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. 2020 Aug 20;10:14018. doi: 10.1038/s41598-020-71051-5

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© The Author(s) 2020

Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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Validation of the double-Flp-In cell clones on genomic and transcriptional level. (a) Simultaneous integration of two vectors my lead to at least four unwanted combinations (red ‘x’) besides the intended one (green tick). (b) Screening of cell clones by multiplex PCR allowed verification of single integration of each vector (EV empty vector; primers as given in Table 1). (c) More precise single PCRs were used to validate correct integration for each cell clone, as shown here using one representative example. (d) OCT1 and CYP2C19 gene expression analysis of double-Flp-In cell clones compared to single transfected cell lines. Results of n = 3 independent experiments are shown. Gene expression was normalized to the OCT1 only or CYP2C19 only transfected cell lines, respectively. Figure (d) was created using GraphPad Prism version 5.01 for Windows, GraphPad Software, La Jolla, California, USA, www.graphpad.com. Protein icons of OCT1 and CYP2C19 were created using The PyMOL Molecular Graphics System, Version 1.3, Schrödinger, LLC, www.pymol.org.