We would like to acknowledge David O'Brien for his support with data analysis, and all the members of the Canto-Soler lab for their critical discussions. This work was supported by NIH grants EY004859 and EY022631 (MVCS), Core Grant EY1765, and an unrestricted departmental grant from Research to Prevent Blindness, Inc.

Tutte le procedure descritte in questo lavoro sono stati eseguiti secondo le linee guida raccomandate dalla cura degli animali e del Comitato uso presso la Johns Hopkins University. 1. Ahead of Time: preparazione di strumenti, reagenti e piatti <ol><li> Preparazione di uova: incubare fecondate uova di gallina bianca Livorno a 37,5 ° C e 60% di umidità relativa per 5 giorni in una incubatrice uovo. NOTA: Un incubatore con capacità di oscillazione può essere utile per la standardizzazione e la sincronizzazione di sviluppo embrionale, ma a dondolo non è strettamente necessario per l&#39;esito di questo protocollo. </li><li> Plasmidi Preparazione: <ol><li> Disegno plasmide per costruire sovraespressione o downregulation gene (ad esempio tramite l&#39;uso della tecnologia RNAi 1,34) come necessario. Includere un gene reporter (come EGFP, RFP o LacZ) nel costrutto, quando possibile. NOTA: Alcuni promotori di uso comune come CMV, possono diventare a tacere dopo il tempo. Il promot CAGer (pollo beta-actina promotore con CMV enhancer) è una buona alternativa, fornendo espressione efficiente a lungo termine del gene di interesse 35-37. </li><li> Preparare una soluzione plasmide ad una concentrazione di 1,5 mg / mL in PBS sterile. OPTIONAL: Preparare la soluzione plasmide prima del tempo e conservarlo congelato fino al momento dell&#39;uso. NOTA: maggiore concentrazione plasmide non comportino un aumento significativo dell&#39;efficienza, mentre l&#39;uso di concentrazioni plasmide più basse riducono l&#39;efficienza di trasfezione 1. </li></ol></li><li> Elettrodi: <ol><li> Per l&#39;uso anodo un oro spessa punta elettrodo (vedi Tabella di materiali specifici / Equipment). Pulire con il 70% di etanolo. NOTA: Isolamento dell&#39;oro punta dell&#39;elettrodo, lasciando circa 0,5 mm a vista, è consigliabile per ridurre la dispersione della corrente elettrica. Informazioni per materiale isolante può essere trovato nella tabella di materiali specifici. </li><li> Fare un catodo di 2,5 mm quadrati bbue filamento, largo 4,5 mm (normalmente utilizzato in estrattori micropipetta). Con l&#39;aiuto di una pinza, svitare la scatola quadrata e raddrizzare filamento in una lunga striscia. Poi, al microscopio dissezione e utilizzando un righello come guida, piegare il filamento in una forma quadrata &#39;U&#39; lunga 3 mm alla base e 2 mm su un lato, con l&#39;altro lato della lunghezza rimanente del filamento (Figura 1 , C - D). Utilizzando pinze, immergere l&#39;elettrodo nel 96% di etanolo e fiamme al sterilizzare. Quindi collegare un cavo elettrico con un piccolo coccodrillo per il braccio più lungo dell&#39;elettrodo. </li></ol></li><li> Elettroporazione Camera Setup: <ol><li> Preparare la camera di elettroporazione tagliando il coperchio di una provetta sterile da 1,5 ml e apposizione, lato piatto verso il basso, ad una piastra di Petri 100 mm usando nastro (Figura 1E). NOTA: altro contenitore in forma di un pozzo di dimensioni simili funzionerebbe; tuttavia, un coperchio provetta è facilmente accessibile e autoclavabile. Il pozzodeve essere sufficiente per ospitare un elettrodo pulcino occhio e catodo grande, ma non così grande da risultare in una perdita di soluzione plasmide. </li></ol></li></ol> 2. Ex ovo Elettroporazione Procedura <ol><li> Preparazione degli strumenti: <ol><li> Sterilizzare gli strumenti di micro-dissezione (2 coppie di grandi pinze Moloney curve per aprire i gusci d&#39;uovo e scavando embrioni, 2 paia di fine-tip curvo pinzette Dumont per la rimozione di RPE, un paio di Bonn dentato forcipe per obiettivo la rimozione e vitreo) per immersione il punte in 96% di etanolo e fiammeggiante. Ambito dissezione Pulire con panni immersi nel 70% di etanolo. Scaldare una bottiglia di soluzione salina bilanciata sterile calcio-magnesio-libero di Hank (CMF-HBSS) in un bagno d&#39;acqua a 37 ° C. Riempire 100 mm sterili piastre Petri a 3/4 con caldo CMF-HBSS. </li><li> Mettere camera di elettroporazione con un altro microscopio dissezione pulito e riempire con 120 ml di soluzione di plasmide. Collegare gli elettrodi agli apparecchi