Name: optimized transfection strategy for expression and electrophysiological recording of recombinant voltage-gated ion channels in hek-293t cells
Uploaded: 2011-01-19T18:00:00
Description: Watch this Scientific Journal Video about Optimized Transfection Strategy for Expression and Electrophysiological Recording of Recombinant Voltage-Gated Ion Channels in HEK-293T Cells at JoVE.com

इस काम के प्राकृतिक विज्ञान और जदएस के लिए डिस्कवरी ऑपरेटिंग अनुदान द्वारा समर्थित किया गया था कनाडा के इंजीनियरिंग रिसर्च () NSERC परिषद, एक NSERC अलेक्जेंडर ग्राहम बेल कनाडा ग्रेजुएट छात्रवृत्ति (डॉक्टरेट) (ए Senatore) और दिल और स्ट्रोक फाउंडेशन अनुदान 6284 # NA में सहायता.

<html> 1. सेल संस्कृति <ol><li> है Dulbecco संशोधित ईगल्स मध्यम (DMEM, सिग्मा Aldrich) के 6mL युक्त मानव भ्रूण गुर्दे 293T कोशिकाओं (HEK-293T, Invitrogen) निकाल 25cm 2 (; Cellstar GREINER जैव - एक ऊतक इलाज संस्कृति) बोतल में पक्षपाती monolayers में बड़े हो रहे हैं 10 क...

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	<li>Thomas, P., Smart, T. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HEK293+cell+line:+A+vehicle+for+the+expression+of+recombinant+proteins.">HEK293 cell line: A vehicle for the expression of recombinant proteins.</a> J. Pharmacological and Toxicological Methods. 51, 187-200 (2005).</li><li>Senatore, A., Spafford, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transient+and+big+are+key+features+of+an+invertebrate+T-type+channel+(LCav3)+from+the+central+nervous+system+of+Lymnaea+stagnalis.">Transient and big are key features of an invertebrate T-type channel (LCav3) from the central nervous system of Lymnaea stagnalis.</a> Journal of Biological Chemistry. 285, 7447-7458 (2010).</li><li>Ogden, D., Stanfield, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patch+clamp+techniques+for+single+channel+and+whole-cell+recording.">Patch clamp techniques for single channel and whole-cell recording.</a> Microelectrode techniques: the Plymouth workshop handbook. , (1987).</li><li>Molleman, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patch+clamping:+an+introductory+guide+to+patch+clamp+electrophysiology.">Patch clamping: an introductory guide to patch clamp electrophysiology.</a> , (2003).</li><li>Peng, S. -. Q., Hajela, R. K., Atchison, W. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluid+flow-induced+increase+in+inward+Ba2++current+expressed+in+HEK293+cells+transiently+transfected+with+human+neuronal+L-type+Ca2+channels.">Fluid flow-induced increase in inward Ba2+ current expressed in HEK293 cells transiently transfected with human neuronal L-type Ca2+channels.</a> Brain Research. 1045, 116-123 (2005).</li></ol>

<html> पूरे सेल में वर्णित प्रोटोकॉल और समाधान का उपयोग धाराओं रिकार्ड करने में सक्षम होने के नाते इंगित करता है कि अभिकर्मक सफल था और वांछित वोल्टेज gated आयन चैनलों या आयन चैनल परिसरों कोशिका झिल्ली में महत...

