The expression of the bacterial nitroreductase gene in COS-7 cells results in protein aggregation. (A) Schematic representation of the coding regions concerning GNTR expression in pGNTR and pGNTRo constructs, respectively. The wild type ntr gene and the codon-optimized ntro gene were N-terminally fused to gfp providing a marker for NTR expression. The bacterial ntr sequence contains several critical codons (red bars) for the expression in mammalian cells. In pGNTRo all critical ntr codons have been adapted to the preferred codon usage in mouse to ensure optimal expression in mammalian cells. (B) Perinuclear structures (white arrowheads) are present in approximately 10% of pGNTR-transfected COS-7 cells, indicating protein aggregation 48 hours after transfection. Protein aggregation was not present in gntro-expressing cells after codon optimization. GFP-labeled nitroreductase (green) and DAPI-stained DNA (blue) are shown in overlay. Scale bar = 50 μm.