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

Das Tier Teil dieser Studie wurde an der Metabolic Research Unit der University of Alberta, Edmonton, Kanada, mit allen tierexperimentellen Verfahren genehmigt (Protokoll # AUP00000131) von der University of Alberta Animal Care und Use Committee, geleitet und Tiere betreut nach an den Canadian Council of Animal Care-Richtlinien (1993). 1. Embryo Produktion, Isolierung von Gesamt-RNA und DNase-Behandlung <ol><li> Für die Tierversuchsprotokolle und Embryo - Entnahme beziehen sich in unseren bisherigen Publikationen 17, 18. </li><li> Pool und Snap einfrieren ähnliche Stufe Embryonen aus jeder Kuh in flüssigem Stickstoff und dann halten bei -80 ° C bis zur RNA-Extraktion. </li><li> Extrahieren Sie die Gesamt - RNA über eine spaltenbasierte RNA - Extraktions - Kits , die RNA aus pico-Skala Proben zu extrahieren ermöglicht (Kit Informationen finden Sie in der Materialliste zur Verfügung). HINWEIS: Empfohlene Kit enthält: Konditionierungspuffer, Extraktion Buffer, 70% Ethanol, Waschpuffer 1, Waschpuffer 2, Elutionspuffer, RNA Reinigungskolonnen mit Sammelröhrchen und Mikrozentrifugenröhrchen. </li><li> In 10 &amp; mgr; l Extraktionspuffer zu Röhrchen mit Embryonen und Inkubation für 30 min bei 42 ° C. Zentrifugenröhrchen bei 800 × g für 2 min Zellextrakt in das Reaktionsgefäß zu sammeln. </li><li> Pipette 250 ul Konditionierungspuffer auf die Reinigungskolonne Filtermembran. Inkubieren der RNA Reinigungskolonne mit Konditionierungspuffer für 5 min bei Raumtemperatur. Zentrifugieren Sie die Reinigungskolonne in dem Sammelröhrchen bei 16.000 × g für 1 min. </li><li> Pipette 10 &amp; mgr; l 70% Ethanol in dem Gemisch aus RNA Extraction Buffer und Embryonen. Gut mischen durch Pipettieren von oben und unten. Pipette-Gemisch in die vorkonditioniert Reinigungskolonne. </li><li> RNA zu binden, zentrifugieren der Reinigungskolonne für 2 min bei 100 xg und sofort von einer Zentrifugation bei 16.000 xg für 30 s folgen, um Strömung durch entfernen. </li><li> Je 10081; L Waschpuffer 1 in die Reinigungskolonne und zentrifugieren für 1 min bei 8.000 x g. HINWEIS: DNase-Behandlung, wenn die Durchführung der reversen Transkription oder Verstärkung nach der RNA-Isolierung empfohlen. </li><li> Digest genomische DNA direkt nach dem Schritt 1.8. HINWEIS: Empfohlene DNase Kit ist in der Materialliste zur Verfügung. Empfohlene Kit enthält: DNase I-Stammlösung und Puffer. </li><li> Bereiten Mischung DNase Inkubation von 5 ul DNase I-Stammlösung mit 35 &amp; mgr; l Puffer zu mischen. Mischen Sie vorsichtig invertieren. </li><li> Pipette, um die 40 &amp; mgr; l DNase Inkubationsmischung direkt in die Reinigungskolonne Membran. für 15 min bei Raumtemperatur inkubieren. </li><li> Pipette 40 &amp; mgr; l Waschpuffer 1 in die Säule Membranreinigung und dann bei 8000 × g für 15 s zentrifugiert. </li><li> Je 100 &amp; mgr; l Waschpuffer 2 in die Reinigungskolonne und zentrifugieren für 1 min bei 8.000 x g. </li><li> Pipette weitere 100 &amp; mgr; l Waschpuffer 2 in die Reinigungskolonne und Zentrifugefür 2 Minuten bei 16.000 x g. </li><li> Übertragen Sie die Reinigungskolonne zu einem neuen 0,5 ml Mikrozentrifugenröhrchen. Pipette 11 ul Elutionspuffer direkt auf die Membran der Reinigungskolonne. Um eine maximale Absorption von Elutionspuffer in die Membran zu gewährleisten, sanft berühren die Spitze der Pipette auf die Oberfläche der Membran, während die Elutionspuffer Abgabe. </li><li> Inkubieren der Säule für 1 min bei Raumtemperatur. Zentrifugieren der Säule für 1 min bei 1000 × g Elution Buffer in der Säule zu verteilen, und dann drehen für 1 min bei 16.000 xg RNA zu eluieren. </li><li> Verwenden Bioanalyzer Instrument nach den Anweisungen des Herstellers , die RNA - Qualität