Name: analysis of the gap junction-dependent transfer of mirna with 3d-frap microscopy
Uploaded: 2017-06-19T14:00:00
Description: Watch this Scientific Journal Video about Analysis of the Gap Junction-dependent Transfer of miRNA with 3D-FRAP Microscopy at JoVE.com

Dette arbejde blev støttet af Forbundsministeriet for Uddannelse og Forskning Tyskland (FKZ 0312138A og FKZ 316159), staten Mecklenburg-Vorpommern med EU&#39;s strukturfonde (ESF / IVWM-B34-0030 / 10 og ESF / IVBM-B35-0010 / 12), DFG (DA1296-1), og German Heart Foundation (F / 01/12). Desuden understøttes RD af FORUN-programmet fra Rostock University Medical Center (889001), DAMP Foundation og BMBF (VIP + 00240).

Alle trin i denne protokol, der involverer neonatale mus, blev udført pr. De etiske retningslinjer for dyrepleje af Rostock University Medical Center. 1. Fremstilling af cellekulturretter og medium til kardiomyocytkultur <ol><li> Coat en cellekulturplade med 0,1% gelatine i PBS og inkuber ved 37 ° C i 4 timer eller ved 4 ° C natten over. Fjern gelatinen og lad det tørre under steril laminar luftstrøm. </li><li> Forbered cellekulturmedium sammensat af 50 ml DMEM suppleret med 10% føta...

<ol>
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<html> MiRNA&#39;er er nøgleaktører i cellulær fysiologi og blev vist til at fungere som signalmolekyler ved at anvende-blandt andet -GJ&#39;er som en vej for intercellulær udveksling 11 , 12 , 22 . Den nuværende protokol præsenterer en in vitro levende cellebilledteknik for at karakterisere denne GJ-afhængige pendling ved anvendelse af fluorescerende miRNA&#39;er i celleklynger. Prot...

Små ikke-kodende RNA&#39;er er vigtige aktører inden for cellegeneregulering. Disse molekyler er sammensat af 20-25 nukleotider, som binder til et specifikt mål-mRNA, hvilket fører til blokering af translation eller til mRNA-nedbrydning 1 , 2 . Genreguleringsprocessen, der gennemføres af små RNA&#39;er, såsom miRNA og siRNA, er en stærkt bevaret mekanisme, som er blevet fundet i mange forskellige arter 3 . Især miRNA-molekyler er af afgørende betydning for en række fysiologiske processer, herunder proliferation, differentiering og regenerering 4 , 5 . Derudover tilskrives dysreguleringen af ​​miRNA-ekspression mange patologiske lidelser. Tilsvarende har miRNA&#39;er vist sig at være egnet som biomarkører til diagnose og som terapeutiske midler til genterapi 6 , 7 </sup&gt;. Gap junctions (GJs) er specialiserede proteinkonstruktioner i plasmamembranen af ​​to tilstødende celler, der tillader diffusionsudveksling af molekyler med en molekylvægt på op til 1 kD. De har vist sig at være vigtige for vævsudvikling, differentiering, celledød og patologiske lidelser såsom cancer eller kardiovaskulær sygdom 8 , 9 , 10 . Flere molekyler er blevet beskrevet som værende i stand til at krydse GJ-kanaler, herunder ioner, metabolitter og nukleotider. Interessant blev GJs også fundet at tilvejebringe en vej til den intercellulære bevægelse af små RNA&#39;er 11 , 12 . Således kan miRNA&#39;er virke ikke kun inden for cellen, hvori de fremstilles, men også inden for modtagerceller. Dette fremhæver rollen som miRNA&#39;er i det intercellulære signaltransduktionssystem. Samtidig viser dataeneAt den mellemliggende intercellulære kommunikation mellem kløften er tæt forbundet med miRNA-funktionen. På grund af den betydelige virkning af miRNA og GJ på vævshomostase, patologi og diagnose vil en omfattende forståelse af GJs funktion og den relaterede intercellulære dynamik i miRNA bidrage til at klarlægge mekanismerne for miRNA-baserede sygdomme og udvikle nye strategier for MiRNA terapier. Afhængig af omfanget af mellemrumskobling kan overførslen af ​​miRNA-molekyler mellem celler være en meget hurtig proces. Derfor kræves en metode, der muliggør visualisering og kvantificering af den hurtige intercellulære bevægelse af disse regulatoriske signalmolekyler. Almindeligvis er flowcytometri og molekylærbiologiske teknikker blevet anvendt til at demonstrere shuttling af små RNA&#39;er 11 , 12 , 13 , 14 . Men i modsætning til FRAP-mikroberKopi, disse tilgange mangler høj tidsmæssig opløsning, hvilket er obligatorisk, når man analyserer udvekslingen af ​​miRNA via GJs. Desuden er FRAP-mikroskopi mindre invasiv og repræsenterer derfor en kraftfuld og ny live-cellebilledteknik til at evaluere den GJ-afhængige molekylveksling i flere celletyper 15 , 16 , 17 . Her præsenterer vi en detaljeret protokol, der beskriver anvendelsen af ​​3D-FRAP for at vurdere miRNA shuttling mellem cardiomyocytter. Til dette formål blev kardiomyocytter transficeret med fluorescensmærkede miRNA. En celle mærket med denne miRNA blev photobleached, og gap-forbindelses-miRNA-tilstrømningen fra tilstødende celler blev registreret på en tidsafhængig måde. Den høje temporale opløsning af FRAP-eksperimenter giver mulighed for at udføre kinetiske undersøgelser for den præcise evaluering af den intercellulære overførsel af miRNA og siRNA mellem levende celler. MoreoEftersom små RNA&#39;er kan udveksles via forskellige mekanismer med meget forskellig kinetik, kan FRAP-mikroskopi bidrage til at præcisere, i hvilket omfang GJ&#39;er er involveret i de respektive shuttling-processer 18 . Derudover kan 3D-FRAP bruges til at undersøge fysiologiske og patologiske ændringer i GJ-permeabilitet og dens indvirkning på små RNA-overførsler 15 , 19 .

