit</td>
			<td>Applied Biosystems</td>
			<td>KIT0505</td>
			<td></td>
		</tr>
		<tr>
			<td>2100 Bioanalyzer Instruments</td>
			<td>Agilent Technologies</td>
			<td>G2940CA</td>
			<td></td>
		</tr>
		<tr>
			<td>RNA Screen Tape</td>
			<td>Agilent Technologies</td>
			<td>5067-5576</td>
			<td></td>
		</tr>
		<tr>
			<td>ULS Fluorescent Labeling Kit</td>
			<td>Kreatech Diagnostics</td>
			<td>EA-021</td>
			<td></td>
		</tr>
		<tr>
			<td>Custom Gene Expression Microarrays</td>
			<td>Agilent Technologies</td>
			<td>G2514F</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;Agilent Gene Expression wash buffer 1</td>
			<td>Agilent Technologies</td>
			<td>Part #5188-5325</td>
			<td></td>
		</tr>
		<tr>
			<td>Agilent Gene Expression wash buffer 2</td>
			<td>Agilent Technologies</td>
			<td>Part #5188-5326</td>
			<td></td>
		</tr>
		<tr>
			<td>2X Hi-RPM Hybridization buffer</td>
			<td>Agilent Technologies</td>
			<td>&#160;Part #5190-0403</td>
			<td></td>
		</tr>
		<tr>
			<td>25X Fragment buffer</td>
			<td>Agilent Technologies</td>
			<td>Part #5185-5974</td>
			<td></td>
		</tr>
		<tr>
			<td>10X GE Blocking Agent</td>
			<td>Agilent Technologies</td>
			<td>Part #5188-5281</td>
			<td></td>
		</tr>
		<tr>
			<td>Stabilization and drying solution</td>
			<td>Agilent Technologies</td>
			<td>&#160;Part #5185-5979</td>
			<td></td>
		</tr>
		<tr>
			<td>Gasket slides enabled by Agilent SureHyb techonolgy</td>
			<td>Agilent Technologies</td>
			<td>G2524-60012</td>
			<td>Pack of 20 gasket slides, 4 microarrays/slide</td>
		</tr>
		<tr>
			<td>Two-Color RNA Spike-In Kit</td>
			<td>Agilent Technologies</td>
			<td>&#160;Cat# 5188-5279</td>
			<td></td>
		</tr>
		<tr>
			<td>GenePix 4000B array scanner</td>
			<td>Molecular Devices</td>
			<td>GENEPIX 4000B-U</td>
			<td></td>
		</tr>
		<tr>
			<td>Ozone Free Box</td>
			<td>BioTray</td>
			<td>OFB_100x200</td>
			<td></td>
		</tr>
		<tr>
			<td>GAL file</td>
			<td>Agilent Technologies</td>
			<td>-</td>
			<td></td>
		</tr>
	</tbody>
</table>

transcriptome profiling of in-vivo produced bovine pre-implantation embryos using two-color microarray platform

Early embryonic loss is a large contributor to infertility in cattle. Moreover, bovine becomes an interesting model to study human preimplantation embryo development due to their similar developmental process. Although genetic factors are known to affect early embryonic development, the discovery of such factors has been a serious challenge. Microarray technology allows quantitative measurement and gene expression profiling of transcript levels on a genome-wide basis. One of the main decisions that have to be made when planning a microarray experiment is whether to use a one- or two-color approach. Two-color design increases technical replication, minimizes variability, improves sensitivity and accuracy as well as allows having loop designs, defining the common reference samples. Although microarray is a powerful biological tool, there are potential pitfalls that can attenuate its power. Hence, in this technical paper we demonstrate an optimized protocol for RNA extraction, amplification, labeling, hybridization of the labeled amplified RNA to the array, array scanning and data analysis using the two-color analysis strategy.

7日目のウシ胚からの総RNAを、増幅のaRNAの代表的な結果を図3に示し、 表1に要約します。 RNAの完全性およびプロファイルは、RNA抽出後に評価することができます。 RNAの品質評価は、バイオアナライザー装置（ 図3A）によって行うことができ、7.0より高いRIN値を有するサンプルのみを...

Watch this Scientific Journal Video about Transcriptome Profiling of In-Vivo Produced Bovine Pre-implantation Embryos Using Two-color Microarray Platform at JoVE.com

Transcriptome Profiling of In-Vivo Produced Bovine Pre-implantation Embryos Using Two-color Microarray Platform

Microarray technology allows quantitative measurement and gene expression profiling of transcripts on a genome-wide basis. Therefore, this protocol provides an optimized technical procedure in a two-color custom made bovine array using Day 7 bovine embryos to demonstrate the feasibility of using low amount of total RNA.

