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Luminescent imaging of β-galactosidase activity in living subjects using sequential reporter-enzyme luminescence

Nature Methods volume 3, pages 295–301 (2006)Cite this article

Abstract

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.

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Figure 1: Luminescent measurement of β-gal activity in living cells using Lugal.
Figure 2: Sensitive and quantitative bioluminescent imaging of β-gal activity in living mice using Lugal.
Figure 3: Luminescent imaging of muscle regeneration over time in transgenic Myf5-nLacZ luc reporter mice.
Figure 4: Luminescent imaging of lymphocyte distribution in vivo using antibodies to CD4 (anti-CD4) conjugated with β-gal.
Figure 5: β-gal–conjuguated antibodies specifically label target cells and remain intact after intravenous injection.

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References

  1. Zhang, W. et al. Rapid in vivo functional analysis of transgenes in mice using whole body imaging of luciferase expression. Transgenic Res. 10, 423–434 (2001).

    Article  CAS  Google Scholar 

  2. Contag, C.H. et al. Photonic detection of bacterial pathogens in living hosts. Mol. Microbiol. 18, 593–603 (1995).

    Article  CAS  Google Scholar 

  3. Contag, P.R., Olomu, I.N., Stevenson, D.K. & Contag, C.H. Bioluminescent indicators in living mammals. Nat. Med. 4, 245–247 (1998).

    Article  CAS  Google Scholar 

  4. Edinger, M. et al. Noninvasive assessment of tumor cell proliferation in animal models. Neoplasia 1, 303–310 (1999).

    Article  CAS  Google Scholar 

  5. Wu, J.C. et al. Molecular imaging of the kinetics of vascular endothelial growth factor gene expression in ischemic myocardium. Circulation 110, 685–691 (2004).

    Article  CAS  Google Scholar 

  6. Gross, S. & Piwnica-Worms, D. Real-time imaging of ligand-induced IKK activation in intact cells and in living mice. Nat. Methods 2, 607–614 (2005).

    Article  CAS  Google Scholar 

  7. Choy, G. et al. Comparison of noninvasive fluorescent and bioluminescent small animal optical imaging. Biotechniques 35, 1022–1030 (2003).

    Article  CAS  Google Scholar 

  8. Troy, T., Jekic-McMullen, D., Sambucetti, L. & Rice, B. Quantitative comparison of the sensitivity of detection of fluorescent and bioluminescent reporters in animal models. Mol. Imaging 3, 9–23 (2004).

    Article  CAS  Google Scholar 

  9. Tang, Y. et al. In vivo tracking of neural progenitor cell migration to glioblastomas. Hum. Gene Ther. 14, 1247–1254 (2003).

    Article  CAS  Google Scholar 

  10. Sweeney, T.J. et al. Visualizing the kinetics of tumor-cell clearance in living animals. Proc. Natl. Acad. Sci. USA 96, 12044–12049 (1999).

    Article  CAS  Google Scholar 

  11. Lin, A.H. et al. Global analysis of Smad2/3-dependent TGF-beta signaling in living mice reveals prominent tissue-specific responses to injury. J. Immunol. 175, 547–554 (2005).

    Article  CAS  Google Scholar 

  12. Welsh, D.K., Yoo, S.H., Liu, A.C., Takahashi, J.S. & Kay, S.A. Bioluminescence imaging of individual fibroblasts reveals persistent, independently phased circadian rhythms of clock gene expression. Curr. Biol. 14, 2289–2295 (2004).

    Article  CAS  Google Scholar 

  13. Luker, K.E., Hutchens, M., Schultz, T., Pekosz, A. & Luker, G.D. Bioluminescence imaging of vaccinia virus: Effects of interferon on viral replication and spread. Virology (2005).

  14. Ciana, P. et al. Engineering of a mouse for the in vivo profiling of estrogen receptor activity. Mol. Endocrinol. 15, 1104–1113 (2001).

    Article  CAS  Google Scholar 

  15. Contag, C.H. & Bachmann, M.H. Advances in in vivo bioluminescence imaging of gene expression. Annu. Rev. Biomed. Eng. 4, 235–260 (2002).

    Article  CAS  Google Scholar 

  16. Ke, S. et al. Near-infrared optical imaging of epidermal growth factor receptor in breast cancer xenografts. Cancer Res. 63, 7870–7875 (2003).

    CAS  PubMed  Google Scholar 

  17. Olafsen, T. et al. Optimizing radiolabeled engineered anti-p185HER2 antibody fragments for in vivo imaging. Cancer Res. 65, 5907–5916 (2005).

    Article  CAS  Google Scholar 

  18. Rubin, R.H., Baltimore, D., Chen, B.K., Wilkinson, R.A. & Fischman, A.J. In vivo tissue distribution of CD4 lymphocytes in mice determined by radioimmunoscintigraphy with an 111In-labeled anti-CD4 monoclonal antibody. Proc. Natl. Acad. Sci. USA 93, 7460–7463 (1996).

    Article  CAS  Google Scholar 

  19. Miska, W. & Geiger, R. 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 (1987).

    CAS  PubMed  Google Scholar 

  20. Geiger, R., Schneider, E., Wallenfels, K. & Miska, W. A new ultrasensitive bioluminogenic enzyme substrate for beta-galactosidase. Biol. Chem. Hoppe Seyler 373, 1187–1191 (1992).

