Abstract
Overexpression of single genes in mammalian cells is widely used to investigate protein function in basic and applied biosciences and in drug research. A better understanding of interactions of two proteins is an important next step in the advancement of our understanding of complex biological systems. However, simultaneous and robust overexpression of two or more genes is challenging. The Flp-In system integrates a vector into cell lines at a specific genomic locus, but has not been used for integration of more than one gene. Here we present a modification of the Flp-In system that enables the simultaneous targeted integration of two genes. We describe the modification and generation of the vectors required and give the complete protocol for transfection and validation of correct genomic integration and expression. We also provide results on the stability and reproducibility, and we functionally validated this approach with a pharmacologically relevant combination of a membrane transporter facilitating drug uptake and an enzyme mediating drug metabolism.