のトランスクリプトームプロファイリング<em&gt;生体内で</em二色マイクロアレイプラットフォームを使用&gt;産ウシ着床前の胚

Early embryonic loss is a large contributor to infertility in cattle. Moreover, bovine becomes an interesting model to study human preimplantation embryo ...

transcriptome-profiling-vivo-produced-bovine-pre-implantation-embryos

Research

JoVE Journal

Developmental Biology

Transcriptome Profiling of In-Vivo Produced Bovine Pre-implantation Embryos Using Two-color Microarray Platform

7.7K ビュー.  University of Alberta. Microarray technology allows quantitative measurement and gene expression profiling of transcripts on a genome-wide basis. Therefore, this protocol provides an optimized technical procedure in a two-color custom made bovine array using Day 7 bovine embryos to demonstrate the feasibility of using low amount of total RNA.

ビデオ: Transcriptome Profiling of In-Vivo Produced Bovine Pre-implantation Embryos Using Two-color Microarray Platform

Measuring Protein Stability in Living Zebrafish Embryos Using Fluorescence Decay After Photoconversion (FDAP)

Protein stability influences many aspects of biology, and measuring the clearance kinetics of proteins can provide important insights into biological systems. In FDAP experiments, the clearance of proteins within living organisms can be measured. A protein of interest is tagged with a photoconvertible fluorescent protein, expressed in vivo and photoconverted, and the decrease in the photoconverted signal over time is monitored. The data is then fitted with an appropriate clearance model to determine the protein half-life. Importantly, the clearance kinetics of protein populations in different compartments of the organism can be examined separately by applying compartmental masks. This approach has been used to determine the intra- and extracellular half-lives of secreted signaling proteins during zebrafish development. Here, we describe a protocol for FDAP experiments in zebrafish embryos. It should be possible to use FDAP to determine the clearance kinetics of any taggable protein in any optically accessible organism.

Measuring Protein Stability in Living Zebrafish Embryos Using Fluorescence Decay After Photoconversion FDAP

Protein levels in cells and tissues are often tightly regulated by the balance of protein production and clearance. Using Fluorescence Decay After Photoconversion (FDAP), the clearance kinetics of proteins can be experimentally measured in vivo.

光変換後の蛍光減衰を使用して生活ゼブラフィッシュ胚におけるタンパク質の安定性の測定（FDAP）

Protein stability influences many aspects of biology, and measuring the clearance kinetics of proteins can provide important insights into biological ...

発生生物学

Contrast Imaging in Mouse Embryos Using High-frequency Ultrasound

Ultrasound contrast-enhanced imaging can convey essential quantitative information regarding tissue vascularity and perfusion and, in targeted applications, facilitate the detection and measure of vascular biomarkers at the molecular level. Within the mouse embryo, this noninvasive technique may be used to uncover basic mechanisms underlying vascular development in the early mouse circulatory system and in genetic models of cardiovascular disease. The mouse embryo also presents as an excellent model for studying the adhesion of microbubbles to angiogenic targets (including vascular endothelial growth factor receptor 2 (VEGFR2) or &#945;v&#946;3) and for assessing the quantitative nature of molecular ultrasound. We therefore developed a method to introduce ultrasound contrast agents into the vasculature of living, isolated embryos. This allows freedom in terms of injection control and positioning, reproducibility of the imaging plane without obstruction and motion, and simplified image analysis and quantification. Late gestational stage (embryonic day (E)16.6 and E17.5) murine embryos were isolated from the uterus, gently exteriorized from the yolk sac and microbubble contrast agents were injected into veins accessible on the chorionic surface of the placental disc. Nonlinear contrast ultrasound imaging was then employed to collect a number of basic perfusion parameters (peak enhancement, wash-in rate and time to peak) and quantify targeted microbubble binding in an endoglin mouse model. We show the successful circulation of microbubbles within living embryos and the utility of this approach in characterizing embryonic vasculature and microbubble behavior.

Here, we present a protocol to inject ultrasound microbubble contrast agents into living, isolated late-gestation stage murine embryos. This method enables the study of perfusion parameters and of vascular molecular markers within the embryo using contrast-enhanced high-frequency ultrasound imaging.

高周波超音波を用いて、マウス胚におけるコントラストイメージング

Ultrasound contrast-enhanced imaging can convey essential quantitative information regarding tissue vascularity and perfusion and, in targeted ...

