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.

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><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.

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

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

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.

Expression von fluoreszierenden Proteinen in<em&gt; Branchiostoma lanceolatum</em&gt; Durch mRNA Injektion in ungedüngte Eizellen

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

Forschung

Entwicklungsbiologie

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.

Lasergestützte Zytoplasmatischen Mikroinjektions in Vieh 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 ...

Genetik

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.

Präzise und Phenol Freie DNA Geschlechtsbestimmung von Tag 30 Schweineembryonen mittels 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, ...

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.

Functional Cloning Mit ein<em&gt; Xenopus</em&gt; Eizellen 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 ...

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.

Quantitative Analyse der Proteinexpression zu studieren Lineage Spezifikation in Maus Präimplantationsembryonen

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.

Verlust- und Gewinn-of-function-Ansatz Frühe Zellschicksal Bestimmungsfaktoren in Preimplantation Maus Embryonen zur Untersuchung

 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.

Ein Instrument zur Analyse der C. elegans Embryonal development

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.

Multiplexed Single Cell mRNA Sequenzierungsanalyse von Mausembryonalzellen

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.

Definition des Programms der mütterlichen mRNA-Translation während der In-vitro-Reifung mit einem 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 ...

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.

Ganze Berge In-situ Hybridisierung von E8.5 bis E11.5 Maus Embryonen

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

Transkriptom Profilieren

Zusammenfassung

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