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. 2009 Oct 15;15(1):55. doi: 10.2478/s11658-009-0034-0

Establishing and functional characterization of an HEK-293 cell line expressing autofluorescently tagged β-actin (pEYFP-ACTIN) and the neurokinin type 1 receptor (NK1-R)

Alenka Hrovat 1, Apolonija Bedina Zavec 2, Azra Pogačnik 1, Robert Frangež 3, Milka Vrecl 1,✉
PMCID: PMC6275802  PMID: 19834649

Abstract

This study focused on establishing and making a comprehensive functional characterization of an HEK-293-transfected cell line that would coexpress the enhanced yellow fluorescent protein-actin (pEYFP-actin) construct and the neurokinin type 1 receptor (NK1-R), which is a member of the seven transmembrane (7TM) receptor family. In the initial selection procedure, the cloning ring technique was used alone, but failed to yield clones with homogenous pEYFP-actin expression. Flow cytometry sorting (FCS) was subsequently used to enrich the pEYFP-actin-expressing subpopulation of cells. The enzyme-linked immunosorbent assay (ELISA), FCS and quantitative real-time reverse transcription/polymerase chain reaction (RT-PCR) were then employed to monitor the passage-dependent effects on transgene expression and to estimate the total β-actin/pEYFP-actin ratio. NK1-R was characterized via radioactive ligand binding and the second messenger assay. The suitability of the pEYFP-actin as a marker of endogenous actin was assessed by colocalizing pEYFP-actin with rhodamine-phalloidine-stained F-actin and by comparing receptor- and jasplakinolide-induced changes in the actin cytoskeleton organization. These experiments demonstrated that: i) both constructs expressed in the generated transfected cell line are functional; ii) the estimated pEYFP-actin: endogenous β-actin ratio is within the limits required for the functional integrity of the actin filaments; and iii) pEYFP-actin and rhodamine-phalloidine-stained F-actin structures colocalize and display comparable reorganization patterns in pharmacologically challenged cells.

Key words: Cytoskeleton, Actin filaments, HEK-293, Neurokinin type 1 receptor, Flow cytometry, Confocal microscopy

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Abbreviations used

7TM

seven transmembrane receptor

Bmax

max. receptor number

BSA

bovine serum albumin

Ct

threshold cycle

DMEM

Dulbecco’s modified Eagles’s Medium

DNA

deoxyribonucleic acid

D-PBS

Dulbecco’s phosphate-buffered saline

ELISA

enzyme-linked immunosorbent assay

F-actin

filamentous actin

FCA

flow cytometry analysis

FCS

flow cytometry sorting

GFP

green fluorescent protein

GPCR

G-protein coupled receptor

HA

hemagglutinin

HANK1-R

N-terminally HA-tagged human neurokinin-1 receptor

HEK-293

human embryonic kidney cells

HIFCS

heat inactivated fetal calf serum

HRP

horseradish peroxidase

IC50

50% inhibitory concentration

IP1

inositol phosphate 1

mRNA

messenger ribonucleic acid

NK1-R

neurokinin type 1 receptor

pEYFP

enhanced yellow fluorescent protein

PN

passage number

RT

reverse transcription

RT-PCR

quantitative real-time reverse transcription / polymerase chain reaction

SP

substance P

TMB

tetramethylbenzidine

WT

wildtype

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