Characterization of Thymic Settling Progenitors in the Mouse Embryo Using In Vivo and In Vitro Assays

Characterizing thymic settling progenitors is important to understand the pre-thymic stages of T cell development, essential to devise strategies for T cell replacement in lymphopenic patients. We studied thymic settling progenitors from murine embryonic day 13 and 18 thymi by two complementary in vitro and in vivo techniques, both based on the &#8220;hanging drop&#8221; method. This method allowed colonizing irradiated fetal thymic lobes with E13 and/or E18 thymic progenitors distinguished by CD45 allotypic markers and thus following their progeny. Colonization with mixed populations allows analyzing cell autonomous differences in biologic properties of the progenitors while colonization with either population removes possible competitive selective pressures. The colonized thymic lobes can also be grafted in immunodeficient male recipient mice allowing the analysis of the mature T cell progeny in vivo, such as population dynamics of the peripheral immune system and colonization of different tissues and organs. Fetal thymic organ cultures revealed that E13 progenitors developed rapidly into all mature CD3+ cells and gave rise to the canonical &#947;&#948; T cell subset, known as dendritic epithelial T cells. In comparison, E18 progenitors have a delayed differentiation and were unable to generate dendritic epithelial T cells. The monitoring of peripheral blood of thymus-grafted CD3-/- mice further showed that E18 thymic settling progenitors generate, with time, larger numbers of mature T cells than their E13 counterparts, a feature that could not be appreciated in the short term fetal thymic organ cultures.

Characterization of Thymic Settling Progenitors in the Mouse Embryo Using In Vivo and In Vitro Assays

This article describes in vivo and in vitro methodology to characterize the thymic settling progenitors by the analysis of the kinetics of generation, phenotype and numbers of their T cell progeny.

胸腺の特性を用いてマウス胚における前駆細胞をセトリング<em&gt;インビボ</em&gt;と<em&gt;インビトロ</em&gt;アッセイ

Characterizing thymic settling progenitors is important to understand the pre-thymic stages of T cell development, essential to devise strategies for T ...

Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos

Cell migration is key to many physiological and pathological conditions, including cancer metastasis. The cellular and molecular bases of cell migration have been thoroughly analyzed in vitro. However, in vivo cell migration somehow differs from in vitro migration, and has proven more difficult to analyze, being less accessible to direct observation and manipulation. This protocol uses the migration of the prospective prechordal plate in the early zebrafish embryo as a model system to study the function of candidate genes in cell migration. Prechordal plate progenitors form a group of cells which, during gastrulation, undergoes a directed migration from the embryonic organizer to the animal pole of the embryo. The proposed protocol uses cell transplantation to create mosaic embryos. This offers the combined advantages of labeling isolated cells, which is key to good imaging, and of limiting gain/loss of function effects to the observed cells, hence ensuring cell-autonomous effects. We describe here how we assessed the function of the TORC2 component Sin1 in cell migration, but the protocol can be used to analyze the function of any candidate gene in controlling cell migration in vivo.

Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos

Combining cell transplantation, cytoskeletal labeling and loss/gain of function approaches, this protocol describes how the migrating zebrafish prospective prechordal plate can be used to analyze the function of a candidate gene in in vivo cell migration.

分析<em&gt;インビボ</emゼブラフィッシュ胚における細胞移植とタイムラプスイメージングを使用して&gt;細胞移動

Cell migration is key to many physiological and pathological conditions, including cancer metastasis. The cellular and molecular bases of cell migration ...

The Power of Simplicity: Sea Urchin Embryos as in Vivo Developmental Models for Studying Complex Cell-to-cell Signaling Network Interactions

Remarkably few cell-to-cell signal transduction pathways are necessary during embryonic development to generate the large variety of cell types and tissues in the adult body form. Yet, each year more components of individual signaling pathways are discovered, and studies indicate that depending on the context there is significant cross-talk among most of these pathways. This complexity makes studying cell-to-cell signaling in any in vivo developmental model system a difficult task. In addition, efficient functional analyses are required to characterize molecules associated with signaling pathways identified from the large data sets generated by next generation differential screens. Here, we illustrate a straightforward method to efficiently identify components of signal transduction pathways governing cell fate and axis specification in sea urchin embryos. The genomic and morphological simplicity of embryos similar to those of the sea urchin make them powerful in vivo developmental models for understanding complex signaling interactions. The methodology described here can be used as a template for identifying novel signal transduction molecules in individual pathways as well as the interactions among the molecules in the various pathways in many other organisms.

The Power of Simplicity: Sea Urchin Embryos as in Vivo Developmental Models for Studying Complex Cell-to-cell Signaling Network Interactions

This video article details a straightforward in vivo methodology that can be used to systematically and efficiently characterize components of complex signaling pathways and regulatory networks in many invertebrate embryos.

