Name: efficient gene transfer in chick retinas for primary cell culture studies: an ex-ovo electroporation approach
Uploaded: 2015-11-02T14:00:00
Description: 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

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.

Alle procedurer, der er beskrevet i dette arbejde blev udført i overensstemmelse med de retningslinjer, der anbefales af Animal Care og brug Udvalg på Johns Hopkins University. 1. Forud for tid: Udarbejdelse af Instrumenter, reagenser og fade <ol><li> Fremstilling af æg: Inkuber befrugtede White Leghorn hønseæg ved 37,5 ° C og 60% relativ fugtighed i 5 dage i et æg inkubator. BEMÆRK: En inkubator med rokkende kapacitet kan være nyttige for standardisering og synkronisering ...

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Den mest kritiske skridt for succes i denne protokol er at vælge den rette fase af embryoner. I tidligere udgivelser, er en række embryonale stadier givet for disse kulturer, typisk defineret af dages inkubation eller embryonale dage (ED); dermed er det normalt antages, at bruge ED 5 til ED 6 embryoner vil give tilsvarende resultater. Men vi har fundet, at på scenen 27 (ED 5), vil transfektionseffektivitet være omkring 22% af den totale cellepopulation, som anført ovenfor; endnu effektivitet vil falde til 16%, hvis...

Dissocierede cellekulturer fra embryonale chick nethinder er ofte blevet brugt til at studere forskellige aspekter af fotoreceptorer cellebiologi, herunder deres overlevelse 2-9, differentiering 10-12, neuritvækst 13, og meget mere. Fordelene ved dette system, der er udviklet i 1980&#39;erne af Ruben Adler og samarbejdspartnere og perfektioneret af hans og andre grupper 14-20, bor i de iboende egenskaber chick som en dyremodel 21. Den store størrelse af chick øjet, selv ved fosterstadiet, giver store mængder materiale for kulturer. Desuden, når kulturer udføres ved anvendelse af embryonale dag (ED) 5 - 6 nethinder, 55 - 80% af deres progenitor celler differentierer som fotoreceptorer 14,15,18,22,23, og da ca. 86% af fotoreceptorerne i dette dyr er kegler 24, disse kulturer er særligt egnede til undersøgelser, der fokuserer på denne celletype. Vi har for nylig udvikPED og karakteriseret en simpel teknik, som muliggør højeffektiv plasmid transfektion af cellerne i disse kulturer, og dermed udvide anvendeligheden af dette system ved at lette genetisk missexpression undersøgelser 1. Udviklingen af ​​denne teknik stammede fra et tomrum i den videnskabelige litteratur af metoder, der vil tilvejebringe et acceptabelt niveau for transfektion at muliggøre undersøgelse af genfunktion i en celle uafhængig måde. Dette skyldes delvist, at primære neuronale kulturer er notorisk svære at transficere 25,26. Nogle af de mest almindeligt anvendte teknikker tidligere tilgængelige til dette formål inkluderet kemiske transfektionsfremgangsmåder såsom lipofektion eller calciumphosphat-medieret transfektion, der resulterer i effektiviteter i størrelsesordenen 3-5% og kan være en betydelig toksicitet 27-32. Selv om anvendelsen af ​​plasmider med en enzymatisk reporter systemet kan omgå problemet med ringe transfektionseffektivitet ved at forstærke signalet, ikke dediskriminere celle-specifikke effekter, og deres resultater er baseret på en lille celle befolkning, der måske ikke er repræsentative for hele. En anden meget anvendt metode i chick, RCAS virusinfektion, er kun for prolifererende celler og dermed ikke egnet til dette primære retinal kultur-system 33. I den nuværende protokol embryoniske kyllinge øjne er tømte i trin 27 (ED 5), det retinale pigmenterede epithelium (RPE) fjernes, og retinal koppen er placeret i et elektroporation kammer fyldt med et plasmid-indeholdende opløsning og elektroporeret under anvendelse af specialfremstillet elektroder, efterfulgt af retinal dissociation og kultur under anvendelse af standardteknikker 21. Efter optimering denne procedure har vi været i stand til konsekvent at opnå transfektionseffektiviteten af ​​størrelsesordenen 22% af det samlede antal celler i kultur og 25% i fotoreceptor befolkningen alene, uden at det går overlevelse og differentiering egenskaberkulturer 1. Her giver vi en detaljeret protokol beskriver alle de vigtige trin i denne procedure for at sikre succes og reproducerbarheden af ​​denne teknik.

