Name: transcriptome profiling of in-vivo produced bovine pre-implantation embryos using two-color microarray platform
Uploaded: 2017-01-30T13:00:00.000+00:00
Duration: 9 min 4 s
Description: Watch this Scientific Journal Video about Transcriptome Profiling of In-Vivo Produced Bovine Pre-implantation Embryos Using Two-color Microarray Platform at JoVE.com

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>

<ol>
	<li>Royal, M. D., Smith, R. F., Friggens, N. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fertility+in+dairy+cows:+bridging+the+gaps.">Fertility in dairy cows: bridging the gaps.</a> Animal. 2 (08), 1101-1103 (2008).</li><li>Diskin, M. G., Murphy, J. J., Sreenan, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryo+survival+in+dairy+cows+managed+under+pastoral+conditions.">Embryo survival in dairy cows managed under pastoral conditions.</a> Anim. Reprod. Sci. 96 (3-4), 297-311 (2006).</li><li>Wrenzycki, C., 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=Effects+of+culture+system+and+protein+supplementation+on+mRNA+expression+in+pre-implantation+bovine+embryos.">Effects of culture system and protein supplementation on mRNA expression in pre-implantation bovine embryos.</a> Hum. Reprod. 16 (5), 893-901 (2001).</li><li>Menezo, Y. J., Herubel, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+and+bovine+models+for+human+IVF.">Mouse and bovine models for human IVF.</a> Reprod. Biomed. Online. 4 (2), 170-175 (2002).</li><li>Schena, M., Shalon, D., Davis, R. W., Brown, P. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+monitoring+of+gene+expression+patterns+with+a+complementary+DNA+microarray.">Quantitative monitoring of gene expression patterns with a complementary DNA microarray.</a> Science. 270 (5235), 467-470 (1995).</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><li>Rhodes, D. R., Chinnaiyan, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrative+analysis+of+the+cancer+transcriptome.">Integrative analysis of the cancer transcriptome.</a> Nat. Genetics. 37, 31-37 (2005).</li><li>Robert, C., 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=Combining+resources+to+obtain+a+comprehensive+survey+of+the+bovine+embryo+transcriptome+through+deep+sequencing+and+microarrays.">Combining resources to obtain a comprehensive survey of the bovine embryo transcriptome through deep sequencing and microarrays.</a> Mol. Reprod. Dev. 78 (9), 651-664 (2011).</li><li>Nygaard, V., Hovig, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Options+available+for+profiling+small+samples:+a+review+of+sample+amplification+technology+when+combined+with+microarray+profiling.">Options available for profiling small samples: a review of sample amplification technology when combined with microarray profiling.</a> Nucleic Acids Res. 34 (3), 996-1014 (2006).</li><li>Kurn, N., Chen, P., Heath, J. D., Kopf-Sill, A., Stephens, K. M., Wang, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+isothermal,+linear+nucleic+acid+amplification+systems+for+highly+multiplexed+applications.">Novel isothermal, linear nucleic acid amplification systems for highly multiplexed applications.</a> Clin Chem. 51 (10), 1973-1981 (2005).</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. 87 (5), 1663-1667 (1990).</li><li>Phillips, J., 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=Antisense+RNA+Amplification:+A+linear+amplification+method+for+analyzing+the+mRNA+population+from+single+living+cells.">Antisense RNA Amplification: A linear amplification method for analyzing the mRNA population from single living cells.</a> Methods. 10 (3), 283-288 (1996).