<table border="1"><tr><td> सामग्री </td><td> कंपनी </td><td> सूचीपत्र संख्या </td><td> टिप्पणी </td></tr><tr><td> प्लाज्मिड pIRES2-EGFP </td><td> Clontech </td><td> 6029-1 </td><td></td></tr><tr><td> सर्किल कांच coverslips </td><td> फिशर साइंटिफिक </td><td> 12-545-80 </td><td> नंबर 1 सर्किलों - 0.13 से 0.17mm मोटी, आकार: 12mm </td></tr><tr><td> एम्पलीफायर </td><td> Axon उपकरण </td><td> Axopatch 200B या Multiclamp 700B </td><td></td></tr><tr><td> डाटा अधिग्रहण प्रणाली </td><td> Axon उपकरण </td><td> Digidata 1440A </td><td></td></tr><tr><td> इलैक्ट्रोफिजियोलॉजी सॉफ्टवेयर </td><td> Axon उपकरण </td><td> pClamp10.1 </td><td></td></tr><tr><td> पिपेट manipulators </td><td> Sutter साधन कं </td><td> MPC-385-2 </td><td></td></tr><tr><td> Epifluorescence उलटा माइक्रोस्कोप </td><td> Zeiss कनाडा </td><td> Axiovert 40 सीएफएल </td><td></td></tr><tr><td> Headstage </td><td> Axon उपकरण </td><td> CV-7B </td><td></td></tr><tr><td> Headstage इलेक्ट्रोड धारक </td><td> Axon उपकरण </td><td> 1-HL-U </td><td></td></tr><tr><td> जमीन इलेक्ट्रोड के लिए microelectrode धारक </td><td> विश्व परिशुद्धता उपकरण </td><td> 15 MEH3RF </td><td></td></tr><tr><td> इलैक्ट्रोफिजियोलॉजी केशिका ट्यूब </td><td> Sutter साधन कं </td><td> BF-150-86-15 </td><td> रेशा, आयुध डिपो 1.5mm, 0.86mm आयुध डिपो, 15cm लंबे समय के साथ borosilicate ग्लास </td></tr><tr><td> / ब्राउन micropipette डांड़ी ज्वलंत </td><td> Sutter साधन कं </td><td> P-97 </td><td></td></tr><tr><td> Micropipette आग पालिशगर </td><td> Narishige </td><td> माइक्रो एमएफ 830 - फोर्ज </td><td></td></tr><tr><td> माइक्रो दवा छिड़काव प्रणाली </td><td> वैज्ञानिक स्वचालित </td><td> SmartSquirt8 Valvelink8.2 </td><td></td></tr><tr><td> नशीली दवाओं के छिड़काव प्रणाली </td><td> वैज्ञानिक स्वचालित </td><td> Valvelink8.2 Teflon वाल्व के साथ </td><td></td></tr></table>

optimized transfection strategy for expression and electrophysiological recording of recombinant voltage-gated ion channels in hek-293t cells

The in vitro expression and electrophysiological recording of recombinant voltage-gated ion channels in cultured human embryonic kidney cells (HEK-293T) is a ubiquitous research strategy. HEK-293T cells must be plated onto glass coverslips at low enough density so that they are not in contact with each other in order to allow for electrophysiological recording without confounding effects due to contact with adjacent cells. Transfected channels must also express with high efficiency at the plasma membrane for whole-cell patch clamp recording of detectable currents above noise levels. Heterologous ion channels often require long incubation periods at 28&deg;C after transfection in order to achieve adequate membrane expression, but there are increasing losses of cell-coverslip adhesion and membrane stability at this temperature. To circumvent this problem, we developed an optimized strategy to transfect and plate HEK-293T cells. This method requires that cells be transfected at a relatively high confluency, and incubated at 28&deg;C for varying incubation periods post-transfection to allow for adequate ion channel protein expression. Transfected cells are then plated onto glass coverslips and incubated at 37&deg;C for several hours, which allows for rigid cell attachment to the coverslips and membrane restabilization. Cells can be recorded shortly after plating, or can be transferred to 28&deg;C for further incubation. We find that the initial incubation at 28&deg;C, after transfection but before plating, is key for the efficient expression of heterologous ion channels that normally do not express well at the plasma membrane. Positively transfected, cultured cells are identified by co-expressed eGFP or eGFP expressed from a bicistronic vector (e.g. pIRES2-EGFP) containing the recombinant ion channel cDNA just upstream of an internal ribosome entry site and an eGFP coding sequence. Whole-cell patch clamp recording requires specialized equipment, plus the crafting of polished recording electrodes and L-shaped ground electrodes from borosilicate glass. Drug delivery to study the pharmacology of ion channels can be achieved by directly micropipetting drugs into the recording dish, or by using microperfusion or gravity flow systems that produce uninterrupted streams of drug solution over recorded cells.

Watch this Scientific Journal Video about Optimized Transfection Strategy for Expression and Electrophysiological Recording of Recombinant Voltage-Gated Ion Channels in HEK-293T Cells at JoVE.com

Optimized Transfection Strategy for Expression and Electrophysiological Recording of Recombinant Voltage-Gated Ion Channels in HEK-293T Cells

अत्यधिक कुशल के लिए विश्वसनीय विधि<em&gt; इन विट्रो में</em&gt; अभिव्यक्ति और पुनः संयोजक वोल्टेज gated सभ्य मानव भ्रूण गुर्दे की कोशिकाओं में आयन चैनल (HEK-293T) के बाद electrophysiological रिकॉर्डिंग.

अभिव्यक्ति और HEK-293T कक्ष में पुनः संयोजक वोल्टेज gated आयन चैनल के electrophysiological रिकॉर्डिंग के लिए अनुकूलित अभिकर्मक रणनीति

The in vitro expression and electrophysiological recording of recombinant voltage-gated ion channels in cultured human embryonic kidney cells (HEK-293T) ...

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