<table border="0" cellpadding="0" cellspacing="0" width="698">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">neonatal NMRI mice</td>
			<td style="width:159px;">Charles River</td>
			<td style="width:119px;"></td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">Gelatin</td>
			<td style="width:159px;">Sigma Aldrich</td>
			<td style="width:119px;">G7041</td>
			<td style="width:179px;">0.1% solution in PBS, sterilized</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">PBS</td>
			<td style="width:159px;">Pan Biotech</td>
			<td style="width:119px;">P04-53500</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Dulbecco&#180;s modified medium</td>
			<td style="width:159px;">Pan Biotech</td>
			<td style="width:119px;">P04-03550</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">Penicillin/Streptomycin</td>
			<td style="width:159px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">15140122</td>
			<td style="width:179px;">100 U/ml, 100&#181;g/ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Fetal bovine serum</td>
			<td style="width:159px;">Pan Biotech</td>
			<td style="width:119px;">P30-3306</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Cell culture plastic</td>
			<td style="width:159px;">TPP</td>
			<td style="width:119px;"></td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">Primary Cardiomyocyte Isolation Kit</td>
			<td style="width:159px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">88281</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:243px;">HBSS</td>
			<td style="width:159px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">88281</td>
			<td style="width:179px;">included in primary cardiomyocyte isolation kit</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">Trypan blue</td>
			<td style="width:159px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">15250061</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:243px;">miRIDIAN Dy547 labeled microRNA Mimic&#160;</td>
			<td style="width:159px;">Dharmacon</td>
			<td style="width:119px;">CP-004500-01-05</td>
			<td style="width:179px;">dissolve in RNAse free water, stock solution 20&#181;M</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:243px;">Sodium phosphate monobasic</td>
			<td style="width:159px;">Sigma</td>
			<td style="width:119px;">S3139</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Sodium phosphate dibasic</td>
			<td style="width:159px;">Carl Roth</td>
			<td style="width:119px;">T877.2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Potassium chloride</td>
			<td style="width:159px;">Sigma</td>
			<td style="width:119px;">P9333</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Magnesium chloride</td>
			<td style="width:159px;">Serva</td>
			<td style="width:119px;">28305.01</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Sodium succinate</td>
			<td style="width:159px;">Carl Roth</td>
			<td style="width:119px;">3195.1</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">0.05% Trypsin/EDTA solution</td>
			<td style="width:159px;">Merck</td>
			<td style="width:119px;">L2153</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">4-well- Glass bottom chamber slides</td>
			<td style="width:159px;">IBIDI</td>
			<td style="width:119px;">80827</td>
			<td style="width:179px;">coat with 0.1% gelatin solution</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">Amaxa Nucleofector II</td>
			<td style="width:159px;">Lonza</td>
			<td style="width:119px;"></td>
			<td style="width:179px;">program G-009 was used for Electroporation&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">LSM 780 ELYRA PS.1 system</td>
			<td style="width:159px;">Zeiss</td>
			<td style="width:119px;"></td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Excel software</td>
			<td style="width:159px;">Microsoft</td>
			<td style="width:119px;"></td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">&#945;-actinin antibody</td>
			<td style="width:159px;">Abcam</td>
			<td style="width:119px;">ab9465</td>
			<td style="width:179px;">dilution 1:200</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Connexin43 antibody</td>
			<td style="width:159px;">Santa Cruz</td>
			<td style="width:119px;">sc-9059</td>
			<td style="width:179px;">dilution 1:200</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">goat anti-mouse Alexa 594 antibody</td>
			<td style="width:159px;">Thermo Fisher Scientific</td>
			<td>A-11005</td>
			<td>dilution 1:300</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">goat anti-rabbit Alexa 488 antibody&#160;</td>
			<td style="width:159px;">Thermo Fisher Scientific</td>
			<td>A-11034</td>
			<td>dilution 1:300</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">Connexin43 siRNA</td>
			<td style="width:159px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">AM16708 ID158724</td>
			<td>&#160;final concentration 250nM</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">Near-IR Live/Dead Cell Stain Kit</td>
			<td style="width:159px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">L10119</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">CellTrace Calcein Red-Orange</td>
			<td style="width:159px;">Thermo Scientific</td>
			<td style="width:119px;">C34851</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">DAPI nuclear stain</td>
			<td style="width:159px;">Thermo Scientific</td>
			<td style="width:119px;">D1306</td>
			<td style="width:179px;"></td>
		</tr>
	</tbody>
</table>