und Quantität 19 zu bewerten. </li><li> Lagern Sie die RNA-Probe bei -80 ° C bis zur Verwendung. </li></ol> 2. RNA-Amplifikation und Markierung für Microarray-Analyse HINWEIS: Zum ersten Mal Benutzer arbeiten mit RNA-Amplifikation und Kennzeichnungsverfahren, verwenden fünf ng spike in RNA aloden tatsächlichen aRNA Proben ng der Qualitätskontrolle Messung der RNA-Amplifikation und Hybridisierung zu überwachen. Der Spike-in RNA - Mix enthält zehn in vitro synthetisierten, polyadenylierte Transkripte in vorbestimmten Verhältnissen. Wenn das Verfahren korrekt durchgeführt, hybridisieren die markierten Transkripte spezifisch nur an komplementäre Kontrollsonden in Arrays und können die Daten abgetastet werden Qualitätskontrollsicherung mit minimaler Selbst- oder Kreuzhybridisierung zu verfolgen. Weitere Details Beschreibung in Bezug auf die mit Spikes-Kontrolle Intensitäten und Normalisierungsverfahren nach der Anordnung und Datenanalyse Scannen 20 zur vorherigen Publikationen verweisen, 21. Das folgende Verfahren wird auf die Routine Microarray-Benutzer gegeben. <ol><li> Bereiten 100 pg hoher Qualität RNA-Probe (RNA Integrity Number (RIN) Wert mindestens&gt; 7,0) in einem Gesamtvolumen von 10 - 11 &amp; mgr; l. </li><li> Amplify die RNA-Proben mit einem Amplifikationskit in der Lage,arbeiten mit Pico-Skala Proben (weniger als 100 pg). HINWEIS: Empfohlene Amplifikationskit Informationen finden Sie in der Materialliste zur Verfügung und folgt den Anweisungen des Herstellers ohne jegliche Änderung. </li><li> Verwenden, um eine fluoreszenzbasierte Quantifizierung des Profils der Größenbereich der amplifizierten aRNA auszuwerten. </li><li> Verwenden Sie das Spektralphotometer Instrument (bezogen auf die Absorption bei 260 und 280 nm), um die Konzentration der amplifizierten aRNA zu messen. </li><li> Lagern Sie den verstärkten aRNA bei -80 ° C bis sie bereit zur Kennzeichnung. </li><li> Verwenden Sie 2 ug amplifizierte RNA für die Fluoreszenzmarkierung gemäß ULS aRNA Labeling Kit Benutzerhandbuch. HINWEIS: Da Cyanine 547 und Cyanine 647 Farbstoff empfindlich auf Photoabbau und Cyanine 647 Farbstoff leicht durch Ozon abgebaut wird, nimmt das Markierungsverfahren in einer minimalen Menge an Licht und unter einer ozonfreien Umgebung. </li><li> Schalten Sie den Ozonfeld (1A) , bis das Niveau des Ozons 0,001 ppm. </li><li&gt; Haben die Reaktionsaufbau innerhalb des Ozons Feld (1B) durch Zugabe von 2 &amp; mgr; l Cyanine 547 oder Cyanine 647, 2 ul Markierungspuffer, und dann auf ein Endvolumen von 20 ul mit RNase-freiem Wasser unter dem Sicherheitslicht einzustellen. Hinzufügen Dunkelkammer Filter auf die Lichtquelle. </li><li> Vorsichtig mischen und die Röhrchen in einem Thermocycler bei 85 ° C für 15 min. Proben auf Eis stellen für mindestens 1 min. Spin down zum Inhalt von Rohren zu sammeln, bevor sie mit RNA-Extraktions-Kits fortfahren, mit Ausnahme Schritte im Zusammenhang mit DNase-Behandlung (1,9 bis 1,11), die Cy-Farbstoff-markierte amplifizierte RNA zu reinigen. </li><li> Verwenden Sie das Spektralphotometer Instrument zu messen und die Konzentration der markierten amplifizierten RNA bestimmen und halten Sie die markierten aRNA bei -80 ° C maximal für 3 Tage vor dem Gebrauch. </li></ol> 3. Microarray-Hybridisierung und Waschen HINWEIS: Die folgenden wichtigen Schritte angepasst sind, nach Zwei-Farben-Biochip-basierten Genexpressionsanalyse-Protokoll (Version6.5, Mai 2010). <ol><li> In gleicher Höhe von 825 von jedem Cyanine ng 547- und Cyanine 647-markierte aRNA und mischen sich mit 25x Fragmentierung und 10x Blockierpuffer. </li><li> Das Gemisch wird bei 60 ° C für 15 min und kühl auf Eis für 1 min. </li><li> In die gleiche Menge von 2x Hybridisierungspuffer und gut mischen. </li><li> Zentrifuge bei 