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...

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Veröffentlicht in:Nature methods Jg. 3; H. 4; S. 295 - 301
Hauptverfasser: Wehrman, Thomas S, von Degenfeld, Georges, Krutzik, Peter O, Nolan, Garry P, Blau, Helen M
Format: Journal Article
Sprache:Englisch
Veröffentlicht: New York Nature Publishing Group US 01.04.2006
Nature Publishing Group
Schlagworte:
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
Online-Zugang:Volltext
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Zusammenfassung: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.
Bibliographie:ObjectType-Article-2
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ISSN:1548-7091
1548-7105
DOI:10.1038/nmeth868