Name: high efficiency, site-specific transfection of adherent cells with sirna using microelectrode arrays (mea)
Uploaded: 2012-09-13T13:00:00
Description: Watch this Scientific Journal Video about High efficiency, Site-specific Transfection of Adherent Cells with siRNA Using Microelectrode Arrays MEA at JoVE.com

1. MEA Forberedelse <ol><li> MMA til brug i elektroporation eksperimenter kan enten fremstilles ved anvendelse af standard fotolitografiske teknik som tidligere beskrevet 18 eller købes direkte fra leverandører af MEA fabrikanter såsom Multi Channel Systems <a href="http://www.multichannelsystems.com" target="_blank">(http:/www.multichannelsystems.com/)</a> og Alpha MED Scientific, Inc. <a href="http://www.med64.com/" target="_blank">( http://www.MED64.com)</a> . De MMA anvendes i vore eksperimenter blev...

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I denne video artikel vil vi demonstrere brugen af ​​MEA til stedspecifik transfektion af HeLa-celler med røræg sekvens af siRNA. En af fordelene ved denne teknologi er dens anvendelighed i forskellige cellelinier, herunder primære cellelinjer. Vores laboratorium har tidligere vist, at anvendelsen af denne teknologi til stedspecifik transfektion af primære hippocampale neuronale kultur fra E18 dage gamle rotter og NIH-3T3-celler med scrambled siRNA sekvenser og GFP-plasmid 18, 19. Vi har også haft su...

<table border="1"><tr><td> Navn på reagenset </td><td> Firma </td><td> Katalognummer </td><td> Kommentarer (valgfrit) </td></tr><tr><td> Cell medier: Advanced MEM L-Glutamin 200 mM Penicillin / streptomycin Føtalt bovint serum </td><td> Gibco / Invitrogen Himedia Laboratories / VWR Lonza Group Ltd Gibco / Invitrogen </td><td> 12492-013 95057-448 09-757F 16000-044 </td><td> Cell media sammensætning: 2% FBS, 2% L-glutamin og 2% Pennstrep i Advance MEM </td></tr><tr><td> Trypsin EDTA </td><td> Mediatech, Inc. </td><td> 25 til 053-CL </td><td></td></tr><tr><td> PBS </td><td> Mediatech, Inc. </td><td> 21-040-CV </td><td></td></tr><tr><td> Alexa 488 og rhodamin mærket krypterede SEQUning siRNA </td><td> Qiagen, Inc. </td><td> 1027292 </td><td></td></tr><tr><td> Elektroporation buffer </td><td> Biorad Laboratories </td><td> 165-2677 </td><td></td></tr><tr><td> Bølgeformsgenerator </td><td> Pragmatisk </td><td> 2414A </td><td> Enhver bølgeform / impulsgeneratoren, der kan levere de ønskede impulser kan anvendes. </td></tr></table>

high efficiency, site-specific transfection of adherent cells with sirna using microelectrode arrays (mea)

Opdagelsen af RNAi vej i eukaryoter og den efterfølgende udvikling af RNAi midler, såsom siRNA og shRNA, har opnået en potent metode til tavshed specifikke gener 1-8 for funktionel genomforskning og sygdomsbehandling. En stor udfordring er involveret i RNAi baserede studier er levering af RNAi midler til målrettede celler. Traditionelle ikke-virale leveringsteknikker, såsom bulk-elektroporering og kemiske transfektionsfremgangsmåder mangler ofte den nødvendige rumlige kontrol med levering og give dårlige transfektionseffektiviteter 9-12. Nylige fremskridt inden for kemiske transfektionsfremgangsmåder, såsom kationiske lipider, kationiske polymerer og nanopartikler har ført til en meget forbedret transfektionseffektiviteter 13. Men disse teknikker stadig undlader at tilbyde præcis rumlig kontrol over leveringen, der kan uhyre gavne miniaturiserede high-throughput teknologier, encellede undersøgelser og efterforskning af celle-celle-interaktioner. nt &quot;&gt; Seneste teknologiske fremskridt inden for gen-levering har gjort det muligt high-throughput transfektion af adhærente celler 14-23, hvoraf størstedelen bruger mikroskala elektroporation. Individuel elektroporation giver præcise spatio-temporale kontrol over levering (op til enkelte celler), og har vist sig at opnå høje virkningsgrader 19, 24-26. Derudover reagerer elektroporering tilgange ikke kræver en lang periode med inkubation (typisk 4 timer) siRNA og DNA-komplekser som nødvendigt kemisk baserede transfektionsfremgangsmåder og føre til direkte optagelse af nøgent siRNA og DNA molekyler ind i cellecytoplasmaet. Som følge genekspression kan opnås så tidligt som seks timer efter transfektion 27. Vores laboratorium har tidligere vist, at anvendelse af mikroelektrode-arrays (MEA) til stedspecifik transfektion i adhærente mammale cellekulturer 17-19. I MEA tilgang, er levering af genetisk nyttelast opnås via lokaliseret mikroskala electroporatiden af ​​celler. En anvendelse af elektrisk puls til udvalgte elektroder skaber lokale elektriske felt der fører til elektroporation af celler, der forefindes i området af de stimulerede elektroder. Den uafhængige kontrol af mikro-elektroder giver rumlige og tidsmæssige kontrol over transfektion og også giver flere transfektionsforsøg baserede eksperimenter, der skal udføres på den samme kultur øger den eksperimentelle gennemløb og reducere kultur-til-kultur variabilitet. Her beskriver vi forsøgsopstillingen og protokollen for målrettet transfektion af adhærente HeLa-celler med en fluorescens mærket kodet sekvens siRNA anvendelse af elektroporation. Den samme protokol kan også anvendes til transfektion af plasmidvektorer. Endvidere kan protokollen beskrevet her kan let udvides til flere forskellige mammale cellelinier med mindre modifikationer. Kommercielle tilgængelighed af multilaterale miljøaftaler med både prædefinerede og custom elektrodemønstre gør denne teknik tilgængelig to fleste forskningslaboratorier med grundlæggende cellekultur udstyr.

Watch this Scientific Journal Video about High efficiency, Site-specific Transfection of Adherent Cells with siRNA Using Microelectrode Arrays MEA at JoVE.com

High efficiency, Site-specific Transfection of Adherent Cells with siRNA Using Microelectrode Arrays MEA

Artiklen indeholder oplysninger om protokollen for stedspecifik transfektion af krypterede sekvens af siRNA i en adhærent pattedyrcellekultur ved hjælp af en mikroelektrode array (MEA).

Høj effektivitet, Site-specifik Transfektion af adhærente celler med siRNA Brug mikroelektrode Arrays (MEA)

The discovery of RNAi pathway in eukaryotes and the subsequent development of RNAi agents, such as siRNA and shRNA, have achieved a potent method for ...

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High efficiency, Site-specific Transfection of Adherent Cells with siRNA Using Microelectrode Arrays (MEA)

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