The authors would like to acknowledge Dr. Robert Price and the staff of the Instrumentation Resource Facility at the University of South Carolina School of Medicine. This work was partially supported by a SPARC Graduate Research Grant from the Office of the Vice President for Research at the University of South Carolina (JDP/VM). In addition this work was supported by Cook Biotech research agreement (JDP) and by FirstString Research Inc (JDP) and NIH 2 P20-RR016434-06 (JDP). In addition, NIH IDeA Networks of Biomedical Research Excellence (INBRE) grant for South Carolina P20GM103499 (JE)

embrioni aviaria non sono considerati animali vertebrati ai sensi della normativa IUCAC. 1. Ottenere embrioni per la chirurgia <ol><li> Incubare 80 - 90 uova di gallina Bovan fecondate (smussato finire) in un umidificata (60%) bilanciere incubatore a 40 ° C per circa 72 ore per ottenere embrioni a Hamilton e Hamburger (HH) fase 17. determinare il numero esatto di uova sulla base di analisi della potenza e il tasso di sopravvivenza degli embrioni 17. Utilizzare i vassoi di uova di plastica per l&#39;incubazione per consentire un&#39;adeguata circolazione dell&#39;aria. </li><li> Dopo questa fase di sviluppo desiderato è raggiunto, versare circa 20 ml di acqua calda (37 ° C) di tampone di Tyrode (integrato con bicarbonato di sodio (1 g / L)) in un x 26 mm piastra di Petri a 100 mm. </li><li> Sterilizzare un guscio d&#39;uovo con il 70% di etanolo. </li><li> rompere delicatamente il guscio con un manico bisturi e rilasciare con cautela il contenuto nel piatto contenente tampone di Tyrode. Scartare gli embrioni se (i) in cui appaiono visivamente anomala (ii)non sono in fase di sviluppo a destra (iii) sono impropriamente orientati sulla tuorlo e / o (iv) si verifica un sanguinamento. Conservare eventuali embrioni non soggetti a un intervento chirurgico subito dopo essere state poste ex ovo a 40 ° C in modo da non compromettere lo sviluppo. Nota: Staging è effettuata sulla base di Hamilton e Hamburger 18. Anomalie di embrioni di pollo è determinata ad occhio nudo e nell&#39;ambito di applicazione dissezione. Assicurarsi che l&#39;embrione ha piegatura appropriata e flessione e vascolare. </li></ol> 2. OFT Banding <ol><li> Tweeze fuori singoli thread lunghi 1 cm da un unico 11/0 nylon sutura chirurgica e formare un nodo sciolto. UV sterilizzare tutti i nodi preformati. </li><li> Sotto un microscopio dissezione, assicurarsi visivamente che il cuore batte a una velocità normale (~ 120 battiti / min). In caso contrario, eliminare l&#39;embrione. </li><li> Appena prima dell&#39;intervento chirurgico, applicare 5 - 6 ml di acqua calda (37 ° C) di tampone di Tyrode alla superficie embrione utilizzando una pipetta di trasferimento. </li><li> Far passare una estremità libera del nodo preformato sotto l&#39;OFT, posizionare la sutura al OFT / ventricolo giunzione (OVJ) (o posizione desiderata), e passare l&#39;estremità libera nel nodo costrizione in tal modo l&#39;OFT comprese le membrane che circondano il cuore. </li><li> Dopo l&#39;intervento chirurgico, bagnare la superficie del tuorlo con 5 - 6 ml di calda (37 ° C) di tampone di Tyrode utilizzando una pipetta di trasferimento. </li><li> Mantenere gli embrioni di controllo ex ovo come gli embrioni a bande; tuttavia non li soggetti alla procedura chirurgica. </li><li> Incubare gli embrioni, per la quantità di tempo desiderato, a 40 ° C in un (60%) incubatrice umidificata. </li><li> Una volta che il punto di tempo desiderato viene raggiunto, asportare l&#39;embrione dal tuorlo con le forbici diritte. Sezionare il cuore con una pinza sottile e utilizzare per diversi studi a valle, come i cambiamenti nella morfologia cardiaca a causa di alterata emodinamica 17. </li></ol> 3. A conferma che l&#39;intervento bendaggio causa un cambiamento nella Emodinamica Nota: La costrizione parziale dovuto all&#39;intervento bande porta ad un aumento della velocità del flusso sanguigno al OVJ. Questo parametro emodinamico è convenientemente valutata utilizzando immagini ecografiche 2D, che viene eseguita nel punto desiderato tempo dell&#39;esperimento. <ol><li> Poiché solo un singolo embrione può essere ripreso alla volta, mantenere tutti gli altri embrioni designati per l&#39;imaging ad ultrasuoni in un incubatore a 40 °. </li><li> Porre la capsula di Petri contenente l&#39;embrione di essere ripreso su una piastra elettrica fissata a 40 ° C. </li><li> Riempire il piatto, fino all&#39;orlo, con acqua calda (37 ° C) del buffer di Tyrode. Versare lentamente la soluzione tampone nel piatto in modo da mantenere intatto il tuorlo. Se, tuttavia, l&#39;integrità del tuorlo è compromessa durante questa fase, scartare l&#39;embrione. </li><li> Orient dell&#39;embrione tale che l&#39;asse centrale dell&#39;embrione è perpendicolare alla sonda ad ultrasuoni che è sospeso da un supporto regolabile. </li><li> Con l&#39;opera macchina ad ultrasuoniTing in B-mode, ottenere un&#39;immagine 2D del cuore pulsante sullo schermo e spostare il palco (piastra elettrica) in modo tale che l&#39;OFT, ventricolo e OVJ sono chiaramente visibili. </li><li> Passare alla modalità pulsato onda (PW), alla frequenza di ripetizione degli impulsi desiderata (ad esempio, 20 kHz) e di ottenere esattamente dati di velocità al OVJ. Una immagine B-mode del cuore pulsante si ottiene sullo schermo. <ol><li> Spostare lo stadio per vedere l&#39;OFT, ventricolo e la OVJ. Utilizzando il software macchina ad ultrasuoni, ottenere misure di velocità esattamente al OVJ. </li></ol></li></ol> Nota: Se la frequenza cardiaca di un embrione diminuisce durante l&#39;imaging, i dati di velocità così ottenuti non devono essere utilizzati per l&#39;analisi. Tutti embrioni utilizzati per misure di velocità non dovrebbero preferibilmente essere utilizzati per altri esperimenti dopo ecografica.

