Construction and evaluation of reporter plasmids for enhancement of inducible gene expression from weak promoters

Abstract

The nuclear factor κB (NF-κB) pathway represents an important signaling pathway which is involved in immune regulation, inflammation and cancer pathogenesis. Monitoring of this pathway is important for Earth-bound applications like therapy of inflammatory and immune diseases and therapy resistance of tumors, but also for space-relevant questions like risk assessment of space radiation exposure and appropriate countermeasure development. Therefore, cellular responses to space-relevant radiation qualities, including NF-κB activation, have to be investigated. In this work, a reporter assay should be constructed, based on (i) a receptor plasmid and (ii) a reporter plasmid. The receptor plasmid carries a NF-κB dependent promoter which controls expression of a chimeric gene coding for fusion protein GAL4VP16, a well known transcriptional enhancer. The reporter plasmid carries binding sites to which the GAL4 binding domains of the fusion protein may attach. An associated minimal thymidine kinase promoter controls expression of a new fluorescent protein, tdTomato. tdTomato, compared to well known fluorescent protein EGFP, shows higher brightness, faster maturation time and a better fluorescence quantum yield. In order to determine the properties of tdTomato in an inducible system, the vector pNF-κBtdTomato was constructed. HEK/293 cells were transfected with this vector, resulting in various stable transfected clones which expressed the fluorescent protein when the NF-κB signal transduction pathway was activated. The tdTomato expression was evaluated in these clones after NF-κB activation was induced with TNF-α and compared with cells in a noninduced state to detect those clones which suit best for subsequent gene expression monitoring experiments, aiming for reporting NF-κB activation after radiation induced DNA double strand breaks. Additionally, HEK/293 cells were transfected with vector ptdTomato-N1 to obtain cells which constitutively express the fluorescent protein. Fluorescence spectra of tdTomato, its precursor DsRed and EGFP were measured to detect excitation- and emission maxima. Stable transfected clones were screened to discover cell lines with a constitutively high expression of the fluorescent protein. Four clones depicting highest fluorescence intensities were evaluated regarding cell line specific fluorescence intensity in dependency on cell number. Two of these clones were used for a cytotoxicity test, where cell growth inhibition after X-ray exposure was determined. Using constitutive fluorescent protein expressing cells enables a fast and convenient test for cytotoxicity of various reagents.