ИСТИНА

Luciferase encoding reporters are widely used for gene expression analysis. The standard procedure involves accumulation of the reporter protein in living cells followed by cell lysis and a single measurement of luciferase activity in the lysate. However, this approach does not allow tracing a dynamics of protein accumulation with high resolution, and thus is not suitable for studying fine details of gene expression and its regulation. We established a method of continuous luciferase measurement in cultured mammalian cells and applied it for studying expression of reporter constructs at the translational level in real time. We compared the translation efficiency and kinetics for several mRNAs encoding two luciferases - firefly and Renilla, depending on substrate variants and concentrations, and tested a number of transfection reagents. We analyzed effects of two modified bases, N1-methylpseudouridine and 5-methylcytidine, within a luciferase transcript, as well as three 5’ cap species, on reporter mRNA expression in various cell lines and primary cells. Then we investigated how the mRNA translational properties changed under conditions of various cellular stresses (oxidative stress, ultraviolet irradiation etc.) The method further allowed us to show that in vitro synthesized transcripts containing an intron can be efficiently spliced after their transfection into proliferating cells. We also tracked the expression kinetics of the luciferase gene delivered by a plasmid DNA transfection and compared it to the direct mRNA delivery. In summary, we developed a highly sensitive method for the continuous measurement of the reporter activity in cultured mammalian cells and showed its applicability for studying different aspects of gene expression. This work was supported by the grant of the Russian Federation government N14.W03.31.0012.