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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)
Cellular & Molecular Biology Letters volume 15, pages 55–69 (2010)
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.
Abbreviations
- 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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Hrovat, A., Zavec, A.B., Pogačnik, A. et al. 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). Cell Mol Biol Lett 15, 55–69 (2010). https://doi.org/10.2478/s11658-009-0034-0
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DOI: https://doi.org/10.2478/s11658-009-0034-0