Controllable Ion Channel Expression through Inducible Transient Transfection

Summary

Studying ion channels through a heterologously expressing system has become a core technique in biomedical research. In this manuscript, we present a time efficient method to achieve tightly controlled ion channel expression by performing transient transfection under the control of an inducible promoter.

Abstract

Transfection, the delivery of foreign nucleic acids into a cell, is a powerful tool in protein research. Through this method, ion channels can be investigated through electrophysiological analysis, biochemical characterization, mutational studies, and their effects on cellular processes. Transient transfections offer a simple protocol in which the protein becomes available for analysis within a few hours to days. Although this method presents a relatively straightforward and time efficient protocol, one of the critical components is calibrating the expression of the gene of interest to physiological relevant levels or levels that are suitable for analysis. To this end, many different approaches that offer the ability to control the expression of the gene of interest have emerged. Several stable cell transfection protocols provide a way to permanently introduce a gene of interest into the cellular genome under the regulation of a tetracycline-controlled transcriptional activation. While this technique produces reliable expression levels, each gene of interest requires a few weeks of skilled work including calibration of a killing curve, selection of cell colonies, and overall more resources. Here we present a protocol that uses transient transfection of the Transient Receptor Potential cation channel subfamily V member 1 (TRPV1) gene in an inducible system as an efficient way to express a protein in a controlled manner which is essential in ion channel analysis. We demonstrate that using this technique, we are able to perform calcium imaging, whole cell, and single channel analysis with controlled channel levels required for each type of data collection with a single transfection. Overall, this provides a replicable technique that can be used to study ion channels structure and function.

Introduction

Heterologously expressing systems is one of the most widely used techniques to study a multitude of cellular functions¹. Their low endogenous protein profile, minimal maintenance requirements, reliable growth, and ability to take up and express foreign DNA have made cell lines such as Human Embryonic Kidney (HEK293) and Chinese Hamster Ovary (CHO) almost essential to biological research^2,3. Areas of research using heterologous systems include membrane proteins, intracellular signaling, and enzymatic activity. Following transfection of foreign DNA into the cell, many different forms of a....

Protocol

1. Ligating the Gene of Interest into the Repressible Site of Vector

1. Obtain an inducible vector such as pcDNA5/FRT/TO or pcDNA4/TO.
2. Analyze the gene of interest for any potentially susceptible restriction sites in the middle of the gene that are also featured in the multiple cloning site of the chosen vector by in silico DNA analysis software⁴.
3. Using standard overlap PCR techniques⁴, flank the gene of interest with two selected restriction enzyme recognition sites (that are not found in the gene of interest), the first inserted before the start codon, and the second after the stop....

Results

To quickly create an inducible expression model, we made use of HEK293 cells that express Tetracycline Repressor protein (TR) (e.g., T-REx-293) and vectors that contain tetracycline operator sequences (TetO) between the CMV promoter and the multiple cloning site (e.g., pcDNA4/TO). When transfected into TREx-293 cells, the expression of the gene of interest in pcDNA4/TO is repressed, as TR binds to TetO. Adding doxycycline to the medium prevents TR-TetO interaction, thus .......

Discussion

Transfection is a widely used protocol for protein expression and research, with many different variations to improve expression consistency and stability. Transient transfection reagents offer a simple, easy to use protocol where the cell and protein of interest can be analyzed within hours to overnight from the time of transfection. Unfortunately this approach can be unpredictable when the mode of analysis requires a consistent level of protein expression, such as single channel recordings in electrophysiology

