Construction & Evaluation of a Reporter Gene Displaying Aldehydes on the Cell Surface

Abstract

Reporter genes are often used to observe expression of promoters, which may change from its natural behaviour as a result of stress or disease states. Reporter genes are useful because they are easily detectable by a variety of imaging methods, including fluorescence microscopy techniques, magnetic resonance imaging, and positron emission tomography. Previously, methyl 5-MeO-N-aminoanthranilate (MMNA) had been synthesized as an aldehyde-conditional fluorophore and was tested in physiological conditions to identify the Aldehydic Load of cells. Thus, it was hypothesized that a reporter protein displaying an aldehyde on the cell surface can be identified by MMNA. This reporter protein would contain a substrate recognition site for formylglycine generating enzyme (FGE) that converts a specific cysteine residue into a formylglycine residue. This will result in production of an aldehyde at the N-terminal of the transmembrane domain of platelet derived growth factor receptor . In this way, the protein product, Aldehyde-presenting FGE-dependent Readout (Alfred), would display an aldehyde on the extracellular surface of the cell. Alfred was expressed in A549 human lung cancer cells using the Tet-On® Inducible System, which allows expression of a gene of interest by use of doxycyclin (dox) as a chemical trigger. Microscopy of Alfred-transfected cells, induced by dox and probed with MMNA, showed no difference in fluorescence between non-transfected and Alfred-transfected cells. The overexpression of FGE to increase thiol-to-aldehyde conversion, and the imaging of cells at longer timepoints (48 and 72 hours) to allow localization of the protein to the cell surface, were attempted. In addition, Alfred was constitutively expressed in another transfection experiment in efforts to increase gene expression. However, these efforts to evaluate Alfred did not improve the microscopy results. Western blotting confirmed FGE overexpression in transgenic cells. Blotting against the Myc-tag in Alfred showed no detected proteins in Alfred-transfected cells. In conjunction with the microscopy images, these results suggest that Alfred is not expressed and cannot be detected as a reporter gene. Comparison to previous works allows the identification of potential approaches to improve Alfred functionality, including the absence of the hemagglutinin epitope, the choice of aldehyde probe used, the choice of cell line used, and the method of analyzing microscopy. Future directives are postulated to identify sources that hinder Alfred expression, and to improve visualization of Alfred over homeostatic aldehydes.