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

La parte animale di questo studio è stato condotto presso l&#39;Unità di Ricerca metabolica della University of Alberta, Edmonton, Canada, con tutte le procedure sperimentali su animali approvato (protocollo # AUP00000131) per l&#39;Università di Alberta cura degli animali e del Comitato uso, e gli animali curati secondo al Consiglio canadese delle linee guida per la cura degli animali (1993). 1. di produzione di embrioni, Isolamento di RNA totale e trattamento DNasi <ol><li> Per i protocolli sperimentali animali e di raccolta degli embrioni riferimento nelle nostre precedenti pubblicazioni 17, 18. </li><li> Piscina e scatto congelare simili embrioni palco da ogni mucca in azoto liquido e poi continuare a -80 ° C fino a quando l&#39;estrazione di RNA. </li><li> Estrarre RNA totale tramite un kit di estrazione di RNA colonna-based che consente di estrarre l&#39;RNA da campioni pico-scala (informazioni kit è disponibile in Lista dei Materiali). NOTA: consigliato kit contiene: condizionata Buffer, estrazione Buffer, il 70% di etanolo, il tampone di lavaggio 1, il tampone di lavaggio 2, tampone di eluizione, colonne di purificazione RNA con tubi di raccolta e tubi microcentrifuga. </li><li> Aggiungere 10 ml Extraction Buffer al tubo contenente gli embrioni e incubare per 30 minuti a 42 ° C. provetta da centrifuga a 800 xg per 2 minuti per raccogliere estratto cellulare nella provetta. </li><li> Pipettare 250 microlitri condizionata tampone sulla membrana del filtro colonna di purificazione. Incubare la colonna di purificazione dell&#39;RNA con condizionata tampone per 5 min a temperatura ambiente. Centrifugare la colonna di purificazione nel tubo di raccolta in 16.000 xg per 1 min. </li><li> Pipettare 10 ml di etanolo al 70% nella miscela di RNA Extraction Buffer ed embrioni. Mescolare bene pipettando su e giù. miscela Pipettare nella colonna di purificazione precondizionato. </li><li> Per legare RNA, centrifugare la colonna di purificazione per 2 min a 100 xg e seguire immediatamente da una centrifugazione a 16.000 xg per 30 s per rimuovere flusso attraverso. </li><li> Dispensare 10081; L di tampone di lavaggio 1 nella colonna di purificazione e centrifugare per 1 min a 8000 x g. NOTA: DNase trattamento è consigliato se si esegue la trascrizione inversa o di amplificazione dopo l&#39;isolamento di RNA. </li><li> Digest DNA genomico dopo passo 1.8. NOTA: consigliato kit DNasi è disponibile in Lista dei Materiali. Kit consigliato contiene: soluzione di riserva DNasi I e Buffer. </li><li> Preparare miscela di incubazione DNase mescolando 5 soluzione madre microlitri DNasi I con 35 microlitri di buffer. Mescolare capovolgendo delicatamente. </li><li> Pipettare il microlitri DNasi miscela 40 di incubazione direttamente nella membrana colonna di purificazione. Incubare a temperatura ambiente per 15 min. </li><li> Pipettare 40 microlitri tampone di lavaggio 1 nella membrana colonna di purificazione e quindi si centrifuga a 8000 xg per 15 s. </li><li> Pipettare 100 microlitri tampone di lavaggio 2 nella colonna di purificazione e centrifugare per 1 min a 8000 x g. </li><li> Pipetta altri 100 microlitri tampone di lavaggio 2 nella colonna di purificazione e centrifugareper 2 minuti a 16.000 x g. </li><li> Trasferire la colonna di purificazione di un nuovo 0.5 mL provetta. Pipettare 11 ml di tampone di eluizione direttamente sulla membrana della colonna di purificazione. Per garantire il massimo assorbimento di Elution Buffer nella membrana, toccare delicatamente la punta della pipetta alla superficie della membrana, mentre l&#39;erogazione del tampone di eluizione. </li><li> Incubare la colonna per 1 min a temperatura ambiente. Centrifugare la colonna per 1 min a 1000 xg per distribuire Elution Buffer nella colonna, e poi girare per 1 min a 16.000 xg per eluire