elettroporazione, rendendoAssicurarsi elettrodo misura è collegato al polo negativo e oro punta dell&#39;elettrodo al polo positivo. </li></ol></li><li> Embrione Collezione: <ol><li> Pulire le uova strofinando conchiglie con salviette bagnate nel 70% di etanolo. Mantenere la procedura sterile per tutto il resto del protocollo per evitare di portare la contaminazione nelle culture. Utilizzando grandi pinze Moloney curve aprire con attenzione un buco nel guscio d&#39;uovo picchiettando delicatamente sulla punta arrotondata di un uovo (sopra la camera d&#39;aria) e poi presa e tirando fuori pezzi di conchiglia. </li><li> Con un altro paio di pinze rimuovere membrane e raccogliere embrione da scavare fuori l&#39;uovo (facendo attenzione a non danneggiare l&#39;embrione). Mettere in un piatto con CMF-HBSS. NOTA: raccogliere il maggior numero di embrioni necessari per ottenere il numero desiderato di tazze retina per elettroporazione come specificato in 2.4.4 </li><li> PUNTO CRITICO: stage gli embrioni secondo Hamburger e Hamilton 38. NOTA: Per ottenere gli embrioni ottimali di efficienzadeve essere di fase 27 (Figura 2A). </li><li> Eutanasia gli embrioni di decapitazione con pinze Moloney. </li></ol></li><li> Ottenere Coppe retina: <ol><li> Usando sottili pinzette Dumont, enucleare con attenzione gli occhi e trasferimento in un nuovo piatto CMF-HBSS. </li><li> Partendo dalla parte posteriore di ciascun occhio, in prossimità della testa del nervo ottico, e utilizzando due pinzette Dumont fine, introdurre una punta di ciascuna coppia di pinzette tra la retina neurale e RPE e accuratamente sezionare la RPE essendo cauto per non danneggiare neurale retina (Figura 2B). NOTA: La mancanza di Ca 2+ e Mg 2+ nella soluzione aiuterà il RPE stacca dalla retina neurale più facilmente. </li></ol></li><li> Elettroporazione: <ol><li> Impostare i parametri di elettroporatore per fornire impulsi di 5 quadrati di 15 volt, 50 msec, durata e intervallo 950 msec. Luogo &#39;U&#39; a forma di elettrodo negativo all&#39;interno della camera di elettroporazione (microcentrifuga coperchio di riempimentoED con 120 ml di soluzione contenente plasmide). </li><li> Usando pinzette Dumont curvi trasferire una tazza retinica alla camera di elettroporazione (scavando, non pressatura), e posizionarlo all&#39;interno dell&#39;elettrodo sagomato &#39;U&#39;, con la parte posteriore della retina verso il fondo dell&#39;elettrodo e la lente rivolta verso l&#39;alto ( Figura 2D). </li><li> Posizionare l&#39;anodo in modo che tocchi la parte anteriore dell&#39;occhio, accanto alla lente, e utilizzando pedale del electroporator fornire gli impulsi elettrici. </li><li> Trasferire tazza retina elettroporate in un piatto Petri con CMF-HBSS e ripetere la procedura di elettroporazione con ciascuno dei restanti tazze retiniche. NOTA: Fino a 6 tazze retina può essere elettroporate con la stessa soluzione plasmide senza una significativa diminuzione efficienza di trasfezione. </li><li> Rimuovere la lente e corpo vitreo di ogni coppa retinica elettroporate da loro presa con Bonn pinza dentata e tirando dolcemente attraverso tegli pupilla apertura tenendo l&#39;occhio in posizione con la parte posteriore delle pinzette Dumont curve. </li><li> Procedere alla dissociazione e la cultura delle cellule della retina elettroporate seguenti procedure standard. NOTA: per un protocollo dettagliato sulla dissociazione e la cultura di cellule retiniche pulcino si riferiscono a Vergara e Canto-Soler 2012, sui file 4 21 In questo protocollo le cellule vengono seminate a bassa densità su 6 pozzetti poliornitina rivestite o 35. piatti -mm (6 x 10 5 cellule / 35 mm di diametro e o piatto). Il mezzo utilizzato è medium 199 con penicillina / glutammina, siero di vitello fetale al 10% (FCS), e acido linoleico-BSA a 100 ug / ml. Le colture vengono mantenute in un umidificata 37 ° C incubatore con 5% di CO 2. Con questa procedura, 5 - 6 x 10 6 cellule in genere può essere ottenuto per ogni fase 27 occhio elettroporate. NOTA: Idealmente, non più di 3 ore dovrebbe passare tra il prelievo e la placcatura delle cellule, per assicurare una buona sopravvivenza cellularee ridurre al minimo lo stress. Una tipica temporizzazione per l&#39;osservazione e l&#39;ulteriore analisi è dopo 4 giorni di coltura, poiché a questo punto le cellule hanno raggiunto una buona differenziazione morfologica e molecolare e la sopravvivenza è ancora alto. </li></ol></li></ol>

