Luminescent imaging of beta-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-galactoside conjugate, must first be cleaved by beta-gal before it can be catalyzed by firefly luciferase (FLuc) to generate light. As a result, luminescence is dependent on beta-gal activity. Using this technology, constitutive beta-gal activity in engineered cells and inducible tissue-specific beta-gal expression in transgenic mice can now be visualized noninvasively 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.
Wehrman
Thomas S
TS
Baxter Laboratory in Genetic Pharmacology, Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California 94305, USA.
von Degenfeld
Georges
G
Krutzik
Peter O
PO
Nolan
Garry P
GP
Blau
Helen M
HM
D000818:Animals; D002384:Catalysis; D002463:Cell Membrane Permeability; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D008027:Light; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D008163:Luminescent Measurements / Q000379:methods*; D008221:Lymphoid Tissue / Q000166:cytology / Q000378:metabolism / Q000648:ultrastructure; D051379:Mice; D008822:Mice, Transgenic; D012680:Sensitivity and Specificity; D001616:beta-Galactosidase / Q000032:analysis* / Q000378:metabolism
D008156:Luciferases; D001616:beta-Galactosidase