Development of a reporter cell line expressing vascular endothelial growth factor receptor 2 and a firefly luciferase reporter gene based on CHO-K1 cells (CHO-KDR/LUC)

Background. Anti-VEGF agents have proven effective in the treatment of ophthalmological and oncological diseases, and their potential applications continue to grow. Limited availability of commercial bioassay systems for determining the biological activity of anti-VEGF drugs necessitates the development of a proprietary, independent reporter cell line.
Objective. To generate a stable reporter cell line expressing vascular endothelial growth factor receptor-2 (KDR) and a firefly luciferase (LUC) reporter gene based on the CHO-K1 suspension cell line.
Methods. CHO-K1 (GosNIIGenetika, Russia) was transfected with two plasmids carrying the KDR gene and the LUC gene in varying ratios. The effect of plasmid ratio was assessed by measuring luciferase activity using Promega Bio-Glo kits (Promega, USA) or buffers containing the D-luciferin substrate (Molecta, Russia). VEGF165 (JSC "Generium", Russia) was added to activate the cell line's reporter response.
Results. The CHO-KDR/LUC reporter cell line was generated by co-transfecting CHO-K1 cells with plasmids encoding the reporter gene (LUC) and the human VEGFR-2 receptor gene (KDR). Maximum luciferase induction was achieved with a KDR plasmid ratio of 25/75 LUC (%) and reached a plateau of 5x105 RLU; the EC50 was 35.19 ng/mL. Analysis of the luciferase response using homologous ligands demonstrated specificity for VEGF165. With increasing cell generations, growth characteristics and the luciferase response to VEGF165 stimulation remained stable.
Conclusion. The CHO-KDR/LUC reporter cell line is suitable for analyzing the biological activity of proteins used in anti-VEGF therapy. Further validation and application will improve the efficiency of developing new therapeutic proteins and biosimilars for VEGF therapy.

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