<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

Keglen fotoreceptor-berigede kulturer afledt af embryonale chick nethinder er blevet et uundværligt redskab for forskere i hele verden studerer biologi retinale neuroner, især fotoreceptorer. Anvendelserne af dette system ud over grundforskning, da de let kan tilpasses til højt gennemløb teknologier for lægemiddeludvikling. Imidlertid har genmanipulation af retinale fotoreceptorer i disse kulturer vist sig at være meget udfordrende, udgør en vigtig begrænsning for anvendeligheden af ​​systemet. Vi har for nylig udviklet og valideret en ex ovo plasmid elektroporation teknik, der øger hastigheden af transfektion af retinale celler i disse kulturer med fem gange sammenlignet med andre aktuelt tilgængelige protokoller 1. Ved denne fremgangsmåde embryonale chick øjne er tømte i trin 27, RPE fjernes, og retinal koppen er placeret i et plasmid-indeholdende opløsning og elektroporeret under anvendelse af let konstrueres kun-lavet elektroder. Nethinder derefter dissocieres og dyrkes under anvendelse af standardprocedurer. Denne teknik kan anvendes til overekspression undersøgelser samt til nedregulering af genekspression, fx via anvendelse af plasmid-drevet RNAi teknologi, der almindeligvis opnå transgen ekspression i 25% af fotoreceptor befolkning. Den video format denne publikation vil gøre denne teknologi let tilgængelig for forskere på området, hvilket muliggør studiet af gen-funktion i primære retinale kulturer. Vi har også inkluderet detaljerede forklaringer om de kritiske trin i denne procedure for et vellykket resultat og reproducerbarhed.

Her præsenterer vi en enkel protokol til plasmidtransfektion ind tømte chick retinale pokaler til efterfølgende dissocieret cellekultur. Transfektion opnås ved elektroporering under anvendelse af let at gøre brugerdefinerede elektroder (figur 1 - 2). De i denne protokol beskrevne parametre er blevet optimeret for at opnå transfektionseffektiviteten der spænder mellem 20 og 27% (med et gennemsnit på 22%) (figur 3D). Bemærk, at for de ovenfor anførte grunde, er disse resultater ...

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

Embryonale chick retina cellekulturer udgør værdifulde værktøjer til undersøgelse af fotoreceptor biologi. Vi har udviklet en effektiv genoverførsel teknik baseret på ex ovo plasmid elektroporation af nethinder før kulturen. Denne teknik forøger transfektionseffektiviteten i øjeblikket tilgængelige protokoller, hvilket gør genmanipulation muligt for dette system.

Effektiv Gene Transfer i Chick nethinder for primær cellekultur Studies: An<em&gt; Ex-ovo</em&gt; Elektroporation Approach

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

Research

JoVE Journal

Developmental Biology

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

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

Optimeret<em&gt; Ex-ovo</em&gt; Dyrkning af kyllingeembryoer til fremskredne stadier af udvikling

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

Culture of Embryonic Mouse Cochlear Explants and Gene Transfer by Electroporation

Kultur af Embryonale Mouse Cochlear Eksplantater og Gene Transfer fra Elektroporation

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

En optogenetic fremgangsmåde for vurdering af Dannelse af neuronal forbindelser i en co-kultur System

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

Forward Genetic Approach to Uncover Stress Resistance Genes in Mice ÃƒÂ¢Ã¢â€šÂ¬Ã¢â‚¬Â A High-throughput Screen in ES Cells

Frem Genetisk tilgang til Afdække Stress resistensgener i mus - En Højkapacitetsforskning skærm i ES-celler

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

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

Ex Vivo Kultur Chick cerebellare Skiver og Rumligt Målrettet Elektroporation af granulat celleforstadier

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

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

Gene Transfer til kylling Auditory Organ med<em&gt; I Ovo</em&gt; Micro-elektroporation

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

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

En roman<em&gt; Ex Ovo</em&gt; Banding Teknik til Alter intrakardielle Hæmodynamik i en Embryonale Kylling System

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

En enzym- og Serum-fri neural stamcelle Culture Model for EMT Investigation Egnet til 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

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

En neddykket Filter Paper Sandwich til Langsigtet<em&gt; Ex Ovo</em&gt; Time-lapse Imaging af Early Chick embryoner

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 ...

Whole-cell Patch-clamp Recordings of Isolated Primary Epithelial Cells from the Epididymis

Hele-celle patch-clamp optagelser af isolerede primære epitelceller fra epididymis

The epididymis is an essential organ for sperm maturation and reproductive health. The epididymal epithelium consists of intricately connected cell types ...