</li><li>Gilbert, I., Scantland, S., Dufort, I., Gordynska, O., Labbe, A., Sirard, M. A., Robert, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+monitoring+of+aRNA+production+during+T7+amplification+to+prevent+the+loss+of+sample+representation+during+microarray+hybridization+sample+preparation.">Real-time monitoring of aRNA production during T7 amplification to prevent the loss of sample representation during microarray hybridization sample preparation.</a> Nucleic Acids Res. 37 (8), 65 (2009).</li><li>Gijlswijk, R. P., Talman, E. G., Jansse, P. J., Snoeijers, S. S., Killian, J., Tanke, H. J., Heetebrij, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Universal+Linkage+System:+versatile+nucleic+acid+labeling+technique.">Universal Linkage System: versatile nucleic acid labeling technique.</a> Expert Re. Mol. Diagn. 1 (1), 81-91 (2001).</li><li>Gilbert, I., Scantland, S., Sylvestre, E. L., Dufort, I., Sirard, M. A., Robert, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Providing+a+stable+methodological+basis+for+comparing+transcript+abundance+of+developing+embryos+using+microarrays.">Providing a stable methodological basis for comparing transcript abundance of developing embryos using microarrays.</a> Mol. Hum. Reprod. 16 (8), 601-616 (2010).</li><li>Tsoi, S., 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=Development+of+a+porcine+(Sus+scofa)+embryo-specific+microarray:+array+annotation+and+validation.">Development of a porcine (Sus scofa) embryo-specific microarray: array annotation and validation.</a> BMC Genomics. 13, 370 (2012).</li><li>Salehi, R., 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=Superovulatory+response+and+embryo+production+in+Holstein+cows+fed+diets+enriched+in+oleic,+linoleic+or+&#945;-linolenic+acid.">Superovulatory response and embryo production in Holstein cows fed diets enriched in oleic, linoleic or &#945;-linolenic acid.</a> Reprod. Fertil. Dev. 26 (1), 218-218 (2013).</li><li>Thangavelu, G., Colazo, M. G., Ambrose, D. J., Oba, M., Okine, E. K., Dyck, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diets+enriched+in+unsaturated+fatty+acids+enhance+early+embryonic+development+in+lactating+Holstein+cows.">Diets enriched in unsaturated fatty acids enhance early embryonic development in lactating Holstein cows.</a> Theriogenology. 68 (7), 949-957 (2007).</li><li>. Agilent 2100 Bioanalyzer User's Guide Available from: <a target="_blank" href="https://www.agilent.com/cs/library/usermanuals/Public/G2946-90004_Vespucci_UG_eBook_(NoSecPack)">https://www.agilent.com/cs/library/usermanuals/Public/G2946-90004_Vespucci_UG_eBook_(NoSecPack)</a> (2016)</li><li>Kerr, K. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extended+analysis+of+benchmark+datasets+for+Agilent+two-color+microarray.">Extended analysis of benchmark datasets for Agilent two-color microarray.</a> BMC Bioinformatics. 8, 371 (2007).</li><li>Zhu, Q., Miecznikowski, J. C., Halfon, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+wholly+defined+Agilent+microarray+spike-in+dataset.">A wholly defined Agilent microarray spike-in dataset.</a> Bioinformatics. 27 (9), 1284-1289 (2011).</li><li>Vallee, M., Gravel, C., Palin, M. F., Reghenas, H., Stothard, P., Wishart, D. S., 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=Identification+of+novel+and+known+oocyte-specific+genes+using+complementary+DNA+subtraction+and+microarray+analysis+in+three+different+species.">Identification of novel and known oocyte-specific genes using complementary DNA subtraction and microarray analysis in three different species.</a> Biol. Reprod. 73 (1), 63-71 (2005).</li><li>Thomas, P. 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><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>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>&amp;nbsp;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>&amp;nbsp;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>&amp;nbsp;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

