Establishing and functional characterization of an HEK-293 cell line expressing autofluorescently tagged beta-actin (pEYFP-ACTIN) and the neurokinin type 1 receptor (NK1-R).
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 beta-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 beta-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.
Hrovat
Alenka
A
Veterinary Faculty, Institute of Anatomy, Histology and Embryology, Gerbiceva 60, SI-1000, Ljubljana, Slovenia.
Zavec
Apolonija Bedina
AB
Pogacnik
Azra
A
Frangez
Robert
R
Vrecl
Milka
M
D000199:Actins / Q000032:analysis / Q000378:metabolism* / Q000648:ultrastructure; D001426:Bacterial Proteins / Q000378:metabolism; D002460:Cell Line; D003599:Cytoskeleton / Q000648:ultrastructure; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D008164:Luminescent Proteins / Q000378:metabolism; D018040:Receptors, Neurokinin-1 / Q000032:analysis / Q000378:metabolism*
D000199:Actins; D001426:Bacterial Proteins; D008164:Luminescent Proteins; D018040:Receptors, Neurokinin-1; C054562:yellow fluorescent protein, Bacteria