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
pcDNA™4/TO Mammalian Expression Vector	ThermoScientific Fisher	V102020
pcDNA™5/TO Mammalian Expression Vector	ThermoScientific Fisher	V103320
PureLink Quick PCR Purification Kit	Invitrogen	K310001
Swift™ MaxPro Thermal Cycler	Esco	n.a
Restriction Enzymes	ThermoScientific Fisher	ER0501
Agarose	Lonza	50004
PureLink Quick Gel Extraction Kit	Invitrogen	K210012
NanoDrop 2000c	ThermoScientific Fisher	ND-2000C
T4 DNA Ligase	ThermoScientific Fisher	EL0011
One Shot® TOP10 Chemically Competent E. coli	ThermoScientific Fisher	C404006
Ampicillin (Sodium), USP Grade	Gold Bio	A-301-5
Tryptone for microbiology	Merck	6.19305E+13
Yeast Extract	BD worldwide	212750
SIF6000R Incubated Shaker	LAB COMPANION	45H118
NucleoSpin®plasmid	Macherey Nagel	740588.25
MS 300V Power Supply	Major Science	MP-300V
Owl™ EasyCast™ B1A Mini Gel Electrophoresis System	ThermoScientific Fisher	B1A
T-REx™-293 cell line	Invitrogen	R710-07
DMEM (1x), liquid (high glucose)	Gibco	41965-039
HindIII-HF®	NEB	R3104S
ApaI	NEB	R0114S
CutSmart® Buffer	NEB	B7204S
pcDNA™6/TR  Mammalian Expression Vector	ThermoScientific Fisher	V102520
Fetal Bovine Serum (FBS), qualified, E.U.-approved, South America origin	Gibco	10270106
HEPES Buffer Solution (1 M)	Biological Industries	03-025-1B
Penicillin-Streptomycin Solution	Biological Industries	03-031-1B
L-Alanyl-L-Glutamine (Stable Glutamine) (200 mM)	Biological Industries	03-022-1B
Heracell™ 150i CO2 Incubator	ThermoScientific Fisher	51026406
MSC-Advantage™ Class II Biological Safety Cabinet	ThermoScientific Fisher	51025411
Blasticidine S hydrochloride	Sigma-Aldrich	15205-25MG
Dulbecco’s Phosphate-buffered Saline (DPBS) Modified, without calcium chloride and magnesium chloride	Sigma-Aldrich	D8537-500ML
Trypsin-EDTA (0.05%), phenol red	Gibco	25300054
Double Neubauer Ruled Metallized Hemacytometer	Hausser Scientific	31000
Opti-MEM I Reduced Serum Medium	Gibco	31985070
TransIT®-LT1 Transfection Reagent	Mirus	MIR 2300
glass coverslips, #1 thickness, 12 mm diameter round	Knittel Glass	GG-12
BioCoat™ Poly-D-Lysine (PDL)	Corning	354210
Water, Cell Culture Grade	Biological Industries	03-055-1A
Doxycycline hyclate	Sigma-Aldrich	D9891-1G
Fura-2, AM ester	Biotium	BTM-50034
Pluronic® F-127	Sigma-Aldrich	P2443-250G
µ-Slide 8 Well	ibidi	80826
(E)-Capsaicin	Tocris	462
Olympus IX70 Fluorescence Microscope	Olympus	n.a
Lambda DG-4 Wavelength Switcher	Sutter Instruments	n.a
EXi Blue Fluorescence Microscopy Camera	QImaging	n.a
MetaFluor Fluorescence Ratio Imaging Software	Molecular Devices	n.a
Thin Walled Borosilicate Tubing	Sutter Instruments	B150-110-7.5HP
Standard Walled Borosilicate Tubing	Sutter Instruments	B150-86-7.5HP
Dimethyl sulfoxide anhydrous	Sigma-Aldrich	276855
P1000 micropipette puller	Sutter Instruments	P-1000
MF-900 Microforge	NARISHIGE	n.a
ValveBank perfusion sysytem	AutoMate Scientific
Digidata® 1440A Low-noise Data Acquisition System	Molecular Devices	n.a
Axopatch 200B Amplifier	Molecular Devices	n.a
pCLAMP 10.6 Software	Molecular Devices	n.a
micromanipulator	Sutter Instruments	MP-225