Reporter gene comparison demonstrates interference of complex body fluids with secreted luciferase activity

M. Neefjes, B.A.C. Housmans, G.G.H. van den Akker, L.W. van Rhijn, T.J.M. Welting*, P.M. van der Kraan

*Corresponding author for this work

Research output: Contribution to journalArticleAcademicpeer-review

Abstract

Reporter gene assays are widely used to study cellular signaling and transcriptional activity. Few studies describe the use of reporter genes for studying cellular responses on complex body fluids, such as urine and blood. Selection of the optimal reporter gene is crucial for study outcome. Here, we compared the characteristics of five reporter genes (Firefly luciferase, stable- and unstable Nano luciferase, secretable Gaussia luciferase and Red Fluorescent Protein) to study complex body fluids. For this comparison, the NF kappa B Response Element (NF kappa B-RE) and Smad Binding Element (SBE) were identically cloned into the five different reporter vectors. Reporter characteristics were evaluated by kinetic and concentration-response measurements in SW1353 and HeLa cell lines. Finally, reporter compatibility with complex body fluids (fetal calf serum, knee joint synovial fluid and human serum) and inter-donor variation were evaluated. Red Fluorescent Protein demonstrated poor inducibility as a reporter gene and slow kinetics compared to luciferases. Intracellularly measured luciferases, such as Firefly luciferase and Nano luciferase, revealed good compatibility with complex body fluids. Secreted Gaussia luciferase appeared to be incompatible with complex body fluids, due to variability in inter-donor signal interference. Unstable Nano luciferase demonstrated clear inducibility, high sensitivity and compatibility with complex body fluids and therefore can be recommended for cellular signaling studies using complex body fluids.
Original languageEnglish
Article number1359
Number of pages13
JournalScientific Reports
Volume11
Issue number1
DOIs
Publication statusPublished - 14 Jan 2021

Keywords

  • GREEN FLUORESCENT PROTEIN
  • FIREFLY LUCIFERASE
  • GFP VARIANTS
  • EXPRESSION
  • CELLS
  • TIME
  • BIOMARKERS
  • KAPPA
  • ASSAY

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