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The authors have nothing to disclose.

Questa tecnica è relativamente semplice e facile da eseguire, tuttavia alcuni punti chiave devono essere tenuti in considerazione in modo da ottenere risultati accurati valle. Gli embrioni devono essere mantenuti ex ovo in una capsula di Petri che contiene tampone di Tyrode di fornire adeguata reidratazione. E &#39;anche importante per idratare il tuorlo post-chirurgia con tampone di Tyrode e per assicurarsi che la camera di incubazione è adeguatamente idratato. Interventi chirurgici non devono essere eseguite su un embrione eventualmente sanguinamento è visibile o il tuorlo è anche un po &#39;rotto. Questo influenza la sopravvivenza degli embrioni, soprattutto per esperimenti a lungo termine. Un&#39;altra causa potenziale di letalità di embrioni, è se la fascia intorno alla OFT è troppo stretto quando applicato prima, in tal modo compromettere in modo significativo la funzione cardiaca. Allo stato attuale, si usa ultrasuoni come strumento di conferma per determinare la variazione di velocità del flusso sanguigno su OFT banding. In futuro, ci sarà confermare con micro-tomografia computerizzata (CT). Abbiamo anche determinare visivamente la tenuta fascia intorno alla OFT in immagini 2D B-mode. È importante sottolineare che non si osservano differenze nella frequenza cardiaca e del flusso volumetrico (calcolata utilizzando CFD) tra embrioni nei banded e gruppo di controllo 17. Questi indicano che la fisiologia del sistema cardiovascolare non è compromesso negli embrioni bande, così convalidando ulteriormente l&#39;intervento strisce. Al punto di tempo desiderato, dopo strisce, diverse analisi possono essere eseguite per capire l&#39;effetto di emodinamica alterati in processi di sviluppo delle valvole critica. Questi includono, ma non sono limitati a, determinando cambiamenti nei livelli di trascrizione di geni regolatori chiave coinvolti nello sviluppo della valvola, determinando la localizzazione dei componenti di ECM e la determinazione della ultrastruttura della OFT. Infine, questa tecnica migliora l&#39;attuale metodo di banding l&#39;OFT nell&#39;embrione all&#39;interno dell&#39;uovo finestra 19. Tale metodo è gravemente ostacolato dalla incapacità di accumulare embrioni necessari per fare analisi molecolari a valle. Questa nuova tecnica può essere utilizzata in esperimenti di soccorso pure. Questa applicazione permette di bande OFT di essere salvati ed embrione permesso di recuperare l&#39;insulto. Nessun metodo corrente può fare questo ed essere ancora in grado di eseguire analisi a valle statisticamente significative. Abbiamo impiegato con successo questa tecnica per cambiare gli stimoli emodinamici nel cuore di embrioni di pollo in una fase molto precoce dello sviluppo (HH 16 - 17). Il protocollo bendaggio potrebbe presentare una sfida quando si esegue l&#39;intervento nelle fasi successive di sviluppo a causa del rischio di intaccare qualsiasi dei vasi sanguigni extraembrionali. Inoltre, negli stadi di sviluppo, è spesso difficile mantenere il sacco vitellino intatti quando il contenuto delle uova vengono trasferiti nella capsula di Petri.

