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.
Highlights
Introduction of restriction sitesCYP2C19_HindIII_fwd CYP2C19_EcoRV_revPrimers used for Validation PCRsIntegration PCR 1 PSV40 Ppuro_r Puni_f Phyr_rMultiple Integration PCR A Puni_f Ppuro_rMultiple Integration PCR B PFRT_f Ppuro_rMultiple Integration PCR C PFRT_f Phyr_r
We introduced a one-nucleotide deletion into the original pcDNA5/Flp recombination target (FRT) vector, which was necessary for correct expression of the hygromycin resistance gene from the promoter of the second vector
We show the stability of the double transfection with two pairs of fluorescent proteins, we compare expression levels of single- to double-transfected genes, and we provide a scientifically relevant example for the application of this method to study the interaction between cell uptake transport and metabolism of the anti-malarial prodrug proguanil (Fig. 1)
Summary
Our goal was to develop a simple and robust protocol to transfect two different genes in a modular concept (Fig. 1)
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