title: Imaging beta-galactosidase activity in vivo using sequential reporter-enzyme luminescence.
abstract: Bioluminescence using the reporter enzyme firefly luciferase (Fluc) and the substrate luciferin enables non-invasive optical imaging of living animals with extremely high sensitivity. This type of analysis enables studies of gene expression, tumor growth, and cell migration over time in live animals that were previously not possible. However, a major limitation of this system is that Fluc activity is restricted to the intracellular environment, which precludes important applications of in vivo imaging such as antibody labeling, or serum protein monitoring. In order to expand the application of bioluminescence imaging to other enzymes, we characterized a sequential reporter-enzyme luminescence (SRL) technology for the in vivo detection of beta-galactosidase (beta-gal) activity. The substrate is a "caged" D-luciferin conjugate that must first be cleaved by beta-gal before it can be catalyzed by Fluc in the final, light-emitting step. Hence, luminescence is dependent on and correlates with beta-gal activity. A variety of experiments were performed in order to validate the system and explore potential new applications. We were able to visualize non-invasively over time constitutive beta-gal activity in engineered cells, as well as inducible tissue-specific beta-gal expression in transgenic mice. Since beta-gal, unlike Fluc, retains full activity outside of cells, we were able to show that antibodies conjugated to the recombinant beta-gal enzyme could be used to detect and localize endogenous cells and extracellular antigens in vivo. In addition, we developed a low-affinity beta-gal complementation system that enables inducible, reversible protein interactions to be monitored in real time in vivo, for example, sequential responses to agonists and antagonists of G-protein-coupled receptors (GPCRs). Thus, using SRL, the exquisite luminescent properties of Fluc can be combined with the advantages of another enzyme. Other substrates have been described that extend the scope to endogenous enzymes, such as cytochromes or caspases, potentially enabling additional unprecedented applications.
authors.affiliation: Cardiovascular Research, Bayer Healthcare AG, Wuppertal, Germany.
authors.forename: Georges
authors.initials: G
authors.lastname: von Degenfeld
authors.forename: Tom S
authors.initials: TS
authors.lastname: Wehrman
authors.forename: Helen M
authors.initials: HM
authors.lastname: Blau
mesh_terms: D000818:Animals; D017930:Genes, Reporter; D049449:Luminescence; D051379:Mice; D008819:Mice, Nude; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism*
chemical_list: D001616:beta-Galactosidase
references.citation: FASEB J. 2007 Dec;21(14):3819-26
references.pmid: 17942828
references.citation: Mol Microbiol. 1995 Nov;18(4):593-603
references.pmid: 8817482
references.citation: Neoplasia. 1999 Oct;1(4):303-10
references.pmid: 10935484
references.citation: Expert Opin Drug Metab Toxicol. 2006 Aug;2(4):629-45
references.pmid: 16859410
references.citation: J Clin Chem Clin Biochem. 1987 Jan;25(1):23-30
references.pmid: 3549962
references.citation: Adv Exp Med Biol. 1989;247B:383-8
references.pmid: 2610082
references.citation: Methods Enzymol. 2002;346:145-57
references.pmid: 11883066
references.citation: Biol Chem Hoppe Seyler. 1992 Dec;373(12):1187-91
references.pmid: 1292503
references.citation: Nat Methods. 2005 Aug;2(8):607-14
references.pmid: 16094386
references.citation: J Biomed Opt. 2005 Jul-Aug;10(4):41210
references.pmid: 16178634
references.citation: Nat Med. 1998 Feb;4(2):245-7
references.pmid: 9461201
references.citation: Nat Methods. 2006 Apr;3(4):295-301
references.pmid: 16554835
references.citation: Curr Protoc Hum Genet. 2002 Feb;Chapter 13:Unit13.4
references.pmid: 18428326
references.citation: Mol Ther. 2005 Jun;11(6):926-31
references.pmid: 15922963
references.citation: Biol Chem Hoppe Seyler. 1988 May;369(5):407-11
references.pmid: 3166746
references.citation: Anal Biochem. 1994 Sep;221(2):329-34
references.pmid: 7810874
references.citation: Circulation. 2004 Aug 10;110(6):685-91
references.pmid: 15302807