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M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+hybrid+promoter-containing+vector+for+direct+cloning+and+enhanced+expression+of+PCR-amplified+ORFs+in+mammalian+cells.">A hybrid promoter-containing vector for direct cloning and enhanced expression of PCR-amplified ORFs in mammalian cells.</a> Mol Biol Rep. 37 (6), 2757-2765 (2009).</li><li>Hamburger, V., Hamilton, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+series+of+normal+stages+in+the+development+of+the+chick+embryo.">A series of normal stages in the development of the chick embryo.</a> Dev Dyn. 195 (4), 231-272 (1992).</li></ol>

The authors declare that they have no competing financial interests or other conflicts of interest.

La fase più critica per il successo di questo protocollo è la selezione della fase appropriata degli embrioni. In pubblicazioni precedenti, una serie di stadi embrionali è dato per queste colture, tipicamente definito dai giorni di incubazione o embrionali giorni (ED); così è di solito presume che l&#39;utilizzo ED 5 a ED 6 embrioni produrrà risultati equivalenti. Tuttavia abbiamo trovato che sulla fase 27 (ED 5), l&#39;efficienza di trasfezione sarà circa il 22% della popolazione cellulare totale, come indicato ...

Colture di cellule dissociate da retine pulcino embrionali sono stati ampiamente utilizzati per studiare vari aspetti della biologia cellulare dei fotorecettori, tra cui la loro sopravvivenza 2-9, differenziazione 10-12, crescita dei neuriti 13, e altro ancora. I vantaggi di questo sistema, sviluppato nel 1980 da Ruben Adler e collaboratori e perfezionati dai suoi e altri gruppi 14-20, risiedono nelle caratteristiche intrinseche del pulcino come un modello animale 21. Le grandi dimensioni dell&#39;occhio pulcino, anche a stadi embrionali, fornisce una grande quantità di materiali per le culture. Inoltre, quando le culture vengono eseguite utilizzando giorno embrionale (ED) 5 - 6 retine, 55-80% dei loro cellule progenitrici differenziarsi come fotorecettori 14,15,18,22,23, e dal momento che circa il 86% dei fotorecettori in questo animale sono coni 24, queste culture sono particolarmente adatti per studi che mettono in questo tipo cellulare. Abbiamo recentemente sviped e caratterizzato una semplice tecnica che consente ad alta efficienza plasmide trasfezione delle cellule in queste culture, ampliando così l&#39;utilità di questo sistema facilitando studi missexpression genetica 1. Lo sviluppo di questa tecnica derivava da un vuoto nella letteratura scientifica di metodi che fornisca un livello accettabile di trasfezione per consentire lo studio della funzione genica in maniera autonoma cella. Ciò è in parte perché colture neuronali primarie sono notoriamente difficili da trasfettare 25,26. Alcune delle tecniche più utilizzate in precedenza disponibili per questo scopo inclusi metodi di transfezione chimici quali lipofezione o calcio trasfezione