Name: controllable ion channel expression through inducible transient transfection
Uploaded: 2017-02-17T15:00:00
Description: Watch this Scientific Journal Video about Controllable Ion Channel Expression through Inducible Transient Transfection at JoVE.com

Dette arbejde blev støttet af Israel Science Foundation [Tilskud 1721/12, 1368/12 og 1444/16] (til AP). AP er tilknyttet Brettler Center og David R. Bloom Center, Farmaceutiske Højskole, Det hebraiske Universitet i Jerusalem.

1. ligering af genet af interesse i det repressible Site of Vector <ol><li> Få en inducerbar vektor såsom pcDNA5 / FRT / TO eller pcDNA4 / TO. </li><li> Analyser genet af interesse for alle potentielt modtagelige restriktionssteder i midten af genet, der ligeledes i den multiple kloningssted i den valgte vektor ved in silico DNA-analyse software 4. </li><li> Anvendelse af standard overlappende PCR-teknikker 4, flankerer genet af interesse med to udvalgte restr...

<ol>
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Transfektion er et meget anvendt protokol for proteinekspression og forskning, med mange forskellige variationer til forbedring ekspression konsistens og stabilitet. Forbigående transfektionsreagenser tilbyder en enkel, let at bruge protokol, hvor cellen og proteinet af interesse kan analyseres i løbet af timer til natten over fra tidspunktet for transfektion. Desværre denne fremgangsmåde kan være uforudsigelig, når den tilstand af analyse kræver et ensartet proteinekspression, såsom enkelt kanal optagelser i el...

Heterologt udtrykker systemer er en af de mest udbredte teknikker til at studere en mangfoldighed af cellulære funktioner 1. Deres lave endogent protein profil, minimal vedligeholdelse, pålidelig vækst, og evne til at optage og udtrykke fremmed DNA har gjort cellelinjer, såsom humane embryonale Kidney (HEK293) og kinesisk hamster ovarie (CHO) næsten afgørende for biologisk forskning 2, 3. Forskningsområder hjælp heterologe systemer omfatter membranproteiner, intracellulær signalering og enzymatisk aktivitet. Efter transfektion af fremmed DNA ind i cellen, kan udføres mange forskellige former for analyse, herunder elektrofysiologi, ratiometrisk calcium imaging, western blot, etc. 4, 5. På grund af den brede vifte af mulige anvendelser for heterologe ekspressionssystemer, mange Forskelnt reagenser og produkter er blevet udviklet til at udnytte disse celler og deres kvaliteter 6. DNA afgivelsessystemer, der transient eller permanent integrere fremmed DNA i celler for at studere exogent protein er blevet en af ​​de mest populære og nyttige værktøjer til biologisk forskning. Mere specifikt er transient transfektion af DNA i en celle i vid udstrækning anvendes som en enkel, ligetil proces, der kræver forholdsvis lidt tid og materialer. Endvidere succesraten af celler, der undergår transfektion er høj 7. Denne teknik er meget pålidelig, når det kombineres med et markørgen, såsom grønt fluorescerende protein (GFP), og kan anvendes til mange forskellige teknikker, såsom calcium imaging og elektrofysiologi 5. Men desværre transient ekspression af DNA i værtsceller kommer med nogle store faldgruber, ikke mindst, at ekspressionsniveauet per celle er upålidelig. Antallet af kopier af plasmid-DNA taget up per celle er ukontrollabel, således at udtrykket mellem individuelle eksperimenter kan variere meget 2. Dette problem bliver signifikant, når enten forsøger at replikere fysiologiske forhold, eller udfører præcise dataindsamlingsteknikker. Som en løsning på de ovennævnte, stabil transfektion protokoller er blevet designet komplikationer, hvor et gen af ​​interesse kan indsættes i genomet af en celle under den stramme kontrol af en inducerbar promotor, såsom en tetracyclin repressor ekspressionssystem, sikrer en enkelt kopi af plasmidet integreres i genomet i hver celle og kun udtrykkes efter induktion af transskription mekanisme, for eksempel i nærvær af doxycyclin. Mens dette løser forhindringer af inkonsekvente protein udtryk niveauer, denne metode mister bekvemmelighed hurtig og relativt simpel protokol af transiente transfektioner. Etablering af en stabil cellelinje tager mindst et par uger i which må man kalibrere en dræbende kurve indstillet af specifikke antibiotika for at bevare proteinekspression og sikre integration af vektoren og dygtigt vælge og vokse cellekolonier. Samlet set tager betydeligt mere tid og kræfter med en lavere succesrate 8. Her introducerer vi en mellemliggende protokol, der trækker på styrkerne i begge de populære transfektion muligheder for at skabe en enkel og effektiv måde at styre ekspressionsniveauerne i enhver inducerbare cellelinje. Samtidig opretholde celler med et inducerbart tet systemet, vi forbigående transficere vores gen af ​​interesse, Transient Receptor Potential kationkanal underfamilien V element 1 (TRPV1), ligeret ind i en vektor, som homologt kan kombinere med repressoren system. På denne måde kan genet indføres i cellerne uden begyndt at udtrykke. Kun ved tilsætning af doxycyclin betyder genet begynde at udtrykke, tillader os at kalibrere niveauet af protein expression ifølge teknikken eller niveauer observeret i fysiologiske betingelser. Vores protokol undgår også langvarige komplikationer forbundet med generering af en stabilt udtrykkende cellelinie. Vi begynder med at vise de skiftende niveauer af TRPV1 aktivering i calcium imaging fra un-induceret gennem fire timers induktion og hvordan stigningen i intracellulære calcium niveauer korrelerer. Vi derefter duplikere protokollen i hele celle konfiguration af patch-clamp teknik, viser den stigende strøm med stigende tid af induktion. Endelig præsenterer vi eksempler på enkelt kanal elektrofysiologi optagelser, og vise, at denne teknik er især nyttig for kontrolleret udtryk, når de søger præcis dataindsamling baseret på individuelle enheder af proteinet. Gennem vores protokol, tilbyder vi en bekvem måde at styre proteinekspression i heterologe systemer samtidig undgå langvarige cellekultur komplikationer, hvilket giver en måde at kontrollere forhold mellem eksperimenter og give more reproducerbare resultater.