Expression of Fluorescent Proteins in Branchiostoma lanceolatum by mRNA Injection into Unfertilized Oocytes

We report here a robust and efficient protocol for the expression of fluorescent proteins after mRNA injection into unfertilized oocytes of the cephalochordate amphioxus, Branchiostoma lanceolatum. We use constructs for membrane and nuclear targeted mCherry and eGFP that have been modified to accommodate amphioxus codon usage and Kozak consensus sequences. We describe the type of injection needles to be used, the immobilization protocol for the unfertilized oocytes, and the overall injection set-up. This technique generates fluorescently labeled embryos, in which the dynamics of cell behaviors during early development can be analyzed using the latest in vivo imaging strategies. The development of a microinjection technique in this amphioxus species will allow live imaging analyses of cell behaviors in the embryo as well as gene-specific manipulations, including gene overexpression and knockdown. Altogether, this protocol will further consolidate the basal chordate amphioxus as an animal model for addressing questions related to the mechanisms of embryonic development and, more importantly, to their evolution.

Expression of Fluorescent Proteins in Branchiostoma lanceolatum by mRNA Injection into Unfertilized Oocytes

We report here the robust and efficient expression of fluorescent proteins after mRNA injection into unfertilized oocytes of Branchiostoma lanceolatum. The development of the microinjection technique in this basal chordate will pave the way for far-reaching technical innovations in this emerging model system, including in vivo imaging and gene-specific manipulations.

蛍光タンパク質の発現において<em&gt; Branchiostoma lanceolatum</em&gt;未受精卵へのmRNA注入による

We report here a robust and efficient protocol for the expression of fluorescent proteins after mRNA injection into unfertilized oocytes of the ...

発生生物学

Laser-assisted Cytoplasmic Microinjection in Livestock Zygotes

Cytoplasmic microinjection into one-cell embryos is a very powerful technique. As an example, it enables the delivery of genome editing tools that can create genetic modifications that will be present in every cell of an adult organism. It can also be used to deliver siRNA, mRNAs or blocking antibodies to study gene function in preimplantation embryos. The conventional technique for microinjecting embryos used in rodents consists of a very thin micropipette that directly penetrates the plasma membrane when advanced into the embryo. When this technique is applied to livestock animals it usually results in low efficiency. This is mainly because in contrast to mice and rats, bovine, ovine, and porcine zygotes have a very dark cytoplasm and a highly elastic plasma membrane that makes visualization during injection and penetration of the plasma membrane hard to achieve. In this protocol, we describe a suitable microinjection method for the delivery of solutions into the cytoplasm of cattle zygotes that has proved to be successful for sheep and pig embryos as well. First, a laser is used to create a hole in the zona pellucida. Then a blunt-end glass micropipette is introduced through the hole and advanced until the tip of the needle reaches about 3/4 into the embryo. Then, the plasma membrane is broken by aspiration of cytoplasmic content inside the needle. Finally, the aspirated cytoplasmic content followed by the solution of interest is injected back into the embryonic cytoplasm. This protocol has been successfully used for the delivery of different solutions into bovine and ovine zygotes with 100% efficiency, minimal lysis, and normal blastocysts development rates.

This protocol shows how to perform cytoplasmic microinjection in farm animal zygotes. This technique can be used to deliver any solution into the one-cell embryo such as genome editing tools to generate knockout animals.

家畜接合体におけるレーザーアシスト細胞質マイクロインジェクション

Cytoplasmic microinjection into one-cell embryos is a very powerful technique. As an example, it enables the delivery of genome editing tools that can ...

遺伝学

Accurate and Phenol Free DNA Sexing of Day 30 Porcine Embryos by PCR

Research into prenatal programming in the pig has shown that the sex of the developing embryo or fetus can influence the developmental outcome. Therefore, the ability to determine an embryo&#39;s sex is necessary in many experiments particularly regarding early development. The present protocol demonstrates an inexpensive, rapid and non-toxic preparation of pig genomic DNA for use with PCR. Day 30 embryos must be humanely collected according to the guidelines established by Institutional Animal Policy and Welfare Committees for the present protocol. The preparation of the whole embryo for this PCR based sexing technique simply involves grinding the frozen embryo to a fine powder using a pre-chilled mortar and pestle. PCR-quality DNA is released from a small amount of embryo powder by applying a hot incubation in an alkaline lysis reagent. Next, the DNA solution is mixed with neutralization buffer and used directly for PCR. Two primer pairs are generated to detect specific sex determining region of the Y- chromosome (SRY) and ZFX region of the X- chromosome with high accuracy and specificity. The same protocol can be applied to other elongated embryos (Day 10 to Day 14) earlier than Day 30. Also, this protocol can be carried with 96-welled plates when screening a large number of embryos, making it feasible for automation and high-throughput sex typing.

This protocol describes an accurate, inexpensive, rapid and non-toxic method to determine the sex of Day 30 porcine embryo using PCR method after grinding an embryo into powder without phenol chloroform extraction and DNA column purification.