fosfato-mediata, che determinano efficienze dell&#39;ordine del 3-5% e possono esercitare una notevole tossicità 27-32. Anche se l&#39;uso di plasmidi con un sistema reporter enzimatico può aggirare il problema della scarsa efficienza di trasfezione amplificando il segnale, non lo fannodiscriminare effetti specifici delle cellule, ed i loro risultati sono basati su una piccola popolazione di cellule che potrebbe non essere rappresentativo dell&#39;intero. Un altro metodo ampiamente usato al pulcino, infezione da virus RCA, è applicabile solo alle cellule proliferanti e quindi non adatto a questo sistema di coltura retinico primario 33. Nel protocollo corrente occhi pulcino embrionale sono enucleati nella fase 27 (ED 5), l&#39;epitelio pigmentato retinico (RPE) viene rimosso, e la coppa retinica è posto in una camera di elettroporazione riempita con una soluzione plasmide contenente e elettroporate con misura elettrodi, seguita da dissociazione della retina e della cultura utilizzando tecniche standard 21. Dopo ottimizzare questa procedura siamo stati in grado di ottenere sempre efficienza di trasfezione dell&#39;ordine del 22% del numero totale di cellule in coltura e 25% nella popolazione fotorecettore sola, senza compromettere le caratteristiche di sopravvivenza e la differenziazione delleculture 1. Qui forniamo un protocollo dettagliata che illustra tutte le fasi importanti di questa procedura al fine di assicurare il successo e la riproducibilità di questa tecnica.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>ECM 830 Electro Square Porator</td>
			<td>BTX/ Harvard Apparatus</td>
			<td>45-0052</td>
			<td></td>
		</tr>
		<tr>
			<td>Genetrode, L-Shaped, 5 mm Gold Tip</td>
			<td>BTX/ Harvard Apparatus</td>
			<td>45-0115 model 512</td>
			<td>Gold tipped electrode used as anode</td>
		</tr>
		<tr>
			<td>Polyimide Tubing</td>
			<td>Vention Medical</td>
			<td>custom made</td>
			<td>Internal Diameter: 0.5mm / wall thickness: 0.15-0.2mm. Used for insulating gold tiped electrode</td>
		</tr>
		<tr>
			<td>2.5mm square box filament, 4.5mm wide</td>
			<td>Sutter Instrument Company</td>
			<td>FB245B</td>
			<td>Used to make cathode electrode</td>
		</tr>
		<tr>
			<td>HBSS, no calcium, no magnesium, no phenol red</td>
			<td>Gibco - Life Technologies</td>
			<td>14175-095</td>
			<td></td>
		</tr>
		<tr>
			<td>Moloney forceps</td>
			<td>Roboz</td>
			<td>RS-8254</td>
			<td>Serrated; Slight Curve; 4.5&#34; Length</td>
		</tr>
		<tr>
			<td>Dumont tweezers 5/45</td>
			<td>Roboz</td>
			<td>RS-5058</td>
			<td>Pattern #5, 45 Degree Angle; .10 X .06mm Tip Size; 109mm Length</td>
		</tr>
		<tr>
			<td>Bonn micro forceps, 1x2 teeth</td>
			<td>Roboz</td>
			<td>RS-5172</td>
			<td>Tying Platform; 1X2 Teeth, 0.12mm Teeth; 3.75&#34; Length; .3mm Tip Width</td>
		</tr>
	</tbody>
</table>

efficient gene transfer in chick retinas for primary cell culture studies: an ex-ovo electroporation approach

