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]

• Autorzy: Hrovat A , Zavec A.B. , Pogacnik A. , Frangez R. , Vrecl M.
• Języki publikacji: EN

Abstrakty

EN

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.

Słowa kluczowe

EN

functional characteristics; HEK-293 cell line; beta-actin; neurokinin-1; neurokinin receptor; cytoskeleton; actin filament; flow cytometry; confocal microscopy

• Wydawca: -
• Czasopismo: Cellular and Molecular Biology Letters
• Rocznik: 2010
• Tom: 15
• Numer: 1
• Opis fizyczny: p.55-69,fig.,ref.

Twórcy

autor

Hrovat A

• Veterinary Faculty, Institute of Anatomy, Histology and Embryology, Gerbiceva 60, SI-1000 Ljubljana, Slovenia

autor

Zavec A.B.

autor

Pogacnik A.

autor

Frangez R.

autor

Vrecl M.

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