PCRによる日30ブタ胚の正確かつフェノールフリーDNA雌雄鑑別

Research into prenatal programming in the pig has shown that the sex of the developing embryo or fetus can influence the developmental outcome. Therefore, ...

Functional Cloning Using a Xenopus Oocyte Expression System

Identification of genes responsible for embryonic induction poses a number of challenges; to name a few, secreted molecules of interest may be low in abundance, may not be secreted but tethered to the signaling cell(s), or may require the presence of binding partners or upstream regulatory molecules. Thus in a search for gene products capable of eliciting an early lens-inductive response in competent ectoderm, we utilized an expression cloning system that would allow identification of paracrine or juxtacrine factors as well as transcriptional or other regulatory proteins. Pools of mRNA were injected into Xenopus oocytes, and responding tissue placed directly on the oocytes and co-cultured. Following functional cloning of ldb1 from a neural plate stage cDNA library based on its ability to elicit the expression of the early lens placode marker foxe3 in lens-competent animal cap ectoderm, we characterized the mRNA expression pattern, and assayed developmental progression following overexpression or knockdown of ldb1. This system is suitable in a very wide variety of contexts where identification of an inducer or its upstream regulatory molecules is sought using a functional response in competent tissue.

Functional Cloning Using a Xenopus Oocyte Expression System

We describe a Xenopus oocyte and animal cap system for the expression cloning of genes capable of inducing a response in competent ectoderm, and discuss techniques for the subsequent analysis of such genes. This system is useful in the functional identification of a wide range of gene products.

用いた機能クローニング<em&gt;アフリカツメガエル</em&gt;卵母細胞発現システム

Identification of genes responsible for embryonic induction poses a number of challenges; to name a few, secreted molecules of interest may be low in ...

Quantitative Analysis of Protein Expression to Study Lineage Specification in Mouse Preimplantation Embryos

This protocol presents a method to perform quantitative, single-cell in situ analyses of protein expression to study lineage specification in mouse preimplantation embryos. The procedures necessary for embryo collection, immunofluorescence, imaging on a confocal microscope, and image segmentation and analysis are described. This method allows quantitation of the expression of multiple nuclear markers and the spatial (XYZ) coordinates of all cells in the embryo. It takes advantage of MINS, an image segmentation software tool specifically developed for the analysis of confocal images of preimplantation embryos and embryonic stem cell (ESC) colonies. MINS carries out unsupervised nuclear segmentation across the X, Y and Z dimensions, and produces information on cell position in three-dimensional space, as well as nuclear fluorescence levels for all channels with minimal user input. While this protocol has been optimized for the analysis of images of preimplantation stage mouse embryos, it can easily be adapted to the analysis of any other samples exhibiting a good signal-to-noise ratio and where high nuclear density poses a hurdle to image segmentation (e.g., expression analysis of embryonic stem cell (ESC) colonies, differentiating cells in culture, embryos of other species or stages, etc.).

This protocol presents a method to perform quantitative, single-cell in situ analysis of protein expression to study lineage specification in mouse preimplantation embryos. The procedures necessary for collection of blastocysts, whole-mount immunofluorescent detection of proteins, imaging of samples on a confocal microscope, and nuclear segmentation and image analysis are described.

マウス着床前胚でリネージュの仕様を研究するためのタンパク質発現の定量分析

This protocol presents a method to perform quantitative, single-cell in situ analyses of protein expression to study lineage specification in mouse ...

Loss- and Gain-of-function Approach to Investigate Early Cell Fate Determinants in Preimplantation Mouse Embryos

Gene silencing and overexpression techniques are instrumental for the identification of genes involved in embryonic development. Direct target gene modification in preimplantation embryos provides a means to study the underlying mechanisms of genes implicated in, for instance, cellular differentiation into the trophectoderm (TE) and the inner cell mass (ICM). Here, we describe a protocol that examines the role of neogenin as an authentic receptor for initial cell fate determination in preimplantation mouse embryos. First, we discuss the experimental manipulations that were used to produce gain and loss of neogenin function by microinjecting neogenin cDNA and shRNA; the effectiveness of this approach was confirmed by a strong correlation between the pair-wise expression levels of either red fluorescent protein (RFP) or green fluorescent protein (GFP) and the immunocytochemical quantification of neogenin expression. Secondly, overexpression of neogenin in preimplantation mouse embryos leads to normal ICM development while neogenin knockdown causes the ICM to develop abnormally, implying that neogenin could be a receptor that relays extracellular cues to drive blastomeres to early cell fates. Given the success of this detailed protocol in investigating the function of a novel embryonic developmental stage-specific receptor, we propose that it has the potential to aid in exploration and identification of other stage-specific genes during embryogenesis.