Abnormally formed outflow valves are the most common type of congenital heart defects 1. However, defective cardiac valve structure and function, even though present at birth, may become symptomatic only in adulthood. In fact, several adult valve diseases can be attributed to a congenital origin. Treatment of such patients often involves replacing defective valves, and, importantly, replaced aortic valves have been shown to have congenital anomalies 2. Given the fact that critical processes involved in valve development begin early during embryogenesis, the importance of better understanding the mechanisms that regulate these events is highlighted. 
The primitive heart tube, which is the first functioning organ in an embryo, exhibits two distinct layers - an endothelial endocardium surrounded by myocardium - separated by extracellular matrix (cardiac jelly) which is mostly produced and secreted by the myocardium 3-5. As development continues, valve primordia (endocardial cushions) are formed, after rightward looping of the embryonic heart, by local expansion of the cardiac jelly at the atrioventricular (AV) canal and the outflow tract (OFT) 4,6. This expansion is mediated by the highly regulated process of epithelial-mesenchymal transition (EMT), during which the cardiac jelly becomes populated by endocardially-derived mesenchymal cells 6. In addition to the mesenchymal population derived through EMT, neural crest cells are also involved in valvulogenesis of the OFT 3. 
Hemodynamic stimuli, such as shear stress, are important epigenetic factors that regulate heart development in the embryo 7,8. Using a 3D in vitro system, we have previously shown shear stress to be a factor influencing the expression and deposition of fibrous extracellular matrix (ECM) proteins in AV and OFT cushions 9,10. Moreover, studies carried out by several researchers have demonstrated that altered blood flow leads to improper valves and septa formation 11-16. Recently, using the novel banding procedure presented here, we have shown that changing hemodynamics in the embryonic chick heart affects the early processes involved in OFT valve formation 17.
The technique described here provides a novel model for altering hemodynamics in the developing chick heart by partially constricting the OFT ex ovo. This reproducible procedure is relatively quick and allows researchers to obtain a sufficient number of embryos/whole hearts/OFT tissue, etc. for downstream analyses including gene expression studies. Moreover, this new model can be used to study 'chronic' effects of altered hemodynamics on OFT valve development.

<table border="0" cellpadding="0" cellspacing="0" width="600">
	<tbody>
		<tr height="105">
			<td height="105" style="height:105px;width:216px;">Fertilized Bovan chicken eggs</td>
			<td style="width:71px;">Clemson University, Clemson, SC</td>
			<td style="width:119px;"></td>
			<td style="width:195px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">11/0 Nylon suture</td>
			<td style="width:71px;">Ashaway</td>
			<td style="width:119px;">S30001</td>
			<td>UV sterilize knots before surgery</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">100 x 26 mm petri dish</td>
			<td style="width:71px;">VWR</td>
			<td style="width:119px;">25387-030</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Transfer pipettes</td>
			<td style="width:71px;">Thermo Scientific&#160;</td>
			<td style="width:119px;">232-20S</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Scalpel handle #3</td>
			<td style="width:71px;">Fine Science Tools</td>
			<td style="width:119px;">91003-12</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Straight scissor</td>
			<td style="width:71px;">Roboz</td>
			<td style="width:119px;">RS-6702</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Dumont #5 fine forceps</td>
			<td style="width:71px;">Fine Science Tools</td>
			<td style="width:119px;">11254-20</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Tyrodes buffer</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">2145-10L</td>
			<td>Filter sterlize before use&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Sodium bicarbonate</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">S233-500</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Vevo 770 Ultrasound Imaging system</td>
			<td style="width:71px;">VisualSonics, Inc.</td>
			<td style="width:119px;">VS-11392</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">708 Ultrasound transducer&#160;</td>
			<td style="width:71px;">VisualSonics, Inc.</td>
			<td style="width:119px;">VS-11171</td>
			<td></td>
		</tr>
	</tbody>
</table>

a novel ex ovo banding technique to alter intracardiac hemodynamics in an embryonic chicken system

The new model presented here can be used to understand the influence of hemodynamics on specific cardiac developmental processes, at the cellular and molecular level. To alter intracardiac hemodynamics, fertilized chicken eggs are incubated in a humidified chamber to obtain embryos of the desired stage (HH17). Once this developmental stage is achieved, the embryo is maintained ex ovo and hemodynamics in the embryonic heart are altered by partially constricting the outflow tract (OFT) with a surgical suture at the junction of the OFT and ventricle (OVJ). Control embryos are also cultured ex ovo but are not subjected to the surgical intervention. Banded and control embryos are then incubated in a humidified incubator for the desired period of time, after which 2D ultrasound is employed to analyze the change in blood flow velocity at the OVJ as a result of OFT banding. Once embryos are maintained ex ovo, it is important to ensure adequate hydration in the incubation chamber so as to prevent drying and eventually embryo death. Using this new banded model, it is now possible to perform analyses of changes in the expression of key players involved in valve development and to understand the role of hemodynamics on cellular responses in vivo, which could not be achieved previously.