Le culture dei fotorecettori arricchito cono derivati ​​da retine pulcino embrionali sono diventati uno strumento indispensabile per i ricercatori di tutto il mondo che studiano la biologia dei neuroni della retina, in particolare fotorecettori. Le applicazioni di questo sistema vanno oltre la ricerca di base, in quanto possono essere facilmente adattate alle alte tecnologie throughput per lo sviluppo di farmaci. Tuttavia, la manipolazione genetica dei fotorecettori retinici in queste culture ha dimostrato di essere molto impegnativo, che costituiscono una limitazione importante per l&#39;utilità del sistema. Abbiamo recentemente sviluppato e validato un ex ovo tecnica plasmide elettroporazione che aumenta il tasso di trasfezione di cellule retiniche in queste culture per cinque volte rispetto ad altri protocolli attualmente disponibili 1. In questo metodo occhi pulcino embrionale sono enucleati nella fase 27, l&#39;RPE viene rimosso, e la coppa retinica viene posto in una soluzione contenente plasmide e elettroporate utilizzando CUSTOM- facilmente costruitoelettrodi in. Le retine sono poi dissociate e coltivate utilizzando le procedure standard. Questa tecnica può essere applicata a studi overespressione nonché alla downregulation dell&#39;espressione genica, per esempio attraverso l&#39;uso della tecnologia RNAi plasmide-driven, comunemente ottenendo l&#39;espressione del transgene nel 25% della popolazione fotorecettori. Il formato video di questa pubblicazione rendere questa tecnologia facilmente accessibile ai ricercatori del settore, che consente lo studio della funzione dei geni in colture primarie della retina. Abbiamo incluso anche spiegazioni dettagliate sulle fasi critiche di questa procedura per un esito positivo e la riproducibilità.

Qui vi presentiamo un semplice protocollo per plasmide trasfezione in enucleate pulcino tazze retina per la successiva coltura cellulare dissociata. Trasfezione si ottiene elettroporazione utilizzando facile fare elettrodi personalizzati (Figure 1 - 2). I parametri descritti in questo protocollo sono stati ottimizzati per ottenere efficienze di trasfezione che variano tra il 20 e il 27% (con una media del 22%) (Figura 3D). Si noti che, per le ragioni sopra esposte, questi risultati sono...

Watch this Scientific Journal Video about Efficient Gene Transfer in Chick Retinas for Primary Cell Culture Studies: An Ex-ovo Electroporation Approach at JoVE.com

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

Pulcino colture di cellule della retina embrionali costituiscono validi strumenti per lo studio della biologia dei fotorecettori. Abbiamo sviluppato una tecnica di trasferimento genico efficace sulla base di ex ovo plasmide elettroporazione delle retine precedenti alla cultura. Questa tecnica aumenta considerevolmente l&#39;efficienza di transfezione su protocolli attualmente disponibili, rendendo la manipolazione genetica fattibile per questo sistema.

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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JoVE Journal

Developmental Biology

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

8.1K Views.  The Johns Hopkins University School of Medicine.  Pulcino colture di cellule della retina embrionali costituiscono validi strumenti per lo studio della biologia dei fotorecettori. Abbiamo sviluppato una tecnica di trasferimento genico efficace sulla base di ex ovo plasmide elettroporazione delle retine precedenti alla cultura. Questa tecnica aumenta considerevolmente l'efficienza di transfezione su protocolli attualmente disponibili, rendendo la manipolazione genetica fattibile per questo sistema. 

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

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 ...

Ricerca

Biologia dello sviluppo

Culture of Embryonic Mouse Cochlear Explants and Gene Transfer by Electroporation

Auditory hair cells located within the mouse organ of Corti detect and transmit sound information to the central nervous system. The mechanosensory hair cells are aligned in one row of inner hair cells and three rows of outer hair cells that extend along the basal to apical axis of the cochlea. The explant culture technique described here provides an efficient method to isolate and maintain cochlear explants from the embryonic mouse inner ear. Also, the morphology and molecular characteristics of sensory hair cells and nonsensory supporting cells within the cochlear explant cultures resemble those observed in vivo and can be studied within its intrinsic cellular environment. The cochlear explants can serve as important experimental tools for the identification and characterization of molecular and genetic pathways that are involved in cellular specification and patterning. Although transgenic mouse models provide an effective approach for gene expression studies, a considerable number of mouse mutants die during embryonic development thereby hindering the analysis and interpretation of developmental phenotypes. The organ of Corti from mutant mice that die before birth can be cultured so that their in vitro development and responses to different factors can be analyzed. Additionally, we describe a technique for electroporating embryonic cochlear explants ex vivo which can be used to downregulate or overexpress specific gene(s) and analyze their potential endogenous function and test whether specific gene product is necessary or sufficient in a given context to influence mammalian cochlear development1-8.

