Advertisement

Imaging β-Galactosidase Activity In Vivo Using Sequential Reporter-Enzyme Luminescence

  • Georges von Degenfeld
  • Tom S. Wehrman
  • Helen M. Blau
Part of the Methods in Molecular Biology™ book series (MIMB, volume 574)

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 β-galactosidase (β-gal) activity. The substrate is a “caged” d-luciferin conjugate that must first be cleaved by β-gal before it can be catalyzed by Fluc in the final, light-emitting step. Hence, luminescence is dependent on and correlates with β-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 β-gal activity in engineered cells, as well as inducible tissue-specific β-gal expression in transgenic mice. Since β-gal, unlike Fluc, retains full activity outside of cells, we were able to show that antibodies conjugated to the recombinant β-gal enzyme could be used to detect and localize endogenous cells and extracellular antigens in vivo. In addition, we developed a low-affinity β-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.

Key words

β-galactosidase luminescent imaging in vivo pharmacology G-protein-coupled receptor 

References

  1. 1.
    Wu, J. C., Chen, I. Y., Wang, Y., Tseng, J R., Chhabra, A., Salek, M., Min, J. J., Fishbein, M. C., Crystal, R., and Gambhir, S. S. (2004) Molecular imaging of the kinetics of vascular endothelial growth factor gene expression in ischemic myocardium. Circulation 110 , 685–691.PubMedCrossRefGoogle Scholar
  2. 2.
    Contag, P. R., Olomu, I. N., Stevenson, D. K., and Contag, C. H. (1998) Bioluminescent indicators in living mammals. Nat Med 4 , 245–247.PubMedCrossRefGoogle Scholar
  3. 3.
    Contag, C. H., Contag, P. R., Mullins, J. I., Spilman, S. D., Stevenson, D. K., and Benaron, D. A. (1995) Photonic detection of bacterial pathogens in living hosts. Mol Microbiol 18 , 593–603.PubMedCrossRefGoogle Scholar
  4. 4.
    Edinger, M., Sweeney, T. J., Tucker, A. A., Olomu, A. B., Negrin, R. S., and Contag, C. H. (1999) Noninvasive assessment of tumor cell proliferation in animal models. Neoplasia 1 , 303–310.PubMedCrossRefGoogle Scholar
  5. 5.
    Gross, S., and Piwnica-Worms, D. (2005) Real-time imaging of ligand-induced IKK activation in intact cells and in living mice. Nat Methods 2 , 607–614.PubMedCrossRefGoogle Scholar
  6. 6.
    Zhao, H., Doyle, T. C., Coquoz, O., Kalish, F., Rice, B. W., and Contag, C. H. (2005) Emission spectra of bioluminescent reporters and interaction with mammalian tissue determine the sensitivity of detection in vivo. J Biomed Opt 10 , 41210.PubMedCrossRefGoogle Scholar
  7. 7.
    Wehrman, T. S., von Degenfeld, G., Krutzik, P. O., Nolan, G. P., and Blau, H. M. (2006) Luminescent imaging of beta-galactosidase activity in living subjects using sequential reporter-enzyme luminescence. Nat Methods 3 , 295–301.PubMedCrossRefGoogle Scholar
  8. 8.
    Geiger, R., Schneider, E., Wallenfels, K., and Miska, W. (1992) A new ultrasensitive bioluminogenic enzyme substrate for beta-galactosidase. Biol Chem Hoppe Seyler 373 , 1187–1191.PubMedCrossRefGoogle Scholar
  9. 9.
    Miska, W., and Geiger, R. (1987) Synthesis and characterization of luciferin derivatives for use in bioluminescence enhanced enzyme immunoassays. New ultrasensitive detection systems for enzyme immunoassays, I. J Clin Chem Clin Biochem 25 , 23–30.PubMedGoogle Scholar
  10. 10.
    Miska, W., and Geiger, R. (1988) A new type of ultrasensitive bioluminogenic enzyme substrates. I. Enzyme substrates with D-luciferin as leaving group. Biol Chem Hoppe Seyler 369 , 407–11.PubMedCrossRefGoogle Scholar
  11. 11.
    Cali, J. J., Ma, D., Sobol, M., Simpson, D. J., Frackman, S., Good, T. D., Daily, W. J., and Liu, D. (2006) Luminogenic cytochrome P450 assays. Expert Opin Drug Metab Toxicol 2 , 629–645.PubMedCrossRefGoogle Scholar
  12. 12.
    Shah, K., Tung, C. H., Breakefield, X. O., and Weissleder, R. (2005) In vivo imaging of S-TRAIL-mediated tumor regression and apoptosis. Mol Ther 11 , 926–931.PubMedCrossRefGoogle Scholar
  13. 13.
    Monsees, T., Miska, W., and Geiger, R. (1994) Synthesis and characterization of a bioluminogenic substrate for alpha-chymotrypsin. Anal Biochem 221, 329–334.PubMedCrossRefGoogle Scholar
  14. 14.
    Geiger, R., and Miska, W. (1989) A new type of ultrasensitive bioluminescence enzyme substrates for kininases. Adv Exp Med Biol 247B, 383–388.Google Scholar
  15. 15.
    von Degenfeld, G., Wehrman, T. S., Hammer, M. M., and Blau, H. M. (2007) A universal technology for monitoring G-protein-coupled receptor activation in vitro and noninvasively in live animals. Faseb J 21 , 3819–3826.CrossRefGoogle Scholar
  16. 16.
    Banfi, A., Springer, M. L., and Blau, H. M. (2002) Myoblast-mediated gene transfer for therapeutic angiogenesis. Methods Enzymol 346 , 145–157.PubMedCrossRefGoogle Scholar
  17. 17.
    Springer, M. L., Rando, T. A., Blau, H. M., Banfi, A., Springer, M. L., and Blau, H. M. (2002) Gene delivery to muscle. Myoblast-mediated gene transfer for therapeutic angiogenesis. Curr Protoc Hum Genet  Chapter 13, Unit 13 4.

Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2009

Authors and Affiliations

  • Georges von Degenfeld
    • 1
    • 2
  • Tom S. Wehrman
    • 3
  • Helen M. Blau
    • 4
  1. 1.Common Mechanism Research, Bayer Schering Pharma AGWuppertalGermany
  2. 2.Department of Experimental CardiologyUniversity of Witten-HerdeckeWuppertalGermany
  3. 3.Discoverx CorpFremontUSA
  4. 4.Baxter Laboratory in Genetic Pharmacology, Department of Microbiology and ImmunologyStanford University School of MedicineStanfordUSA

Personalised recommendations