The goal of this protocol is to describe a loss- and gain-of function method that is applicable to identify neogenin as a stage-specific receptor that leads to trophectoderm and inner cell mass differentiation in preimplantation mouse embryos.

loss-が及び着床前マウス胚の初期の細胞運命決定要因を調査するための機能獲得型のアプローチ

 Gene silencing and overexpression techniques are instrumental for the identification of genes involved in embryonic development. Direct target gene ...

A Semi-high-throughput Imaging Method and Data Visualization Toolkit to Analyze C. elegans Embryonic Development

C. elegans is the premier system for the systematic analysis of cell fate specification and morphogenetic events during embryonic development. One challenge is that embryogenesis dynamically unfolds over a period of about 13 h; this half day-long timescale has constrained the scope of experiments by limiting the number of embryos that can be imaged. Here, we describe a semi-high-throughput protocol that allows for the simultaneous 3D time-lapse imaging of development in 80&#8211;100 embryos at moderate time resolution, from up to 14 different conditions, in a single overnight run. The protocol is straightforward and can be implemented by any laboratory with access to a microscope with point visiting capacity. The utility of this protocol is demonstrated by using it to image two custom-built strains expressing fluorescent markers optimized to visualize key aspects of germ-layer specification and morphogenesis. To analyze the data, a custom program that crops individual embryos out of a broader field of view in all channels, z-steps, and timepoints and saves the sequences for each embryo into a separate tiff stack was built. The program, which includes a user-friendly graphical user interface (GUI), streamlines data processing by isolating, pre-processing, and uniformly orienting individual embryos in preparation for visualization or automated analysis. Also supplied is an ImageJ macro that compiles individual embryo data into a multi-panel file that displays maximum intensity fluorescence projection and brightfield images for each embryo at each time point. The protocols and tools described herein were validated by using them to characterize embryonic development following knock-down of 40 previously described developmental genes; this analysis visualized previously annotated developmental phenotypes and revealed new ones. In summary, this work details a semi-high-throughput imaging method coupled with a cropping program and ImageJ visualization tool that, when combined with strains expressing informative fluorescent markers, greatly accelerates experiments to analyze embryonic development.

A Semi-high-throughput Imaging Method and Data Visualization Toolkit to Analyze C. elegans Embryonic Development

This work describes a semi-high-throughput protocol that allows simultaneous 3D time-lapse imaging of embryogenesis in 80&#8211;100 C. elegans embryos in a single overnight run. Additionally, image processing and visualization tools are included to streamline data analysis. The combination of these methods with custom reporter strains enables detailed monitoring of embryogenesis.

C.エレガンス胚発生を解析するセミハイスループットイメージング法とデータ可視化ツールキット

C. elegans is the premier system for the systematic analysis of cell fate specification and morphogenetic events during embryonic development. One ...

Multiplexed Single Cell mRNA Sequencing Analysis of Mouse Embryonic Cells

Single cell mRNA sequencing has made significant progress in the last several years and has become an important tool in the field of developmental biology. It has been successfully used to identify rare cell populations, discover novel marker genes, and decode spatial and temporal developmental information. The single cell method has also evolved from the microfluidic based Fluidigm C1 technology to the droplet-based solutions in the last two to three years. Here we used the heart as an example to demonstrate how to profile the mouse embryonic tissue cells using the droplet based scRNA-Seq method. In addition, we have integrated two strategies into the workflow to profile multiple samples in a single experiment. Using one of the integrated methods, we have simultaneously profiled more than 9,000 cells from eight heart samples. These methods will be valuable to the developmental biology field by providing a cost-effective way to simultaneously profile single cells from different genetic backgrounds, developmental stages, or anatomical locations.