Mostrato nella Figura 1 sono strumenti necessari per OFT bande raccomandato. La capsula di Petri (Figura 1A) contenente l&#39;embrione ex ovo deve essere sufficientemente profonda in modo da non distruggere l&#39;embrione quando coperto con il coperchio. Capsule di Petri profondi (Figura 1C) dovrebbero essere utilizzati anche per l&#39;imaging ad ultrasuoni per consentire un adeguato volume di tampone di Tyrode da versare in cima al tuorlo. Una singola 1 centimetro filo (Figura 2A) è tweezed fuori da un 11/0 sutura di nylon (Figura 1A) e legato in un nodo sciolto (Figura 1B). Tutti i nodi preformati sono sterilizzati ai raggi UV. La figura 3A mostra una camera rocker umidificata cui uova di pollo fecondate sono incubate fino alla fase desiderata. La figura 3B mostra un incubatore dell&#39;embrione, dove hanno legato e gli embrioni di controllo sono mantenuti ex ovo </em&gt; per la quantità di tempo desiderato. Entrambi i tipi incubatori sono mantenuti a 40 ° C. Mostrato in figura 4A è un embrione HH17 sopra il tuorlo ex ovo. Figura 4B è una rappresentazione schematica che mostra la posizione della sutura (banda) al OVJ di un embrione HH17. Figura 4C è una immagine rappresentativa di un embrione fasciato ripreso 48 ore dopo l&#39;intervento chirurgico. L&#39;effetto dell&#39;intervento bande sulla velocità del flusso di sangue attraverso il sito costrizione viene misurata mediante ecografia 2D. Come previsto, la velocità alla OVJ è maggiore nell&#39;embrione bande rispetto al controllo (Figura 5). Abbiamo recentemente eseguito fluidodinamica computazionale analisi (CFD) su cuori bande e di controllo in corrispondenza del punto di tempo di 24 ore che mostra gli effetti della OFT strisce sulla velocità del flusso sanguigno e di taglio della parete dello stress 17. &lt;p class = &quot;jove_content&quot; fo: keep-together.within-page = &quot;1&quot;&gt; <img alt="Figura 1" src="/files/ftp_upload/53955/53955fig1.jpg" /> Figura 1. strumenti chirurgici. (A) 11/0 nylon di sutura. (B) piastra di Petri contenente nodi pre-formato. (C) profonda capsula di Petri per la coltura degli embrioni ex ovo e ecografia. (D) Trasferimento pipetta. (E) Scalpel handle per rompere il guscio dell&#39;uovo. (F) forbici dritte per embrione escissione per gli studi post-operatorio. (G) una pinza sottile <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53955/53955fig1large.jpg" target="_blank">clicca qui per vedere una versione più grande di questa figura.</a> <img alt="figura 2" src="/files/ftp_upload/53955/53955fig2.jpg" /> Figura 2. sutura per OFT Banding. (A) Un unico filo 1 centimetro tweezed fuori da una sutura. <strong&gt; (B) nodo pre-formata. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53955/53955fig2large.jpg" target="_blank">Fai clic qui per vedere una versione più grande di questa figura.</a> <img alt="Figura 3" src="/files/ftp_upload/53955/53955fig3.jpg" /> Figura 3. incubazione Chambers. (A) umidificata incubatore bilanciere per le uova fecondate. (B) camera umidificata per la cultura ex ovo di embrioni bande e di controllo. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53955/53955fig3large.jpg" target="_blank">Cliccate qui per vedere una versione più grande di questa figura.</a> <img alt="Figura 4" src="/files/ftp_upload/53955/53955fig4.jpg" /> Figura 4. OFT Banding. (A) Tutta la HH17 embrione di pollo mantenuto ex ovo, bar scala = 10 mm (B) Rappresentazione schematica raffigurante la costrizionesito (rosso). (C) fasciato embrione ripreso 48 ore dopo l&#39;OFT strisce, bar scala = 0,5 mm. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53955/53955fig4large.jpg" target="_blank">Clicca qui per vedere una versione più grande di questa figura.</a> <img alt="Figura 5" src="/files/ftp_upload/53955/53955fig5.jpg" /> Figura 5. Ultrasound-derivato Velocity misurazioni. Profili di velocità rappresentativo di controllo (A) e (B) fasciato embrioni ottenuti in un punto nel tempo di 1 ora. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53955/53955fig5large.jpg" target="_blank">Si prega di cliccare qui per vedere una versione più grande di questa figura.</a>

Watch this Scientific Journal Video about A Novel Ex Ovo Banding Technique to Alter Intracardiac Hemodynamics in an Embryonic Chicken System at JoVE.com

A Novel Ex Ovo Banding Technique to Alter Intracardiac Hemodynamics in an Embryonic Chicken System

For the first time we present here a reproducible banding procedure to alter hemodynamics in the developing heart ex ovo. This is achieved by partially constricting the outflow tract (OFT).