13000 rpm, dann ist die Mischung fertig für die Hybridisierung. </li><li> Last 100 &amp; mgr; l aus der Mischung auf das Array Rutsche und versiegeln Sie die Folie im Inneren der Hybridisierungskammer. </li><li> Laden die zusammengesetzte Kammer in den Hybridisierungsofen für 17 h bei 65 ° C bei 10 Umdrehungen pro Minute im Ofen zu drehen. </li><li> Waschen Sie die Arrays gemäß Protokoll mit Ozonschutzbehandlung durch in Stabilisierung Tauchen und Trocknen Lösung für 30 s. </li><li> Entfernen Sie die Felder aus der Lösung und stellen Sie sicher, dass der Array-Oberfläche ohne Staubpartikel trocken ist </li></ol> 4. Scanning Microarray Slide <ol><li> Halten Sie die Arrays in dunklen Ort und schalten Sie scanner bis es gebrauchsfertig (15 Minuten Wartezeit). </li><li> Legen Sie das Array-Barcode auf der linken Seite, in den Scanner und Scanvorgang zu starten. Führen Sie die Analyse vor Ort nach dem Software-Benutzerhandbuch und speichern die Daten als (GPR) Dateiformat. </li></ol> 5. Statistische Analyse <ol><li> Laden Sie die Software FlexArray http://genomequebec.mcgill.ca/FlexArray/license.php Klicken Sie auf &quot;Import Rohdaten&quot; und laden Sie die GPR - Dateien in FlexArray. </li><li> Führen Sie die Normalisierung und die statistische Analyse bei Bedarf durch die Anweisung folgen. Hinweis: Berechnen Sie den Grenzwert für positive Punktauswahl in dieser Studie , wie es zuvor 22 beschrieben wurde. </li><li> Wählen Sie das Drop-Down-Handbuch aus dem Grundstück Betrachter des FlexArray alle Signale, die die hybridisierten Spots und Hintergrund zu sehen. Hinweis: Ein ähnliches Bild von Spike bei den Kontrollen nach der Hybridisierung auf dem Microarray kann in früheren Studie 8 zu sehen. </li><li> Wählen Sie einLöss Normalisierung innerhalb des Arrays durch ein Quantil zwischen Prozessanordnungen Normalisierung folgende von der FlexArray Drop-Down-Handbuch entsprechend. </li><li> Exportieren Sie alle normalisierten Daten von FlexArray in eine Excel zur weiteren Verwendung einreichen. </li></ol> 6. Bioinformatik-Analyse Die ursprüngliche Anmerkung der Sondensequenzen von Rinderembryonen spezifische Transkripte (BESTv1) Microarray zuvor 8 beschrieben worden ist . In dieser Studie, 20.403 einzigartige Gene Symbole (GS) wurden (erhalten <a href="http://ecsource.jove.com/files/ftp_upload/53754/Supplement_file_1.xlsx">Supplement File1</a> ) durch das EmbryoGENE Labor - Informations - Management - System (LIMS) ELMA (http://elma.embryo-gene.ca). Eine Liste von 6765 einzigartige Gene ( <a href="http://ecsource.jove.com/files/ftp_upload/53754/Supplement_file_2.xlsx">Supplement File2</a> ) wurde auf die alle positiven Signale nach Microarray - Normalisierungsprozess (Schritt 5 ausgewählt und entsprach0,5) aus Rinder Tag 7 Embryonen Genexpressionsprofil. Positive Signalschwelle wurde berechnet basierend auf durchlässigen Veröffentlichung 16 von der Signalintensität eines A - Wert. <ol><li> Zur Funktionsanalyse mit PANTHER (Protein - Analyse durch evolutionäre Beziehungen) Klassifizierungssystem 23, 24, führen Sie über den folgenden Link (http://www.pantherdb.org/about.jsp). </li><li> Laden Sie eine Liste von 6765 embryospezifischen GS spezifischen biologischen Prozess zu identifizieren , während der embryonalen Entwicklung mit Hilfe von statistischen Überrepräsentierung Test und verwenden Standardeinstellung von Bos taurus (alle Gene in der Datenbank) als Referenzliste 25. HINWEIS: Dies ermöglicht die Identifizierung von entwicklungsbezogenen Prozesse von den GO Begriffe, die statistisch über- oder unterdrückten eine binomische Test. </li><li> Stellen Sie P-Wert Schwelle bei &lt;0,05 als Software-Standard. Die Daten können heruntergeladen und Exportet in Excel - Datei zur weiteren Auswahl 16. </li></ol>

