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 NFκB Response Element (NFκB-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.
Highlights
Reporter gene assays are widely used to study cellular signaling and transcriptional activity
This resulted in the following tmax characteristics: stable Nano luciferase (NLuc; tmax = 12 h), unstable Nano luciferase (NLucP; tmax = 6 h), Firefly luciferase (FFLuc; tmax = 8 h), Gaussia luciferase (GLuc; tmax = 8 h) and tdTomato (tmax = 12 h) (Fig. 1)
Since GLuc was heavily inhibited by complex body fluids, we evaluated whether this was (1) directly caused by interference of the GLuc enzyme by the complex body fluid, (2) whether inhibition depends on the type of body fluid, and (3) whether this differs between individual donors of such body fluids
Summary
Reporter gene assays are widely used to study cellular signaling and transcriptional activity. 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. Red Fluorescent Protein demonstrated poor inducibility as a reporter gene and slow kinetics compared to luciferases Measured luciferases, such as Firefly luciferase and Nano luciferase, revealed good compatibility with complex body fluids. By using transcription factor-binding response elements coupled to a reporter gene, activation of signaling pathways can be studied upon stimulation Measuring such responses induced by a complex body fluid can be challenging due to viscosity, ionic strength and the proteolytic milieu of the sample[1,2]. In general cellular autofluorescence is lower for the emission wavelength of the red channel, favoring a red fluorescent protein such as tdTomato[12]
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