Here we presented a multiplexed single cell mRNA sequencing method to profile gene expression in mouse embryonic tissues. The droplet-based single cell mRNA sequencing (scRNA-Seq) method in combination with multiplexing strategies can profile single cells from multiple samples simultaneously, which significantly reduces reagent costs and minimizes experimental batch effects.

マウス胚細胞の多重化単細胞mRNAシーケンシング解析

Single cell mRNA sequencing has made significant progress in the last several years and has become an important tool in the field of developmental ...

Defining the Program of Maternal mRNA Translation during In vitro Maturation using a Single Oocyte Reporter Assay

Events associated with oocyte nuclear maturation have been well described. However, much less is known about the molecular pathways and processes that take place in the cytoplasm in preparation for fertilization and acquisition of totipotency. During oocyte maturation, changes in gene expression depend exclusively on the translation and degradation of maternal messenger RNAs (mRNAs) rather than on transcription. Execution of the translational program, therefore, plays a key role in establishing oocyte developmental competence to sustain embryo development. This paper is part of a focus on defining the program of maternal mRNA translation that takes place during meiotic maturation and at the oocyte-to-zygote transition. In this method paper, a strategy is presented to study the regulation of translation of target mRNAs during in vitro oocyte maturation. Here, a Ypet reporter is fused to the 3&#39; untranslated region (UTR) of the gene of interest and then micro-injected into oocytes together with polyadenylated mRNA encoding for mCherry to control for injected volume. By using time-lapse microscopy to measure reporter accumulation, translation rates are calculated at different transitions during oocyte meiotic maturation. Here, the protocols for oocyte isolation and injection, time-lapse recording, and data analysis have been described, using the Ypet/interleukin-7 (IL-7)-3&#39; UTR reporter as an example.

This protocol describes a reporter assay to study the regulation of mRNA translation in single oocytes during in vitro maturation.

単一の卵母細胞レポーターアッセイを用いたインビトロ成熟時の母体母体翻訳プログラムの定義

Events associated with oocyte nuclear maturation have been well described. However, much less is known about the molecular pathways and processes that ...

Whole Mount in Situ Hybridization of E8.5 to E11.5 Mouse Embryos

Whole mount in situ hybridization is a very informative approach for defining gene expression patterns in embryos. The in situ hybridization procedures are lengthy and technically demanding with multiple important steps that collectively contribute to the quality of the final result. This protocol describes in detail several key quality control steps for optimizing probe labeling and performance. Overall, our protocol provides a detailed description of the critical steps necessary to reproducibly obtain high quality results. First, we describe the generation of digoxygenin (DIG) labeled RNA probes via in vitro transcription of DNA templates generated by PCR. We describe three critical quality control assays to determine the amount, integrity and specific activity of the DIG-labeled probes. These steps are important for generating a probe of sufficient sensitivity to detect endogenous mRNAs in a whole mouse embryo. In addition, we describe methods for the fixation and storage of E8.5-E11.5 day old mouse embryos for in situ hybridization. Then, we describe detailed methods for limited proteinase K digestion of the rehydrated embryos followed by the details of the hybridization conditions, post-hybridization washes and RNase treatment to remove non-specific probe hybridization. An AP-conjugated antibody is used to visualize the labeled probe and reveal the expression pattern of the endogenous transcript. Representative results are shown from successful experiments and typical suboptimal experiments.

Whole Mount in Situ Hybridization of E8.5 to E11.5 Mouse Embryos

This whole mount in situ hybridization protocol discusses critical steps that ensure reproducible high quality results for gene expression studies in E8.5-E11.5 day old mouse embryos.

全体のマウント<em&gt;上皮内</emE11.5マウス胚へのE8.5&gt;ハイブリダイゼーション

Whole mount in situ hybridization is a very informative approach for defining gene expression patterns in embryos. The in situ hybridization procedures ...

のトランスクリプトームプロファイリング

要約

さらに動画を探す

この動画の章

蛍光タンパク質の発現において