A New approach to SNAP‐tag technology as a possible imaging tool for translational research

The SNAP‐tag is a modified protein designed from the DNA repair protein O 6 ‐alkylguanine‐DNA‐alkyltransferase that recognizes benzylguanine derivatives and covalently attaches to the tag (Johnsson, 2003). To advance this method for in vivo and translational research we need better ways to modify target cells as well as improvements in probe design. We developed a retroviral vector bearing SNAP‐ADRB2 protein and Green Fluorescent Protein (GFP) for the generation of permanently transduced cancer cell lines. The SNAP‐ADRB2 sequence from a commercial plasmid was cloned into p28z‐IRES‐hsvTKGFP. The chimeric antigen receptor p28z of this plasmid was replaced with SNAP‐ADRB2 to construct the final vector SNAP‐ADRB2iTG. Supernatants from H29 virus producer cells were collected at 48 and 72 hours following transfection and used to permanently transduce C6 (rat glioma) and U87 (human glioma) cells in culture. Vector presence in the C6 and U87 cells was confirmed by GFP imaging and expression analysis using flow cytometry. Functionality of the SNAP‐tag system was assessed by in vitro labeling with fluorescent Surface‐549 substrate. The externalized SNAP protein was confirmed by fluorescent microscopy after in vitro labeling, which demonstrated a localized signal on the membrane of target cells. GFP and SNAP fluorescence in target cells persisted for at least two weeks. We have successfully created a new retroviral vector that allowed the generation of human and rat cancer cell lines with constitutive expression of the SNAP‐tag that could be used for in vitro and in vivo imaging. Further development of probes for this system could open the possibility of translational applications.