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.
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We would like to thank M. Hammer and M.J. Merchant for technical assistance. The work was funded by a grant of the Deutsche Forschungsgemeinschaft (DE740-1/1) to G.v.D. and grants from the US National Institutes of Health (AG09521, AG20961, HL65572, HD18179) and the Baxter Foundation to H.M.B.
H.M.B. is a major stockholder in a company that might have a gain or loss financially through publication of this manuscript.
H.M.B., T.S.W. and G.vD. are inventors of the SRL technology; a patent is pending.
Lugal and luciferin show biodistribution to major organs following intraperitoneal injection in 2 separate mice. (PDF 232 kb)
Comparison of bioluminescent and fluorescent imaging of β-gal activity. (PDF 45 kb)
Luminescent imaging of β-gal labeled antibodies. (PDF 50 kb)
Colocalization of injected anti-CD4 antibody with TCR-β chain. (PDF 49 kb)
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A hemicyanine-based colorimetric turn-on fluorescent probe for β-galactosidase activity detection and application in living cells
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