    Article  CAS  Google Scholar 

  21. Miska, W. & Geiger, R. A new type of ultrasensitive bioluminogenic enzyme substrates. I. Enzyme substrates with D-luciferin as leaving group. Biol. Chem. Hoppe Seyler 369, 407–411 (1988).

    Article  CAS  Google Scholar 

  22. Masuda-Nishimura, I., Fukuda, S., Sano, A., Kasai, K. & Tatsumi, H. Development of a rapid positive/absent test for coliforms using sensitive bioluminescence assay. Lett. Appl. Microbiol. 30, 130–135 (2000).

    Article  CAS  Google Scholar 

  23. Yang, X., Janatova, J. & Andrade, J.D. Homogeneous enzyme immunoassay modified for application to luminescence-based biosensors. Anal. Biochem. 336, 102–107 (2005).

    Article  CAS  Google Scholar 

  24. Tung, C.H. et al. In vivo imaging of beta-galactosidase activity using far red fluorescent switch. Cancer Res. 64, 1579–1583 (2004).

    Article  CAS  Google Scholar 

  25. Cui, C., Wani, M.A., Wight, D., Kopchick, J. & Stambrook, P.J. Reporter genes in transgenic mice. Transgenic Res. 3, 182–194 (1994).

    Article  CAS  Google Scholar 

  26. Jeong, J. & McMahon, A.P. Growth and pattern of the mammalian neural tube are governed by partially overlapping feedback activities of the hedgehog antagonists patched 1 and Hhip1. Development 132, 143–154 (2005).

    Article  CAS  Google Scholar 

  27. Candido, E.P. & Jones, D. Transgenic Caenorhabditis elegans strains as biosensors. Trends Biotechnol. 14, 125–129 (1996).

    Article  CAS  Google Scholar 

  28. Tajbakhsh, S. et al. Gene targeting the myf-5 locus with nlacZ reveals expression of this myogenic factor in mature skeletal muscle fibres as well as early embryonic muscle. Dev. Dyn. 206, 291–300 (1996).

    Article  CAS  Google Scholar 

  29. Cao, Y.A. et al. Shifting foci of hematopoiesis during reconstitution from single stem cells. Proc. Natl. Acad. Sci. USA 101, 221–226 (2004).

    Article  CAS  Google Scholar 

  30. Cooper, R.N. et al. In vivo satellite cell activation via Myf5 and MyoD in regenerating mouse skeletal muscle. J. Cell Sci. 112, 2895–2901 (1999).

    CAS  PubMed  Google Scholar 

  31. Mendler, L., Zador, E., Dux, L. & Wuytack, F. mRNA levels of myogenic regulatory factors in rat slow and fast muscles regenerating from notexin-induced necrosis. Neuromuscul. Disord. 8, 533–541 (1998).

    Article  CAS  Google Scholar 

  32. Shah, K., Tung, C.H., Breakefield, X.O. & Weissleder, R. In vivo imaging of S-TRAIL-mediated tumor regression and apoptosis. Mol. Ther. 11, 926–931 (2005).

    Article  CAS  Google Scholar 

  33. Cao, Y.A. et al. Molecular imaging using labeled donor tissues reveals patterns of engraftment, rejection, and survival in transplantation. Transplantation 80, 134–139 (2005).

    Article  Google Scholar 

  34. Palermo, A.T., Labarge, M.A., Doyonnas, R., Pomerantz, J. & Blau, H.M. Bone marrow contribution to skeletal muscle: a physiological response to stress. Dev. Biol. 279, 336–344 (2005).

    Article  CAS  Google Scholar 

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Acknowledgements

We would like to thank M. Hammer and M.J. Merchant for technical assistance. The work was funded by a grant of the Deutsche Forschungsgemeinschaft (DE740-1/1) to G.v.D. and grants from the US National Institutes of Health (AG09521, AG20961, HL65572, HD18179) and the Baxter Foundation to H.M.B.

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Author notes

  1. Thomas S Wehrman and Georges von Degenfeld: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Microbiology and Immunology, Baxter Laboratory in Genetic Pharmacology, Stanford University School of Medicine, Stanford, 94305, California, USA

    Thomas S Wehrman, Georges von Degenfeld, Peter O Krutzik, Garry P Nolan & Helen M Blau

  2. Department of Molecular Pharmacology, Stanford University School of Medicine, Stanford, 94305, California, USA

    Peter O Krutzik & Helen M Blau

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  1. Thomas S Wehrman
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  2. Georges von Degenfeld
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Corresponding author

Correspondence to Helen M Blau.

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Competing interests

H.M.B. is a major stockholder in a company that might have a gain or loss financially through publication of this manuscript.

H.M.B., T.S.W. and G.vD. are inventors of the SRL technology; a patent is pending.

Supplementary information

Supplementary Fig. 1

Lugal and luciferin show biodistribution to major organs following intraperitoneal injection in 2 separate mice. (PDF 232 kb)

Supplementary Fig. 2

Comparison of bioluminescent and fluorescent imaging of β-gal activity. (PDF 45 kb)

Supplementary Fig. 3

Luminescent imaging of β-gal labeled antibodies. (PDF 50 kb)

Supplementary Fig. 4

Colocalization of injected anti-CD4 antibody with TCR-β chain. (PDF 49 kb)

Supplementary Methods (DOC 32 kb)

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Wehrman, T., von Degenfeld, G., Krutzik, P. et al. Luminescent imaging of β-galactosidase activity in living subjects using sequential reporter-enzyme luminescence. Nat Methods 3, 295–301 (2006). https://doi.org/10.1038/nmeth868

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