RNA. </li><li> Utilizzare strumento bioanalizzatore secondo le istruzioni del produttore per valutare la qualità e la quantità di RNA 19. </li><li> Conservare il campione di RNA a -80 ° C fino al momento dell&#39;uso. </li></ol> 2. RNA di amplificazione e di etichettatura per l&#39;analisi di microarray NOTA: Per l&#39;utente prima volta a lavorare con le procedure di amplificazione di RNA e di etichettatura, utilizzare cinque ng di spike-in RNA along con i campioni reali ARNA per monitorare la misura di controllo della qualità dell&#39;amplificazione RNA e ibridazione. Il picco-in mix RNA contiene dieci in vitro di sintesi, trascrizioni poliadenilato in rapporti prestabiliti. Quando la procedura eseguita correttamente, le trascrizioni etichettati specificamente ibridano solo per sonde di controllo complementari matrici ei dati possono essere acquisiti per monitorare la garanzia di controllo della qualità con sé il minimo o cross-ibridazione. Maggiori dettagli descrizione per quanto riguarda la procedura di spillo-in intensità di controllo e di normalizzazione dopo la scansione l&#39;array e analisi dei dati è fare riferimento a precedenti pubblicazioni 20, 21. La procedura seguente viene data agli utenti di microarray di routine. <ol><li> Preparare alta qualità (valore numerico RNA Integrity (RIN) almeno&gt; 7.0) 100 pg campione di RNA in un volume totale di 10 - 11 microlitri. </li><li> Amplificare i campioni di RNA con un kit di amplificazione in grado dilavorare con i campioni pico-scala (non più di 100 pg). NOTA: Consigliato informazioni kit di amplificazione è disponibile in Lista dei Materiali e segue le istruzioni del produttore, senza alcuna modifica. </li><li> Utilizzare una quantificazione basato sulla fluorescenza per valutare il profilo della gamma di dimensioni del ARNA amplificato. </li><li> Utilizzare lo strumento spettrofotometro (basato sul assorbanza a 260 e 280 nm) per misurare la concentrazione della ARNA amplificato. </li><li> Conservare il ARNA amplificato a -80 ° C fino al momento per l&#39;etichettatura. </li><li> Utilizzare 2 mg di RNA amplificato per l&#39;etichettatura fluorescente secondo le ULS ARNA manuale d&#39;uso Kit di etichettatura. NOTA: Poiché Cyanine 547 e 647 Cyanine colorante sono sensibili a foto-degradazione e Cyanine 647 colorante è facilmente degradato dall&#39;ozono, la procedura di etichettatura avviene in una quantità minima di luce e sotto un ambiente privo di ozono. </li><li> Attiva la casella di ozono (Figura 1A) finché il livello di ozono è 0,001 ppm. </li><li&gt; eseguire la configurazione reazione all&#39;interno della scatola di ozono (Figura 1B) con l&#39;aggiunta di 2 ml di Cyanine 547 o Cyanine 647, 2 ml di tampone di etichettatura, e quindi regolare ad un volume finale di 20 ml con acqua RNase-free sotto la luce di sicurezza. Aggiungi filtro camera oscura alla sorgente luminosa. </li><li> Miscelare delicatamente e incubare le provette in un termociclatore a 85 ° C per 15 min. I campioni in ghiaccio per almeno 1 min. Spin down per raccogliere il contenuto dei tubi prima di procedere con il kit di estrazione di RNA, salvo passaggi relativi al trattamento DNasi (1,9-1,11), per ripulire l&#39;RNA amplificato Cy-dye-etichettati. </li><li> Utilizzare lo strumento spettrofotometro per misurare e determinare la concentrazione di RNA amplificato marcato e mantenere il ARNA etichettati a -80 ° C al massimo per 3 giorni prima dell&#39;uso. </li></ol> 3. microarray ibridazione e di lavaggio NOTA: Le seguenti passi importanti sono adattate in base a due colori per microarray a base di analisi di espressione genica Protocol (versione6.5, maggio 2010). <ol><li> Aggiungere la stessa quantità di 825 ng da ciascuna Cyanine 547- e 647 Cyanine marcati ARNA e mescolare con 25x e 10x frammentazione buffer di blocco. </li><li> Riscaldare il miscuglio a 60 ° C per 15 minuti e raffreddare in