Un romanzo<em&gt; Ex Ovo</em&gt; Tecnica Banding alterare intracardiaca Emodinamica in un sistema embrionali di pollo

The new model presented here can be used to understand the influence of hemodynamics on specific cardiac developmental processes, at the cellular and ...

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Nanyang Technological University

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JoVE Journal

Developmental Biology

A Novel Ex Ovo Banding Technique to Alter Intracardiac Hemodynamics in an Embryonic Chicken System

6.5K Views.  School of Medicine, University of South Carolina. For the first time we present here a reproducible banding procedure to alter hemodynamics in the developing heart ex ovo. This is achieved by partially constricting the outflow tract (OFT). 

Video: A Novel Ex Ovo Banding Technique to Alter Intracardiac Hemodynamics in an Embryonic Chicken System

Efficient Gene Transfer in Chick Retinas for Primary Cell Culture Studies: An Ex-ovo Electroporation Approach

The cone photoreceptor-enriched cultures derived from embryonic chick retinas have become an indispensable tool for researchers around the world studying the biology of retinal neurons, particularly photoreceptors. The applications of this system go beyond basic research, as they can easily be adapted to high throughput technologies for drug development. However, genetic manipulation of retinal photoreceptors in these cultures has proven to be very challenging, posing an important limitation to the usefulness of the system. We have recently developed and validated an ex&#160;ovo plasmid electroporation technique that increases the rate of transfection of retinal cells in these cultures by five-fold compared to other currently available protocols1. In this method embryonic chick eyes are enucleated at stage 27, the RPE is removed, and the retinal cup is placed in a plasmid-containing solution and electroporated using easily constructed custom-made electrodes. The retinas are then dissociated and cultured using standard procedures. This technique can be applied to overexpression studies as well as to the downregulation of gene expression, for example via the use of plasmid-driven RNAi technology, commonly achieving transgene expression in 25% of the photoreceptor population. The video format of the present publication will make this technology easily accessible to researchers in the field, enabling the study of gene function in primary retinal cultures. We have also included detailed explanations of the critical steps of this procedure for a successful outcome and reproducibility.

Efficient Gene Transfer in Chick Retinas for Primary Cell Culture Studies: An Ex-ovo Electroporation Approach

Embryonic chick retinal cell cultures constitute valuable tools for the study of photoreceptor biology. We have developed an efficient gene transfer technique based on ex&#160;ovo plasmid electroporation of the retinas prior to culture. This technique considerably increases transfection efficiencies over currently available protocols, making genetic manipulation feasible for this system.

Trasferimento efficiente Gene in Chick retine per cellulari primarie Cultura Studies: An<em&gt; Ex-ovo</em&gt; Elettroporazione Approach

The cone photoreceptor-enriched cultures derived from embryonic chick retinas have become an indispensable tool for researchers around the world studying ...

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Biologia dello sviluppo

Optimized Ex-ovo Culturing of Chick Embryos to Advanced Stages of Development

Research in anatomy, embryology, and developmental biology has largely relied on the use of model organisms. In order to study development in live embryos model organisms, such as the chicken, are often used. The chicken is an excellent model organism due to its low cost and minimal maintenance, however they present observational challenges because they are enclosed in an opaque eggshell. In order to properly view the embryo as it develops, the shell must be windowed or removed. Both windowing and ex ovo techniques have been developed to assist researchers in the study of embryonic development. However, each of the methods has limitations and challenges. Here, we present a simple, optimized ex ovo culture technique for chicken embryos that enables the observation of embryonic development from stage HH 19 into late stages of development (HH 40), when many organs have developed. This technique is easy to adopt in both undergraduate classes and more advanced research laboratories where embryo manipulations are conducted.

Optimized Ex-ovo Culturing of Chick Embryos to Advanced Stages of Development

Viewing and accessing the chicken embryo during development can be challenging. We have developed an ex ovo method that is simple, cost effective, and can easily be used in a classroom or research setting. This method provides access to the embryo into late stages of embryonic development (HH 40).

Ottimizzato<em&gt; Ex-ovo</em&gt; Coltura di Chick embrioni a stadi avanzati di sviluppo

Research in anatomy, embryology, and developmental biology has largely relied on the use of model organisms. In order to study development in live embryos ...

