Optimization of the Doxycycline-Induced Gene Expression System in HCT116 and A549 Cells
摘要
Objective: The doxycycline (Dox)-inducible gene expression system, which allows for the overexpression of a gene of interest under the control of the Tet-operator, is a practical and widely used approach in both in vitro and in vivo studies. However, recent observations indicate the possibility of antiproliferative and/or cytotoxic effects of doxycycline in certain cell systems. Consequently, the results of studies using Dox-inducible expression systems may be misinterpreted or confounded by the side effects of doxycycline itself. Methods and Results: In this study, we determined the lowest concentration of doxycycline required for efficient induction of the gene of interest under the control of the Tet-operator and analyzed its impact on the proliferation and viability of two human tumor cell lines HCT116 and A549, which are widely used in scientific research. Conclusions: Our findings highlight the importance of including additional controls in experiments using Dox-inducible expression systems. Specifically, the effects of the applied doxycycline concentrations should be assessed in cells that do not carry the Dox-inducible gene. This step is critical to avoid artifacts arising from the direct effects of doxycycline on the studied parameters.