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		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">(E)-Capsaicin</td>
			<td style="width:153px;">Tocris</td>
			<td align="right" style="width:119px;">462</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Olympus IX70 Fluorescence Microscope</td>
			<td style="width:153px;">Olympus</td>
			<td style="width:119px;">n.a</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Lambda DG-4 Wavelength Switcher</td>
			<td style="width:153px;">Sutter Instruments</td>
			<td style="width:119px;">n.a</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">EXi Blue Fluorescence Microscopy Camera</td>
			<td style="width:153px;">QImaging</td>
			<td style="width:119px;">n.a</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">MetaFluor Fluorescence Ratio Imaging Software</td>
			<td style="width:153px;">Molecular Devices</td>
			<td style="width:119px;">n.a</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Thin Walled Borosilicate Tubing</td>
			<td style="width:153px;">Sutter Instruments</td>
			<td style="width:119px;">B150-110-7.5HP</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Standard Walled Borosilicate Tubing</td>
			<td style="width:153px;">Sutter Instruments</td>
			<td style="width:119px;">B150-86-7.5HP</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Dimethyl sulfoxide anhydrous</td>
			<td style="width:153px;">Sigma-Aldrich</td>
			<td align="right" style="width:119px;">276855</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">P1000 micropipette puller</td>
			<td style="width:153px;">Sutter Instruments</td>
			<td style="width:119px;">P-1000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">MF-900 Microforge</td>
			<td style="width:153px;">NARISHIGE</td>
			<td style="width:119px;">n.a</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">ValveBank perfusion sysytem</td>
			<td style="width:153px;">AutoMate Scientific</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Digidata&#174; 1440A Low-noise Data Acquisition System</td>
			<td style="width:153px;">Molecular Devices</td>
			<td style="width:119px;">n.a</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Axopatch 200B Amplifier</td>
			<td style="width:153px;">Molecular Devices</td>
			<td style="width:119px;">n.a</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">pCLAMP 10.6 Software</td>
			<td style="width:153px;">Molecular Devices</td>
			<td style="width:119px;">n.a</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">micromanipulator</td>
			<td style="width:153px;">Sutter Instruments</td>
			<td style="width:119px;">MP-225</td>
			<td></td>
		</tr>
	</tbody>
</table>