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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.

Das erste Problem, Microarray-Analyse unter Verwendung von Tag 7 Rinderembryonen durchzuführen, ist nicht ausreichende Mengen an qualitativ hochwertigen RNA immer Genexpression für das Studium. Traditionellen Phenol / Chloroform-RNA-Extraktion und Ethanol-Fällungsverfahren ist nicht für den Tag 7 Embryonen empfohlen, was zu einer geringen Ausbeute und mögliche Überbleibsel phenol inhibierende RNA-Amplifikationsreaktion. Stattdessen wird eine Standard-Spalte-basierte Methode ist besser Gesamt-RNA zu isolieren und d...

Frühe embryonale Verlust in hoher produzierenden Milchkühe ist eine der größten Herausforderungen in der Milchindustrie 1, 2. Bovine hat sich ein interessantes Modell menschlichen preimplantation Embryoentwicklung zu studieren aufgrund ihrer ähnlichen Entwicklungsprozess 3, 4. Jedoch ist mehr Forschung erforderlich besseres Verständnis über Gene in bovinen frühen Embryonalentwicklung beteiligt zu haben. Nach zwanzig Jahren seit Technologie der ersten Microarray 1995 entwickelte 5, 6 die Entwicklung anspruchsvollerer Sonde Herstellungstechnologie reduziert Druckfehler und Variabilität von Array - Chips innerhalb und zwischen verschiedenen Microarray - Plattformen. Verbesserte Mikroarray - Technologie führte zu einer weit Anwendung dieser Technologie in der klinischen Forschung 7 und in jüngster Zeit in frühen Embryos quality Bewertung 8. Die große Menge an benötigten Materialien für Microarray-Technologie ist der Hauptgrund, warum der Microarray-Technologie zunächst eine Reihe von Forschungsgebieten wie der frühen Embryonalentwicklung zu betreten gescheitert. In jüngerer Zeit wurden Verfahren RNA - Amplifikation verbessert RNA linear zu amplifizieren bis 9 Mikrogramm Ebene von Unter Nanogramm- Ausgangs - RNA - Material. Es gibt mehrere kommerzielle RNA Amplifizierung Kits auf dem Markt erhältlich; jedoch sind die immer beliebter gut entwickelten Kits im Zusammenhang mit Ribo-Einzel Primer isothermen Amplifikation 10 und T7 - Promotor 11 Methoden angetrieben. Die beliebtesten Antisense - RNA - Amplifikation verwendet in - vitro - Transkription mit einem Primer Oligo - dT - Verknüpfung mit einem T7 - Promotor am 5&#39; - Ende 12. Diese Technologie ermöglicht es, das Beste aus repräsentativen Antisense-Transkripte nach linearer Verstärkung f Aufrechterhaltungoder Arrays Hybridisierung 13. Dieses Verfahren wurde angepasst Picogramm Ebene der Gesamt - RNA aus Rinderembryo 8 extrahiert zu amplifizieren. Universal - Linkage System (ULS) ist das Markierungsverfahren , die direkt die DNA oder RNA verstärkt mit Platin-gebundenen Fluoreszenzfarbstoff entweder Cyanine 547 oder Cyanine 647, durch eine koordinative Bindung an N7 - Position von Guanin 14 bildet enthält. Diese Methode wurde in Embryonen Forschung angepasst stabiler amplifizierten aRNA ohne Modifikation zu erzeugen im Vergleich zu dem aminoallyl modifizierten aRNA durch enzymatische Verfahren 15 erzeugt. Sowohl Einzel- als Farbstoff und zwei Farbstoffe Markierungsverfahren wurden mit Universal Linkage System in Microarray angepasst. Eine große Microarray - Vergleiche Studie war , dass es eine gute Korrelation der Datenqualität ist zwischen den Ein- und Zwei-Farben - Array - Plattformen 6. Vor kurzem beide T7-Promotor-Laufwerkn - Antisense - RNA - Amplifikation und ULS Markierungsverfahren wurden eine zuverlässigere Protokoll bereitzustellen entwickelt eine ausreichende Menge von hoher Qualität zu erzeugen , gekennzeichnet aRNA Materialien für Mikroarray - Hybridisierungs 8, 16. Daher stellt diese Studie ein Protokoll in Zweifarb-Mikroarray mit Tag 7 Rinderembryonen als Beispiel beteiligt einige der wichtigen Schritte von der RNA-Extraktion bis zur Datenanalyse zu demonstrieren.