An Optogenetic Approach for Assessing Formation of Neuronal Connections in a Co-culture System

Here we describe a protocol to generate a co-culture consisting of 2 different neuronal populations. Induced pluripotent stem cells (iPSCs) are reprogrammed from human fibroblasts using episomal vectors. Colonies of iPSCs can be observed 30 days after initiation of fibroblast reprogramming. Pluripotent colonies are manually picked and grown in neural induction medium to permit differentiation into neural progenitor cells (NPCs). iPSCs rapidly convert into neuroepithelial cells within 1 week and retain the capability to self-renew when maintained at a high culture density. Primary mouse NPCs are differentiated into astrocytes by exposure to a serum-containing medium for 7 days and form a monolayer upon which embryonic day 18 (E18) rat cortical neurons (transfected with channelrhodopsin-2 (ChR2)) are added. Human NPCs tagged with the fluorescent protein, tandem dimer Tomato (tdTomato), are then seeded onto the astrocyte/cortical neuron culture the following day and allowed to differentiate for 28 to 35 days. We demonstrate that this system forms synaptic connections between iPSC-derived neurons and cortical neurons, evident from an increase in the frequency of synaptic currents upon photostimulation of the cortical neurons. This co-culture system provides a novel platform for evaluating the ability of iPSC-derived neurons to create synaptic connections with other neuronal populations.

A protocol to generate a co-culture system consisting of neurons derived from induced pluripotent stem cells (iPSCs), primary cortical neurons and astrocytes is described. This co-culture system allows detection of the formation of synaptic contacts and circuits between new, iPSC-derived neurons and pre-existing cortical neurons expressing channelrhodopsin-2.

Un approccio per la valutazione optogenetic Formazione di connessioni neuronali in un sistema di co-coltura

Here we describe a protocol to generate a co-culture consisting of 2 different neuronal populations. Induced pluripotent stem cells (iPSCs) are ...

Generating CRISPR/Cas9 Mediated Monoallelic Deletions to Study Enhancer Function in Mouse Embryonic Stem Cells

Enhancers control cell identity by regulating tissue-specific gene expression in a position and orientation independent manner. These enhancers are often located distally from the regulated gene in intergenic regions or even within the body of another gene. The position independent nature of enhancer activity makes it difficult to match enhancers with the genes they regulate. Deletion of an enhancer region provides direct evidence for enhancer activity and is the gold standard to reveal an enhancer's role in endogenous gene transcription. Conventional homologous recombination based deletion methods have been surpassed by recent advances in genome editing technology which enable rapid and precisely located changes to the genomes of numerous model organisms. CRISPR/Cas9 mediated genome editing can be used to manipulate the genome in many cell types and organisms rapidly and cost effectively, due to the ease with which Cas9 can be targeted to the genome by a guide RNA from a bespoke expression plasmid. Homozygous deletion of essential gene regulatory elements might lead to lethality or alter cellular phenotype whereas monoallelic deletion of transcriptional enhancers allows for the study of cis-regulation of gene expression without this confounding issue. Presented here is a protocol for CRISPR/Cas9 mediated deletion in F1 mouse embryonic stem (ES) cells (Mus musculus129 x Mus castaneus). Monoallelic deletion, screening and expression analysis is facilitated by single nucleotide polymorphisms (SNP) between the two alleles which occur on average every 125 bp in these cells.

Experimental validation of enhancer activity is best approached by loss-of-function analysis. Presented here is an efficient protocol that uses CRISPR/Cas9 mediated deletion to study allele-specific regulation of gene transcription in F1 ES cells which contain a hybrid genome (Mus musculus129 x Mus castaneus).

Generazione CRISPR / Cas9 Mediated monoallelic eliminazioni per studiare la funzione Enhancer nel topo cellule staminali embrionali

Enhancers control cell identity by regulating tissue-specific gene expression in a position and orientation independent manner. These enhancers are often ...

Technique to Target Microinjection to the Developing Xenopus Kidney

The embryonic kidney of Xenopus&#160;laevis (frog), the pronephros, consists of a single nephron, and can be used as a model for kidney disease. Xenopus embryos are large, develop externally, and can be easily manipulated by microinjection or surgical procedures. In addition, fate maps have been established for early Xenopus embryos. Targeted microinjection into the individual blastomere that will eventually give rise to an organ or tissue of interest can be used to selectively overexpress or knock down gene expression within this restricted region, decreasing secondary effects in the rest of the developing embryo. In this protocol, we describe how to utilize established Xenopus fate maps to target the developing Xenopus kidney (the pronephros), through microinjection into specific blastomere of 4- and 8-cell embryos. Injection of lineage tracers allows verification of the specific targeting of the injection. After embryos have developed to stage 38 - 40, whole-mount immunostaining is used to visualize pronephric development, and the contribution by targeted cells to the pronephros can be assessed. The same technique can be adapted to target other tissue types in addition to the pronephros.

Technique to Target Microinjection to the Developing Xenopus Kidney

Here, we present a protocol to use fate maps and lineage tracers to target injections into individual blastomeres that give rise to the kidney of Xenopus laevis embryos.

Tecnica a Target microiniezione per lo sviluppo<em&gt; Xenopus</em&gt; Kidney

The embryonic kidney of Xenopus&#160;laevis (frog), the pronephros, consists of a single nephron, and can be used as a model for kidney disease. Xenopus ...