シンプルの力：ウニ胚として<em&gt; in vivoで</em&gt;複雑な細胞間シグナル伝達ネットワークの相互作用を研究するための発達モデル

Remarkably few cell-to-cell signal transduction pathways are necessary during embryonic development to generate the large variety of cell types and ...

In Vivo Imaging of Transgenic Gene Expression in Individual Retinal Progenitors in Chimeric Zebrafish Embryos to Study Cell Nonautonomous Influences

The genetic and technical strengths have made the zebrafish vertebrate a key model organism in which the consequences of gene manipulations can be traced in vivo throughout the rapid developmental period. Multiple processes can be studied including cell proliferation, gene expression, cell migration and morphogenesis. Importantly, the generation of chimeras through transplantations can be easily performed, allowing mosaic labeling and tracking of individual cells under the influence of the host environment. For example, by combining functional gene manipulations of the host embryo (e.g., through morpholino microinjection) and live imaging, the effects of extrinsic, cell nonautonomous signals (provided by the genetically modified environment) on individual transplanted donor cells can be assessed. Here we demonstrate how this approach is used to compare the onset of fluorescent transgene expression as a proxy for the timing of cell fate determination in different genetic host environments.
In this article, we provide the protocol for microinjecting zebrafish embryos to mark donor cells and to cause gene knockdown in host embryos, a description of the transplantation technique used to generate chimeric embryos, and the protocol for preparing and running in vivo time-lapse confocal imaging of multiple embryos. In particular, performing multiposition imaging is crucial when comparing timing of events such as the onset of gene expression. This requires data collection from multiple control and experimental embryos processed simultaneously. Such an approach can easily be extended for studies of extrinsic influences in any organ or tissue of choice accessible to live imaging, provided that transplantations can be targeted easily according to established embryonic fate maps.

In Vivo Imaging of Transgenic Gene Expression in Individual Retinal Progenitors in Chimeric Zebrafish Embryos to Study Cell Nonautonomous Influences

Live tracking of individual WT retinal progenitors in distinct genetic backgrounds allows for the assessment of the contribution of cell non-autonomous signaling during neurogenesis. Here, a combination of gene knockdown, chimera generation via embryo transplantation and in vivo time-lapse confocal imaging was utilized for this purpose.

細胞非自律影響を研究するためのキメラゼブラフィッシュ胚における個々の網膜前駆細胞におけるトランスジェニック遺伝子発現のin vivoイメージング

The genetic and technical strengths have made the zebrafish vertebrate a key model organism in which the consequences of gene manipulations can be traced ...

Fast and Efficient Expression of Multiple Proteins in Avian Embryos Using mRNA Electroporation

We report that mRNA electroporation permits fluorescent proteins to label cells in living quail embryos more quickly and broadly than DNA electroporation. The high transfection efficiency permits at least 4 distinct mRNAs to be co-transfected with ~87% efficiency. Most of the electroporated mRNAs are degraded during the first 2 h post-electroporation, permitting time-sensitive experiments to be carried out in the developing embryo. Finally, we describe how to dynamically image live embryos electroporated with mRNAs that encode various subcellular targeted fluorescent proteins.

We report messenger RNA (mRNA) electroporation as a method that permits fast and efficient expression of multiple proteins in the quail embryo model system. This method can be used to fluorescently label cells and record their in vivo movements by time-lapse microscopy shortly after electroporation.

mRNAエレクトロポレーションを用いて鳥類胚における複数のタンパク質の迅速かつ効率的な発現

We report that mRNA electroporation permits fluorescent proteins to label cells in living quail embryos more quickly and broadly than DNA electroporation. ...

Three-dimensional Angiogenesis Assay System using Co-culture Spheroids Formed by Endothelial Colony Forming Cells and Mesenchymal Stem Cells

