Abstract
BACKGROUND: Optimization of gene therapy protocols requires accurate and non-invasive quantification of vector delivery and gene expression. To facilitate non-invasive imaging of gene expression, we have genetically engineered 'artificial receptors', i.e. membrane proteins that bind (99m)Tc-oxotechnetate ((99m)TcOT) via transchelation from a complex with glucoheptonate. The latter is a component of a widely used clinical imaging kit.
METHODS: The engineered marker proteins were designed as type I and II membrane proteins and consisted of (1) an (99m)TcOT-binding domain, metallothionein (MT), and (2) a membrane-anchoring domain. Engineered constructs were used for transfection of COS-1 and 293 cells; the expression of mRNA was verified by RT-PCR.
RESULTS: Immunofluorescent analysis, cell fractionation and immunoblotting revealed expression of marker proteins on plasma membrane. Transfection of cells resulted in strong positive staining of plasma membrane with anti-His-tag antibodies. Scintigraphic imaging in vitro confirmed the ability of transfected cells to bind (99m)TcOT. The fraction of bound radioactivity reached a peak (3.53%) when 0.93 MBq (99m)TcOT was added to transfected COS-1 cells. The experiment-to-control signal ratio was equal to 32 at the same added dose.
CONCLUSIONS: (1) Both types of engineered 'artificial receptors' were expressed on the surface of eukaryotic cells; (2) marker proteins were functional in binding (99m)TcOT; and (3) type II membrane proteins were more efficient in binding (99m)TcOT than type I proteins. We anticipate that the developed approach could be useful for 'tagging' transfected cells with (99m)TcOT enabling imaging of tracking in vivo transduced cells or cell therapies.