Gene Transfer into the Chicken Auditory Organ by In Ovo Micro-electroporation

Chicken embryos are ideal model systems for studying embryonic development as manipulations of gene function can be conducted with relative ease in ovo. The inner ear auditory sensory organ is critical for our ability to hear. It houses a highly specialized sensory epithelium that consists of mechano-transducing hair cells (HCs) and surrounding glial-like supporting cells (SCs). Despite structural differences in the auditory organs, molecular mechanisms regulating the development of the auditory organ are evolutionarily conserved between mammals and aves. In ovo electroporation is largely limited to early stages at E1 - E3. Due to the relative late development of the auditory organ at E5, manipulations of the auditory organ by in ovo electroporation past E3 are difficult due to the advanced development of the chicken embryo at later stages. The method presented here is a transient gene transfer method for targeting genes of interest at stage E4 - E4.5 in the developing chicken auditory sensory organ via in ovo micro-electroporation. This method is applicable for gain- and loss-of-functions with conventional plasmid DNA-based expression vectors and can be combined with in ovo cell proliferation assay by adding EdU (5-ethynyl-2&#180;-deoxyuridine) to the whole embryo at the time of electroporation. The use of green or red fluorescent protein (GFP or RFP) expression plasmids allows the experimenter to quickly determine whether the electroporation successfully targeted the auditory portion of the developing inner ear. In this method paper, representative examples of GFP electroporated specimens are illustrated; embryos were harvested 18 - 96 hr&#160;after electroporation and targeting of GFP to the pro-sensory area of the auditory organ was confirmed by RNA in situ hybridization. The method paper also provides an optimized protocol for the use of the thymidine analog EdU to analyze cell proliferation; an example of an EdU based cell proliferation assay that combines immuno-labeling and click EdU chemistry is provided.

Gene Transfer into the Chicken Auditory Organ by In Ovo Micro-electroporation

The auditory organ mediates hearing. Here we present a modified in ovo micro-electroporation method optimized for studying auditory progenitor cell proliferation and differentiation in the developing chicken auditory organ.

Gene Transfer nel pollo Auditory Organ<em&gt; In Ovo</em&gt; Micro-elettroporazione

Chicken embryos are ideal model systems for studying embryonic development as manipulations of gene function can be conducted with relative ease in ovo. ...

A Submerged Filter Paper Sandwich for Long-term Ex Ovo Time-lapse Imaging of Early Chick Embryos

Due to its availability, low cost, flat geometry, and transparency, the ex ovo chick embryo has become a major vertebrate animal model for the study of morphogenetic events, such as gastrulation2, neurulation3-5, somitogenesis6, heart bending7,8, and brain formation9-13, during early embryogenesis. Key to understanding morphogenetic processes is to follow them dynamically by time-lapse imaging. The acquisition of time-lapse movies of chick embryogenesis ex ovo has been limited either to short time windows or to the need for an incubator to control temperature and humidity around the embryo14. Here, we present a new technique to culture chick embryos ex ovo for high-resolution time-lapse imaging using transmitted light microscopy. The submerged filter paper sandwich is a variant of the well-established filter paper carrier technique (EC-culture)1 and allows for the culturing of chick embryos without the need for a climate chamber. The embryo is sandwiched between two identical filter paper carriers and is kept fully submerged in a simple, temperature-controlled medium covered by a layer of light mineral oil. Starting from the primitive streak stage (Hamburger-Hamilton stage 5, HH5)15 up to at least the 28-somite stage (HH16)15, embryos can be cultured with either their ventral or dorsal side up. This allows the acquisition of time-lapse movies covering about 30 hr of embryonic development. Representative time-lapse frames and movies are shown. Embryos are compared morphologically to an embryo cultured in the standard EC-culture.
The submerged filter paper sandwich provides a stable environment to study early dorsal and ventral morphogenetic processes. It also allows for live fluorescence imaging and micromanipulations, such as microsurgery, bead implantation, microinjection, gene silencing, and electroporation, and has a strong potential to be combined with immersion objectives for laser-based imaging (including light-sheet microscopy).

A Submerged Filter Paper Sandwich for Long-term Ex Ovo Time-lapse Imaging of Early Chick Embryos

Here, we present a new technique to culture chick embryos ex ovo for time-lapse imaging using transmitted light and fluorescence microscopy. The submerged filter paper sandwich is a variant of the well-established filter paper carrier technique1 that allows for the culturing of chick embryos fully-submerged in a liquid culture medium.

Un sommerso Sandwich carta da filtro per il lungo termine<em&gt; Ex Ovo</em&gt; Time-lapse imaging di Chick primi embrioni

Due to its availability, low cost, flat geometry, and transparency, the ex ovo chick embryo has become a major vertebrate animal model for the study of ...