Studies in the field of angiogenesis have been aggressively growing in the last few decades with the recognition that angiogenesis is a hallmark of more than 50 different pathological conditions, such as rheumatoid arthritis, oculopathy, cardiovascular diseases, and tumor metastasis. During angiogenesis drug development, it is crucial to use in vitro assay systems with appropriate cell types and proper conditions to reflect the physiologic angiogenesis process. To overcome limitations of current in vitro angiogenesis assay systems using mainly endothelial cells, we developed a 3-dimensional (3D) co-culture spheroid sprouting assay system. Co-culture spheroids were produced by two human vascular cell precursors, endothelial colony forming cells (ECFCs) and mesenchymal stem cells (MSCs) with a ratio of 5 to 1. ECFCs+MSCs spheroids were embedded into type I collagen matrix to mimic the in vivo extracellular environment. A real-time cell recorder was utilized to continuously observe the progression of angiogenic sprouting from spheroids for 24 h. Live cell fluorescent labeling technique was also applied to tract the localization of each cell type during sprout formation. Angiogenic potential was quantified by counting the number of sprouts and measuring the cumulative length of sprouts generated from the individual spheroids. Five randomly-selected spheroids were analyzed per experimental group. Comparison experiments demonstrated that ECFCs+MSCs spheroids showed greater sprout number and cumulative sprout length compared with ECFCs-only spheroids. Bevacizumab, an FDA-approved angiogenesis inhibitor, was tested with the newly-developed co-culture spheroid assay system to verify its potential to screen anti-angiogenic drugs. The IC50 value for ECFCs+MSCs spheroids compared to the ECFCs-only spheroids was closer to the effective plasma concentration of bevacizumab obtained from the xenograft tumor mouse model. The present study suggests that the 3D ECFCs+MSCs spheroid angiogenesis assay system is relevant to physiologic angiogenesis, and can predict an effective plasma concentration in advance of animal experiments.

Three-dimensional co-culture spheroid angiogenesis assay system is designed to mimic the physiologic angiogenesis. Co-culture spheroids are formed by two human vascular cell precursors, ECFCs and MSCs, and embedded in collagen gel. The new system is effective for evaluating angiogenic modulators, and provides more relevant information to the in vivo study.

内皮コロニー形成細胞と間葉系幹細胞によって形成された共培養スフェロイドを用いた3次元血管新生アッセイ系

Studies in the field of angiogenesis have been aggressively growing in the last few decades with the recognition that angiogenesis is a hallmark of more ...

Quantification of Ethanol Levels in Zebrafish Embryos Using Head Space Gas Chromatography

Fetal Alcohol Spectrum Disorders (FASD) describe a highly variable continuum of ethanol-induced developmental defects, including facial dysmorphologies and neurological impairments. With a complex pathology, FASD affects approximately 1 in 100 children born in the United States each year. Due to the highly variable nature of FASD, animal models have proven critical in our current mechanistic understanding of ethanol-induced development defects. An increasing number of laboratories has focused on using zebrafish to examine ethanol-induced developmental defects. Zebrafish produce large numbers of externally fertilized, genetically tractable, translucent embryos. This allows researchers to precisely control timing and dosage of ethanol exposure in multiple genetic contexts and quantify the impact of embryonic ethanol exposure through live imaging techniques. This, combined with the high degree of conservation of both genetics and development with humans, has proven zebrafish to be a powerful model in which to study the mechanistic basis of ethanol teratogenicity. However, ethanol exposure regimens have varied between different zebrafish studies, which has confounded the interpretation of zebrafish data across these studies. Here is a protocol to quantify ethanol concentrations in zebrafish embryos using head space gas chromatography.

This work describes a protocol to quantify ethanol levels in a zebrafish embryo using head space gas chromatography from proper exposure methods to embryo processing and ethanol analysis.

ヘッドスペースガスクロマトグラフィーを用いたゼブラフィッシュ胚におけるエタノール濃度の定量

Fetal Alcohol Spectrum Disorders (FASD) describe a highly variable continuum of ethanol-induced developmental defects, including facial dysmorphologies ...

Analysis of Congenital Heart Defects in Mouse Embryos Using Qualitative and Quantitative Histological Methods

Congenital heart defects (CHD) are the most common type of birth defect in humans, affecting up to 1% of all live births. However, the underlying causes for CHD are still poorly understood. The developing mouse constitutes a valuable model for the study of CHD, because cardiac developmental programs between mice and humans are highly conserved. The protocol describes in detail how to produce mouse embryos of the desired gestational stage, methods to isolate and preserve the heart for downstream processing, quantitative methods to identify common types of CHD by histology (e.g., ventricular septal defects, atrial septal defects, patent ductus arteriosus), and quantitative histomorphometry methods to measure common muscular compaction phenotypes. These methods articulate all the steps involved in sample preparation, collection, and analysis, allowing scientists to correctly and reproducibly measure CHD.

In this protocol, we describe procedures to qualitatively and quantitatively analyze developmental phenotypes in mice associated with congenital heart defects.

定性・定量的組織学的手法を用いたマウス胚における先天性心不全の解析

Congenital heart defects (CHD) are the most common type of birth defect in humans, affecting up to 1% of all live births. However, the underlying causes ...