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

• Published in: Nature methods Vol. 3; no. 4; pp. 295 - 301
• Main Authors: Wehrman, Thomas S, von Degenfeld, Georges, Krutzik, Peter O, Nolan, Garry P, Blau, Helen M
• Format: Journal Article
• Language: English
• Published: New York Nature Publishing Group US 01.04.2006; Nature Publishing Group
• Subjects: Analysis; Animals; Beta galactosidases; beta-Galactosidase - analysis; beta-Galactosidase - metabolism; Bioinformatics; Biological Microscopy; Biological Techniques; Bioluminescence; Biomedical and Life Sciences; Biomedical Engineering/Biotechnology; Catalysis; Cell Membrane Permeability; Drugs; Enzymatic analysis; Gene expression; Gene Expression Regulation; Genes, Reporter; Health aspects; Life Sciences; Light; Luciferases - genetics; Luciferases - metabolism; Luminescent Measurements - methods; Lymphoid Tissue - cytology; Lymphoid Tissue - metabolism; Lymphoid Tissue - ultrastructure; Methods; Mice; Mice, Transgenic; Proteomics; Sensitivity and Specificity; United States
• ISSN: 1548-7091; 1548-7105
• DOI: 10.1038/nmeth868

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.