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

This work was supported by a Discovery Operating Grant to JDS from the Natural Science and Engineering Research Council (NSERC) of Canada, an NSERC Alexander Graham Bell Canada Graduate Scholarships (doctoral) (to A. Senatore) and the Heart and Stroke Foundation, Grant-In-Aid NA#6284.

1. Cell Culture
<ol>
 <li>Human embryonic kidney 293T cells (HEK-293T, Invitrogen) are grown in adherent monolayers in vented 25cm2 flasks (tissue culture treated; Cellstar; greiner bio-one) containing 6mL of Dulbecco's Modified Eagles Medium (DMEM; Sigma Aldrich) supplemented with 10% v/v fetal bovine serum (FBS; Sigma Aldrich), 1% sodium pyruvate, and 250 &mu;g/mL penicillin/streptomycin at 37&deg;C with 5% CO2). 
 
 Cells are incubated in CO2 incubat...

<ol>
	<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>

Being able to record whole-cell currents using the described protocols and solutions indicates that the transfection was successful and that the desired voltage-gated ion channels or ion channel complexes are present with significant quantities at the cell membrane. Should the cells be positively transfected (i.e. fluorescent green) but not have recordable currents, additional time at 28&deg;C may be required to allow for the channel protein to be properly packaged and inserted into the cell membrane. Note that prolong...

<table border="1">
 <tr>
 <td>Material</td>
 <td>Company</td>
 <td>Catalogue Number</td>
 <td>Comment</td>
 </tr>
 <tr>
 <td>pIRES2-EGFP plasmid</td>
 <td>Clontech</td>
 <td>6029-1</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Circle glass coverslips</td>
 <td>Fisher Scientific</td>
 <td>12-545-80</td>
 <td>Circles No. 1 - 0.13 to 0.17mm thick, Size: 12mm</td>
 </tr>
 <tr>
 <td>Amplifier</td>
 <td>Axon Instruments</td>
 <td>Axopatch 200B or Multiclamp 700B</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Data Acquisition System</td>
 <td>Axon Instruments </td>
 <td>Digidata 1440A</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Electrophysiology Software</td>
 <td>Axon Instruments</td>
 <td>pClamp10.1</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Pipette manipulators</td>
 <td>Sutter Instrument Co.</td>
 <td>MPC-385-2</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Epifluorescence inverted microscope</td>
 <td>Zeiss Canada</td>
 <td>Axiovert 40 CFL</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Headstage</td>
 <td>Axon Instruments</td>
 <td>CV-7B</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Headstage electrode holder</td>
 <td>Axon Instruments</td>
 <td>1-HL-U</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Microelectrode holder for ground electrode</td>
 <td>World Precision Instruments</td>
 <td>MEH3RF 15</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Electrophysiology capillary tubes</td>
 <td>Sutter Instrument Co.</td>
 <td>BF-150-86-15</td>
 <td>Borosilicate glass with filament, O.D. 1.5mm, O.D. 0.86mm, 15cm long</td>
 </tr>
 <tr>
 <td>Flaming/Brown Micropipette Puller</td>
 <td>Sutter Instrument Co.</td>
 <td>P-97</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Micropipette fire polisher</td>
 <td>Narishige</td>
 <td>Micro Forge MF-830</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Micro drug perfusion system</td>
 <td>AutoMate Scientific</td>
 <td>SmartSquirt8 Valvelink8.2</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Drug perfusion system</td>
 <td>AutoMate Scientific</td>
 <td>Valvelink8.2 with Teflon valves</td>
 <td>&nbsp;</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

Reliable method for highly efficient in vitro expression and subsequent electrophysiological recording of recombinant voltage-gated ion channels in cultured human embryonic kidney cells (HEK-293T).

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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