We present a method that describes isolation and culture of cochlear explants from embryonic mouse inner ear. We also demonstrate a method for gene transfer into cochlear explants via square-wave electroporation. The in vitro explant culture coupled with gene transfer technique enables researchers to study the effects of altering gene expression during development.

Cultura della embrionali di topo cocleari espianti e Gene Transfer con elettroporazione

Auditory hair cells located within the mouse organ of Corti detect and transmit sound information to the central nervous system. The mechanosensory hair ...

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 ...

Forward Genetic Approach to Uncover Stress Resistance Genes in Mice &#8212; A High-throughput Screen in ES Cells

Phenotype-driven genetic screens in mice is a powerful technique to uncover gene functions, but are often hampered by extremely high costs, which severely limits its potential. We describe here the use of mouse embryonic stem (ES) cells as surrogate cells to screen for a phenotype of interest and subsequently introduce these cells into a host embryo to develop into a living mouse carrying the phenotype. This method provides (1) a cost effective, high-throughput platform for genetic screen in mammalian cells; (2) a rapid way to identify the mutated genes and verify causality; and (3) a short-cut to develop mouse mutants directly from these selected ES cells for whole animal studies. We demonstrated the use of paraquat (PQ) to select resistant mutants and identify mutations that confer oxidative stress resistance. Other stressors or cytotoxic compounds may also be used to screen for resistant mutants to uncover novel genetic determinants of a variety of cellular stress resistance.

Stress resistance is one of the hallmarks for longevity and is known to be genetically governed. Here, we developed an unbiased high-throughput method to screen for mutations that confer stress resistance in ES cells with which to develop mouse models for longevity studies.

Approccio genetico in avanti per scoprire la resistenza allo stress geni nei topi - Uno Schermo high-throughput in cellule ES

Phenotype-driven genetic screens in mice is a powerful technique to uncover gene functions, but are often hampered by extremely high costs, which severely ...

Ex Vivo Culture of Chick Cerebellar Slices and Spatially Targeted Electroporation of Granule Cell Precursors

The cerebellar external granule layer (EGL) is the site of the largest transit amplification in the developing brain, and an excellent model for studying neuronal proliferation and differentiation. In addition, evolutionary modifications of its proliferative capability have been responsible for the dramatic expansion of cerebellar size in the amniotes, making the cerebellum an excellent model for evo-devo studies of the vertebrate brain. The constituent cells of the EGL, cerebellar granule progenitors, also represent a significant cell of origin for medulloblastoma, the most prevalent paediatric neuronal tumour. Following transit amplification, granule precursors migrate radially into the internal granular layer of the cerebellum where they represent the largest neuronal population in the mature mammalian brain. In chick, the peak of EGL proliferation occurs towards the end of the second week of gestation. In order to target genetic modification to this layer at the peak of proliferation, we have developed a method for genetic manipulation through ex vivo electroporation of cerebellum slices from embryonic Day 14 chick embryos. This method recapitulates several important aspects of in vivo granule neuron development and will be useful in generating a thorough understanding of cerebellar granule cell proliferation and differentiation, and thus of cerebellum development, evolution and disease.

Ex Vivo Culture of Chick Cerebellar Slices and Spatially Targeted Electroporation of Granule Cell Precursors

The cerebellar external granule layer is the site of the largest transit amplification in the developing brain. Here, we present a protocol to target genetic modification to this layer at the peak of proliferation using ex vivo electroporation and culture of cerebellar slices from embryonic Day 14 chick embryos.

Ex Cultura Vivo del Chick cerebellare fette e spazialmente mirata elettroporazione di granuli precursori delle cellule

The cerebellar external granule layer (EGL) is the site of the largest transit amplification in the developing brain, and an excellent model for studying ...

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 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.

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 ...