analysis of the gap junction-dependent transfer of mirna with 3d-frap microscopy

Små antisense-RNA&#39;er, som miRNA og siRNA, spiller en vigtig rolle i cellulær fysiologi og patologi og kan desuden anvendes som terapeutiske midler til behandling af flere sygdomme. Udviklingen af ​​nye, innovative strategier for miRNA / siRNA-terapi er baseret på en omfattende viden om de underliggende mekanismer. Nylige data tyder på, at små RNA&#39;er udveksles mellem celler på en gabsforbindelsesafhængig måde og derved inducerer genregulerende virkninger i recipientcellen. Molekylærbiologiske teknikker og flowcytometrisk analyse anvendes almindeligt til at studere intercellulær udveksling af miRNA. Imidlertid tilvejebringer disse metoder ikke høj tidsmæssig opløsning, hvilket er nødvendigt, når man studerer gab-forbindelsesfluxet af molekyler. Derfor, for at undersøge virkningen af ​​miRNA / siRNA som intercellulære signalmolekyler, er der brug for nye værktøjer, som vil muliggøre analysen af ​​disse små RNA&#39;er på cellulært niveau. Den foreliggende protokol beskriver apPlication af tredimensionel fluorescensgenvinding efter photobleaching (3D-FRAP) mikroskopi for at belyse kløften forbindelsesafhængig udveksling af miRNA molekyler mellem hjerteceller. Det er vigtigt, at denne ligefremme og ikke-invasive levendecellebilledfremgangsmåde gør det muligt at visualisere og kvantificere mellemrumskrydsningen af ​​fluorescensmærkede små RNA&#39;er i realtid med høj spatio-temporal opløsning. Dataene opnået ved 3D-FRAP bekræfter en ny vej af intercellulær genregulering, hvor små RNA&#39;er virker som signalmolekyler inden for det intercellulære netværk.

Her præsenterer vi 3D-FRAP-mikroskopi som en ikke-invasiv teknik til at studere gapforbindelseshukling af fluorescerende miRNA inden for neonatale kardiomyocytter. De isolerede kardiomyocytter afslørede det typiske striberede a-actinin-mønster og indeholdt store plaques af Cx43 langs cellecellekanterne ( Figur 1A , hvide pilehoveder), hvilket tillod den høje intercellulære flux af molekyler. Renheden af ​​isolerede kardiomyocytter blev vurderet ved ...

Watch this Scientific Journal Video about Analysis of the Gap Junction-dependent Transfer of miRNA with 3D-FRAP Microscopy at JoVE.com

Analysis of the Gap Junction-dependent Transfer of miRNA with 3D-FRAP Microscopy

Her beskriver vi anvendelsen af ​​tredimensionel fluorescensgenvinding efter photobleaching (3D-FRAP) til analysen af ​​den mellemrumsafhængige pendling af miRNA. I modsætning til almindeligt anvendte metoder tillader 3D-FRAP kvantificering af den intercellulære overførsel af små RNA&#39;er i realtid med høj spatio-temporal opløsning.

