Journal
NATURE METHODS
Volume 3, Issue 4, Pages 295-301Publisher
NATURE PUBLISHING GROUP
DOI: 10.1038/NMETH868
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Funding
- NHLBI NIH HHS [HL65572] Funding Source: Medline
- NIA NIH HHS [AG09521, AG20961] Funding Source: Medline
- NICHD NIH HHS [HD18179] Funding Source: Medline
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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-gatactoside conjugate, must first be cleaved by beta-gaL before it can be catalyzed by firefly Luciferase (FLuc) to generate tight. As a result, luminescence is dependent on beta-gal activity. Using this technology, constitutive beta-gal activity in engineered Celts and inducible tissue-specific beta-gal expression in transgenic mice can now be visualized noninvasivety 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.
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