ghiaccio per 1 min. </li><li> Aggiungere la stessa quantità di tampone di ibridazione 2x e mescolare bene. </li><li> Centrifugare a 13.000 rpm, quindi la miscela è pronta per l&#39;ibridazione. </li><li> Carica 100 microlitri dalla miscela sul vetrino matrice e sigillare la slitta all&#39;interno della camera di ibridazione. </li><li> Caricare la camera montata nel forno di ibridazione per 17 ore a 65 ° C rotante a 10 rpm in forno. </li><li> Lavare le matrici secondo protocollo con trattamento protettivo di ozono mediante immersione in stabilizzazione ed essiccazione soluzione per 30 s. </li><li> Rimuovere le matrici dalla soluzione e assicurarsi che la superficie array è asciutta, senza particelle di polvere </li></ol> 4. La scansione microarray diapositive <ol><li> Mantenere gli array in luogo buio e accendere scanner finché è pronto per l&#39;uso (15 min tempo di attesa). </li><li> Caricare il codice a barre serie a sinistra, nello scanner e avviare la scansione. Fare l&#39;analisi loco secondo il manuale d&#39;uso del software e salvare i dati come (GPR) formato di file. </li></ol> 5. Analisi statistica <ol><li> Scaricare il software FlexArray http://genomequebec.mcgill.ca/FlexArray/license.php Clicca su &quot;dati grezzi di importazione&quot; e caricare i file GPR in FlexArray. </li><li> Eseguire la normalizzazione e l&#39;analisi statistica, se necessario, seguendo le istruzioni. Nota: Calcolare la soglia per la selezione positiva posto in questo studio come è stato descritto in precedenza 22. </li><li> Selezionare il menu a tendina dal visualizzatore trama del FlexArray per visualizzare tutti i punti ibridate e segnali di fondo. Nota: Un quadro analogo di spillo nei controlli dopo l&#39;ibridazione di microarray può essere visto in precedente studio 8. </li><li> Seleziona unnormalizzazione Loess all&#39;interno dell&#39;array seguito da un quantile tra processo di normalizzazione array dal FlexArray discesa manuale di conseguenza. </li><li> Esportare tutti i dati normalizzati da FlexArray in un file Excel per un ulteriore utilizzo. </li></ol> 6. Analisi Bioinformatica L&#39;annotazione originale delle sequenze di scansione da trascrizioni specifici embrione bovino (BESTv1) microarray è stato descritto in precedenza 8. Nel presente studio, 20,403 Simboli Gene unici (GS) sono stati ottenuti ( <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53754/Supplement_file_1.xlsx">Supplemento File1</a> ) attraverso il EmbryoGENE Laboratory Information Management System (LIMS) ELMA (http://elma.embryo-gene.ca). Un elenco di 6.765 geni unici ( <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53754/Supplement_file_2.xlsx">Supplemento File2</a> ) è stato selezionato e corrispondeva ai segnali tutti positivi dopo il processo di normalizzazione microarray (punto 5.5) Bovine 7 ° giorno gene embrioni profilo di espressione. Soglia di segnale positivo è stato calcolato sulla base di pubblicazione pervio 16 dalla intensità di un valore di un segnale. <ol><li> Per eseguire l&#39;analisi funzionale con PANTHER (Protein analisi tramite evolutivi Relationships) Sistema di Classificazione 23, 24, aprire il seguente link (http://www.pantherdb.org/about.jsp). </li><li> Carica un elenco di 6.765 GS-specific di embrioni per identificare specifici processi biologici durante lo sviluppo embrionale utilizzando il test sovrarappresentazione statistiche e di utilizzare l&#39;impostazione predefinita di Bos taurus (tutti i geni nel database) come elenco di riferimento 25. NOTA: Questo permette l&#39;identificazione dei processi di sviluppo legati dai termini GO che sono statisticamente sovra o sotto-espressi utilizzando un test binomiale. </li><li> Set soglia P-value &lt;0.05 di default del software. I dati possono essere scaricati e expoari in file di Excel per ulteriore selezione 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.