Analyse af Gap Junction-afhængig Overførsel af miRNA med 3D-FRAP Microscopy

Small antisense RNAs, like miRNA and siRNA, play an important role in cellular physiology and pathology and, moreover, can be used as therapeutic agents ...

Research

JoVE Journal

Biology

The Xenopus Oocyte Cut-open Vaseline Gap Voltage-clamp Technique With Fluorometry

The Xenopus Oocyte Cut-open Vaseline Gap Voltage-clamp Technique With Fluorometry

Den<em&gt; Xenopus</em&gt; Oocyte Cut-open Vaseline Gap Spænding-clamp teknik med fluorometri

The cut-open oocyte Vaseline gap (COVG) voltage clamp technique allows for analysis of electrophysiological and kinetic properties of heterologous ion ...

Detection of miRNA Targets in High-throughput Using the 3'LIFE Assay

Påvisning af miRNA mål i High-throughput Brug af 3&#39;LIFE Assay

Luminescent Identification of Functional Elements in 3&#8217;UTRs (3&#8217;LIFE) allows the rapid identification of targets of specific miRNAs within an ...

Real-time Imaging of Plant Cell Surface Dynamics with Variable-angle Epifluorescence Microscopy

Real-time Imaging af Plant Cell Surface Dynamics med variabel vinkel epifluorescens Microscopy

A plant&#8217;s cell surface is its interface for perceiving environmental cues; it responds with cell biological changes such as membrane trafficking and ...

Open-source Single-particle Analysis for Super-resolution Microscopy with VirusMapper

Open source Single-partikel Analyse for Super-resolution Microscopy med VirusMapper

Super-resolution fluorescence microscopy is currently revolutionizing cell biology research. Its capacity to break the resolution limit of around 300 nm ...

Biological Sample Preparation by High-pressure Freezing, Microwave-assisted Contrast Enhancement, and Minimal Resin Embedding for Volume Imaging

Biologiske prøveforberedelse højtryks frysebehandling, mikrobølgeovn-assisteret kontrastforbedring og Minimal harpiks indlejring for volumen Imaging

The described sample preparation technique is designed to combine the best quality of ultrastructural preservation with the most suitable contrast for the ...

Tethered Bilayer Lipid Membranes to Monitor Heat Transfer between Gold Nanoparticles and Lipid Membranes

Tøjrede Bilayer Lipid Membraner til at overvåge varmeoverførsel mellem Gold Nanopartikler og Lipid Membraner

Here we report a protocol to investigate the heat transfer between irradiated gold nanoparticles (GNPs) and bilayer lipid membranes by electrochemistry ...

Application of Passive Head Motion to Generate Defined Accelerations at the Heads of Rodents

Anvendelse af passiv hovedbevægelse til at generere definerede accelerationer ved gnaverhovederne på gnavere

Exercise is widely recognized as effective for various diseases and physical disorders, including those related to brain dysfunction. However, molecular ...

Measurement of Liver Stiffness Using Atomic Force Microscopy Coupled with Polarization Microscopy

Måling af leverstivhed ved hjælp af atomkraftmikroskopi kombineret med polarisationsmikroskopi

Matrix stiffening has been recognized as one of the key drivers of the progression of liver fibrosis. It has profound effects on various aspects of cell ...

Fluorescence Lifetime Macro Imager for Biomedical Applications

Fluorescens levetid makro imager til biomedicinske applikationer

This paper presents a new photoluminescence lifetime imager designed to map the molecular oxygen (O2) concentration in different phosphorescent samples ...

Imaging of Mitochondrial Peroxide in Living Yeast Cells Using mtHyper7

Imaging of mtHyPer7, a Ratiometric Biosensor for Mitochondrial Peroxide, in Living Yeast Cells

Advancing Mitochondrial Research Â– mtHyper7 Biosensor for Subcellular Analysis (Video) | JoVE

Billeddannelse af mtHyPer7, en ratiometrisk biosensor for mitokondrieperoxid, i levende gærceller

Mitochondrial dysfunction, or functional alteration, is found in many diseases and conditions, including neurodegenerative and musculoskeletal disorders, ...