Luminescent imaging of β-galactosidase activity in living subjects using sequential reporter-enzyme luminescence

We generated a sequential reporter-enzyme luminescence (SRL) technology for in vivo detection of beta-galactosidase (beta-gal) activity. The substrate, a caged D-Luciferin-gatactoside conjugate, must first be cleaved by beta-gaL before it can be catalyzed by firefly Luciferase (FLuc) to generate tight. As a result, luminescence is dependent on beta-gal activity. Using this technology, constitutive beta-gal activity in engineered Celts and inducible tissue-specific beta-gal expression in transgenic mice can now be visualized noninvasivety over time. A substantial advantage of beta-gal as a bioluminescent probe is that the enzyme retains full activity outside of cells, unlike FLuc, which requires intracellular cofactors. As a result, antibodies conjugated to the recombinant beta-gal enzyme can be used to detect endogenous cells and extracellular antigens in vivo. Thus, coupling the properties of FLuc to the advantages of beta-gal permits bioluminescent imaging applications that previously were not possible.