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 β-galactosidase (β-gal) activity. The substrate, a caged D -luciferin–galactoside conjugate, must first be cleaved by β-gal before it can be catalyzed by firefly luciferase (FLuc) to generate light. As a result, luminescence is dependent on β-gal activity. Using this technology, constitutive β-gal activity in engineered cells and inducible tissue-specific β-gal expression in transgenic mice can now be visualized noninvasively over time. A substantial advantage of β-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 β-gal enzyme can be used to detect endogenous cells and extracellular antigens in vivo . Thus, coupling the properties of FLuc to the advantages of β-gal permits bioluminescent imaging applications that previously were not possible.