controllable ion channel expression through inducible transient transfection

Transfektion levering af fremmede nukleinsyrer i en celle, er et effektivt værktøj i protein forskning. Gennem denne metode, kan ionkanaler undersøges gennem elektrofysiologisk analyse, biokemisk karakterisering, mutationsmønstre undersøgelser, og deres virkninger på cellulære processer. Transiente transfektioner tilbyder en enkel protokol, hvor proteinet bliver tilgængelig for analyse i løbet af få timer til dage. Selv om denne fremgangsmåde giver en forholdsvis enkel og tidsbesparende protokol, er en af ​​de kritiske komponenter kalibrering ekspressionen af ​​genet af interesse til fysiologiske relevante niveauer eller niveauer, der er egnet til analyse. Til dette formål har mange forskellige tilgange, der tilbyder muligheden for at styre ekspressionen af ​​genet af interesse opstået. Adskillige stabile celle transfektionsprotokoller giver en måde at permanent indføre et gen af ​​interesse i det cellulære genom under regulering af et tetracyclin-kontrolleret transcriptional aktivering. Mens denne teknik producerer pålidelige ekspressionsniveauerne, hvert gen af ​​interesse kræver et par ugers faglært arbejde, herunder kalibrering af en dræbende kurve, udvælgelse af celle kolonier, og generelt flere ressourcer. Her præsenterer vi en protokol, som anvender transient transfektion af Transient Receptor Potential kationkanal underfamilien V element 1 (TRPV1) gen i et inducerbart system, som en effektiv måde til at udtrykke et protein i en kontrolleret måde, som er væsentlig i ionkanal analyse. Vi viser, at bruge denne teknik, er vi i stand til at udføre calcium imaging, hel celle, og en enkelt kanal analyse med kontrollerede kanalniveauer kræves for hver enkelt af indsamling af data med en enkelt transfektion. Samlet giver dette en replikerbar teknik, der kan anvendes til at studere ionkanaler struktur og funktion.

For hurtigt at oprette en inducerbar ekspression model, gjorde vi brug af HEK293-celler, der udtrykker Tetracyklin repressorproteinet (TR) (f.eks T-Rex-293) og vektorer, der indeholder tetracyklin operator sekvenser (TetO) mellem CMV-promotoren og det multiple kloningssted (f.eks pcDNA4 / TO). Når transficeret ind Trex-293-celler, er ekspressionen af ​​genet af interesse i pcDNA4 / TO undertrykt, som TR binder til TetO. Tilføjelse doxycyclin til mediet forhindrer ...

Watch this Scientific Journal Video about Controllable Ion Channel Expression through Inducible Transient Transfection at JoVE.com

Controllable Ion Channel Expression through Inducible Transient Transfection

Studere ionkanaler gennem et heterologt udtrykker system er blevet en central teknik i biomedicinsk forskning. I dette manuskript, præsenterer vi en tid effektiv metode til at opnå tæt kontrolleret ionkanal-ekspression ved at udføre transient transfektion under kontrol af en inducerbar promotor.

Kontrollerbar Ion Channel Expression gennem Inducible Transient transfektion

Transfection, the delivery of foreign nucleic acids into a cell, is a powerful tool in protein research. Through this method, ion channels can be ...

Research

JoVE Journal

Genetics

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