Il primo problema di effettuare analisi di microarray usando Giorno 7 embrioni di bovini non è sempre una quantità sufficiente di RNA di alta qualità per lo studio dell&#39;espressione genica. estrazione di RNA fenolo Tradizionale / cloroformio e metodo di precipitazione in etanolo non è raccomandato per Giorno 7 embrioni, con conseguente bassa resa e la possibile fenolo avanzi di inibire la reazione di amplificazione di RNA. Invece, un metodo basato su colonne standard è preferibile isolare l&#39;RNA totale e quin...

All&#39;inizio perdita embrionale nelle vacche ad alta produzione di latte è una delle principali sfide del settore lattiero-caseario 1, 2. Bovina è diventato un modello interessante per studiare lo sviluppo preimpianto dell&#39;embrione umano a causa del loro processo di sviluppo simile a 3, 4. Tuttavia, più ricerca è necessaria per avere una migliore comprensione di geni coinvolti nello sviluppo embrionale precoce bovina. Dopo venti anni dalla prima tecnologia microarray sviluppato nel 1995 5, lo sviluppo di più sofisticate tecnologie sonda fabbricazione errori di stampa ridotti e variabilità dei chip a matrice all&#39;interno e tra le diverse piattaforme di microarray 6. La tecnologia microarray migliorata determinato un ampiamente l&#39;applicazione di questa tecnologia nel campo della ricerca clinica 7 e, più recentemente, in embrione precoce qvalutazione ualità 8. La grande quantità di materiale necessario per la tecnologia microarray è la ragione principale per cui la tecnologia microarray inizialmente non è riuscito a entrare in un certo numero di campi di ricerca come sviluppo embrionale precoce. Più di recente, i metodi di amplificazione di RNA sono stati migliorati per amplificare linearmente RNA fino al livello microgrammi da sub-nanogrammo materiale di partenza di RNA 9. Ci sono diversi kit RNA amplificazione commerciali disponibili sul mercato; tuttavia, il più popolare kit ben sviluppati sono legati alla Ribo-Single Primer isotermica di amplificazione 10 e T7 promotore guidato 11 metodi. Il più popolare di amplificazione RNA antisenso utilizza nella trascrizione vitro con un oligo dT fondo il collegamento a un promotore T7 a 5 &#39;12. Questa tecnologia consente di mantenere più di trascritti rappresentativi anti-senso dopo amplificazione lineare fo array ibridazione 13. Questo metodo è stato adattato per amplificare il livello picogrammi di RNA totale estratto da embrioni bovini 8. Universale Linkage System (ULS) è il metodo di etichettatura che incorpora direttamente il DNA o RNA amplificato con colorante fluorescente in platino-linked sia Cyanine 547 o 647 Cyanine, formando un legame coordinativo sulla posizione N7 della guanina 14. Questo metodo è stato adattato nella ricerca embrioni per generare più stabile amplificato ARNA senza modifiche rispetto al aminoallyl modificati su ARNA generata dal metodo enzimatico 15. metodi sia singolo colorante ed etichettatura due coloranti sono stati adattati utilizzando universale Linkage System nella microarray. Un ampio studio confronti microarray è che vi è una buona correlazione della qualità dei dati tra le piattaforme di array uno o due colori 6. Recentemente, sia in auto promotore T7Metodi n amplificazione RNA antisenso ed etichettatura ULS sono stati sviluppati per fornire un protocollo più affidabile per generare una quantità sufficiente di alta qualità etichettati materiali ARNA per microarray ibridazione 8, 16. Pertanto, questo studio fornisce un protocollo per dimostrare alcune importanti passi da estrazione di RNA per l&#39;analisi dei dati coinvolti in microarray due colori usando Giorno 7 embrioni bovini come esempio.

<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.

Un risultato rappresentativo di RNA totale e amplificato aRNA dal Giorno 7 embrioni di bovini è mostrato in figura 3 e riassunti nella Tabella 1. integrità dell&#39;RNA e profilo possono essere valutate dopo l&#39;estrazione di RNA. Valutazione della qualità di RNA potrebbe essere fatto per strumento Bioanalyzer (figura 3A) e solo i campioni con un valore superiore a 7,0 RI...

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.

Trascrittoma Profiling di<em&gt; In-Vivo</em&gt; Embrioni bovini pre-impianto prodotto con piattaforma microarray a due colori

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 Views.  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.

Video: 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.

Misurare Protein Stabilità in Living embrioni di zebrafish Usando fluorescenza Decay Dopo Fotoconversione (FDAP)

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

Ricerca

Biologia dello sviluppo

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 embrioni di topo Utilizzo ad alta frequenza Ultrasound

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.

Caratterizzazione di timica assestamento Progenitori nell&#39;embrione mouse Uso<em&gt; In Vivo</em&gt; E<em&gt; In Vitro</em&gt; Saggi

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.

analizzare<em&gt; In Vivo</em&gt; Migrazione cellulare utilizzando Trapianti cellulari e Time-lapse imaging in embrioni di zebrafish

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.

La forza della semplicità: Sea Urchin embrioni<em&gt; In Vivo</em&gt; Modelli di sviluppo per lo studio delle cellule cellula-Complex segnalazione di rete Interazioni

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.

Imaging in vivo del transgenico espressione genica nei singoli retinica Progenitori in embrioni di zebrafish Chimerico da Cell studio Influenze non autonomo

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.

Espressione rapida ed efficiente di più proteine negli embrioni aviari utilizzando l'elettroporazione dell'mRNA

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.

Sistema di analisi dell'angiogenesi tridimensionale utilizzando Gli spheroidi co-culturali formati da cellule formanti colonia endoteliale e cellule staminali mesenchiche

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.

Quantificazione dei livelli di etanolo negli embrioni di pesce zebra utilizzando la cromatografia dello spazio della testa

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.

Analisi dei difetti cardiaci congeniti negli embrioni dei topi utilizzando metodi istologici qualitativi e quantitativi

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 ...