<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.

Ein repräsentatives Ergebnis der Gesamt - RNA und verstärkt aRNA von Tag 7 Rinderembryonen ist in Abbildung 3 und zusammengefasst in Tabelle 1 dargestellt. RNA-Integrität und das Profil kann nach der RNA-Extraktion beurteilt werden. Qualitätsbewertung von RNA könnte durch Bioanalyzer (3A) und nur diesen Proben mit RIN - Wert höher als 7,0 sind qualifiziert werden verwend...

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.

Transkriptom Profilieren<em&gt; In-Vivo</em&gt; Produziert Bovine Präimplantationsembryonen Mit Zwei-Farben-Microarray-Plattform

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

Universidad San Sebastian

Research

JoVE Journal

Developmental Biology

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

7.7K Aufrufe.  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.

Serie: 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.

Mess Protein Stabilität in Wohnzebrafischembryonen unter Einsatz von Fluoreszenz Decay Nach Photokonversion (FDAP)

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

Forschung

Entwicklungsbiologie

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.

Contrast Imaging in Maus Embryonen Verwendung von Hochfrequenz-Ultraschall

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.

Charakterisierung von Thymic Einschwingzeit Vorläufern in der Mouse Embryo Verwendung<em&gt; In Vivo</em&gt; Und<em&gt; In-vitro-</em&gt; Assays

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.

Analyzing<em&gt; In Vivo</em&gt; Zellmigration unter Verwendung von Zelltransplantationen und Zeitraffer-Imaging in Zebrafischembryonen

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.

Die Kraft der Einfachheit: Seeigel Embry als<em&gt; In Vivo</em&gt; Entwicklungsmodelle für komplexe Zell-zu-Zell-Signalnetzwerk Wechselwirkungen Studieren

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.

In - vivo - Bildgebung von Expression Transgene Gene in einzelnen Retinal Vorläufern in Chimäre Zebrabärblingembryonen Studie Zellnichtautonome Einflüsse

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.

Schnelle und effiziente Expression mehrerer Proteine in Aviären Embryonen mit mRNA-Elektroporation

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.

Dreidimensionales Angiogenese-Assay-System mit Co-Culture-Spheroiden, die von endotheliaalen Koloniebildenden Zellen und mesenchymalen Stammzellen gebildet werden

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.

Quantifizierung des Ethanolgehalts in Zebrafisch-Embryonen mittels Head Space Gas Chromatographie

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.

Analyse von angeborenen Herzfehlern bei Mausembryonen mit qualitativen und quantitativen histologischen Methoden

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 ...