In Ovo Electroporation in the Chicken Auditory Brainstem

Electroporation is a method that introduces genes of interest into biologically relevant organisms like the chicken embryo. It is long established that the chicken embryo is an effective research model for studying basic biological functions of auditory system development. More recently, the chicken embryo has become particularly valuable in studying gene expression, regulation and function associated with hearing. In ovo electroporation can be used to target auditory brainstem regions responsible for highly specialized auditory functions. These regions include the chicken nucleus magnocellularis (NM) and nucleus laminaris (NL). NM and NL neurons arise from distinct precursors of rhombomeres 5 and 6 (R5/R6). Here, we present in ovo electroporation of plasmid-encoded genes to study gene-related properties in these regions. We show a method for spatial and temporal control of gene expression that promote either gain or loss of functional phenotypes. By targeting auditory neural progenitor regions associated with R5/R6, we show plasmid transfection in NM and NL. Temporal regulation of gene expression can be achieved by adopting a tet-on vector system. This is a drug inducible procedure that expresses the genes of interest in the presence of doxycycline (Dox). The in ovo electroporation technique &#8211; together with either biochemical, pharmacological, and or in vivo functional assays &#8211; provides an innovative approach to study auditory neuron development and associated pathophysiological phenomena.

In Ovo Electroporation in the Chicken Auditory Brainstem

Auditory brainstem neurons of avians and mammals are specialized for fast neural encoding, a fundamental process for normal hearing functions. These neurons arise from distinct precursors of embryonic hindbrain. We present techniques utilizing electroporation to express genes in the hindbrain of chicken embryos to study gene function during auditory development.

In Ovo Electroporation nel Brainstem Auditory del pollo

Electroporation is a method that introduces genes of interest into biologically relevant organisms like the chicken embryo. It is long established that ...

A Novel Mammary Fat Pad Transplantation Technique to Visualize the Vessel Generation of Vascular Endothelial Stem Cells

Endothelial cells (ECs) are the fundamental building blocks of the vascular architecture and mediate vascular growth and remodeling to ensure proper vessel development and homeostasis. However, studies on endothelial lineage hierarchy remain elusive due to the lack of tools to gain access as well as to directly evaluate their behavior in vivo. To address this shortcoming, a new tissue model to study angiogenesis using the mammary fat pad has been developed. The mammary gland develops mostly in the postnatal stages, including puberty and pregnancy, during which robust epithelium proliferation is accompanied by extensive vascular remodeling. Mammary fat pads provide space, matrix, and rich angiogenic stimuli from the growing mammary epithelium. Furthermore, mammary fat pads are located outside the peritoneal cavity, making them an easily accessible grafting site for assessing the angiogenic potential of exogenous cells. This work also describes an efficient tracing approach using fluorescent reporter mice to specifically label the targeted population of vascular endothelial stem cells (VESCs) in vivo. This lineage tracing method, coupled with subsequent tissue whole-mount microscopy, enable the direct visualization of targeted cells and their descendants, through which the proliferation capability can be quantified and the differentiation commitment can be fate-mapped. Using these methods, a population of bipotent protein C receptor (Procr) expressing VESCs has recently been identified in multiple vascular systems. Procr+ VESCs, giving rise to both new ECs and pericytes, actively contribute to angiogenesis during development, homeostasis, and injury repair. Overall, this manuscript describes a new mammary fat pad transplantation and in vivo lineage tracing techniques that can be used to evaluate the stem cell properties of VESCs.

This work demonstrates a novel approach to assess the proliferation, differentiation, and vessel-forming potential of vascular endothelial stem cells (VESCs) through mammary fat pad transplantation followed by whole-mount tissue preparation for microscopic observation. A lineage tracing strategy to investigate the behavior of VESCs in vivo is also presented.

Una tecnica novel di trapianto di grassi del grasso mammario per visualizzare la generazione di vascelli delle cellule staminali endoteliali vascolari

Endothelial cells (ECs) are the fundamental building blocks of the vascular architecture and mediate vascular growth and remodeling to ensure proper ...

Organotypic Culture Method to Study the Development Of Embryonic Chicken Tissues

The embryonic chicken is commonly used as a reliable model organism for vertebrate development. Its accessibility and short incubation period makes it ideal for experimentation. Currently, the study of these developmental pathways in the chicken embryo is conducted by applying inhibitors and drugs at localized sites and at low concentrations using a variety of methods. In vitro tissue&#160;culturing is a technique that enables the study of tissues separated from the host organism, while simultaneously bypassing many of the physical limitations present when working with whole embryos, such as the susceptibility of embryos to high doses of potentially lethal chemicals. Here, we present an organotypic culturing protocol for culturing the embryonic chicken half head in vitro, which presents new opportunities for the examination of developmental processes beyond the currently established methods.

Here, we present an organotypic culturing protocol to grow embryonic chicken organs in vitro. Using this method, the development of embryonic chicken tissue can be studied, while maintaining a high degree of control over the culture environment.

Metodo di coltura organotypic per studiare lo sviluppo dei tessuti embrionali di pollo

The embryonic chicken is commonly used as a reliable model organism for vertebrate development. Its accessibility and short incubation period makes it ...