An Enzyme- and Serum-free Neural Stem Cell Culture Model for EMT Investigation Suited for Drug Discovery

Epithelial to mesenchymal transition (EMT) describes the process of epithelium transdifferentiating into mesenchyme. EMT is a fundamental process during embryonic development that also commonly occurs in glioblastoma, the most frequent malignant brain tumor. EMT has also been observed in multiple carcinomas outside the brain including breast cancer, lung cancer, colon cancer, gastric cancer. EMT is centrally linked to malignancy by promoting migration, invasion and metastasis formation. The mechanisms of EMT induction are not fully understood. Here we describe an in vitro system for standardized isolation of cortical neural stem cells (NSCs) and subsequent EMT-induction. This system provides the flexibility to use either single cells or explant culture. In this system, rat or mouse embryonic forebrain NSCs are cultured in a defined medium, devoid of serum and enzymes. The NSCs expressed Olig2 and Sox10, two transcription factors observed in oligodendrocyte precursor cells (OPCs). Using this system, interactions between FGF-, BMP- and TGF&#946;-signaling involving Zeb1, Zeb2, and Twist2 were observed where TGF&#946;-activation significantly enhanced cell migration, suggesting a synergistic BMP-/TGF&#946;-interaction. The results point to a network of FGF-, BMP- and TGF&#946;-signaling to be involved in EMT induction and maintenance. This model system is relevant to investigate EMT in vitro. It is cost-efficient and shows high reproducibility. It also allows for the comparison of different compounds with respect to their migration responses (quantitative distance measurement), and high-throughput screening of compounds to inhibit or enhance EMT (qualitative measurement). The model is therefore well suited to test drug libraries for substances affecting EMT.

Epithelial to mesenchymal transition (EMT) allows cancers to become invasive. To investigate EMT, a neural stem cell (NSC)-based in vitro model devoid of serum and enzymes is described. This standardized system allows quantitative and qualitative assessment of cell migration, gene and protein expression. The model is suited for drug discovery.

Un modello enzima e Neural Stem Cell Culture siero-libero per EMT Investigation Adatto per Drug Discovery

Epithelial to mesenchymal transition (EMT) describes the process of epithelium transdifferentiating into mesenchyme. EMT is a fundamental process during ...

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 ...

CRISPR-mediated Loss of Function Analysis in Cerebellar Granule Cells Using In Utero Electroporation-based Gene Transfer

Brain malformation is often caused by genetic mutations. Deciphering the mutations in patient-derived tissues has identified potential causative factors of the diseases. To validate the contribution of a dysfunction of the mutated genes to disease development, the generation of animal models carrying the mutations is one obvious approach. While germline genetically engineered mouse models (GEMMs) are popular biological tools and exhibit reproducible results, it is restricted by time and costs. Meanwhile, non-germline GEMMs often enable exploring gene function in a more feasible manner. Since some brain diseases (e.g., brain tumors) appear to result from somatic but not germline mutations, non-germline chimeric mouse models, in which normal and abnormal cells coexist, could be helpful for disease-relevant analysis. In this study, we report a method for the induction of CRISPR-mediated somatic mutations in the cerebellum. Specifically, we utilized conditional knock-in mice, in which Cas9 and GFP are chronically activated by the CAG (CMV enhancer/chicken &#223;-actin) promoter after Cre-mediated recombination of the genome. The self-designed single-guide RNAs (sgRNAs) and the Cre recombinase sequence, both encoded in a single plasmid construct, were delivered into cerebellar stem/progenitor cells at an embryonic stage using in utero electroporation. Consequently, transfected cells and their daughter cells were labeled with green fluorescent protein (GFP), thus facilitating further phenotypic analyses. Hence, this method is not only showing electroporation-based gene delivery into embryonic cerebellar cells but also proposing a novel quantitative approach to assess CRISPR-mediated loss-of-function phenotypes.

CRISPR-mediated Loss of Function Analysis in Cerebellar Granule Cells Using In Utero Electroporation-based Gene Transfer

Conventional loss-of-function studies of genes using knockout animals have often been costly and time-consuming. Electroporation-based CRISPR-mediated somatic mutagenesis is a powerful tool to understand gene functions in vivo. Here, we report a method to analyze knockout phenotypes in proliferating cells of the cerebellum.

CRISPR-mediata di perdita di funzione di analisi in granuli cerebellari cellule utilizzando nell'Utero basati su elettroporazione trasferimento del Gene

Brain malformation is often caused by genetic mutations. Deciphering the mutations in patient-derived tissues has identified potential causative factors ...

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