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Gene expression imaging by enzymatic catalysis of a fluorescent probe via membrane-anchored β-glucuronidase

Gene Therapy volume 14, pages 565–574 (2007)Cite this article

Abstract

Development of nonimmunogenic and specific reporter genes to monitor gene expression in vivo is important for the optimization of gene therapy protocols. We developed a membrane-anchored form of mouse β-glucuronidase (mβG) as a reporter gene to hydrolyze a nonfluorescent glucuronide probe (fluorescein di-β-D-glucuronide, (FDGlcU) to a highly fluorescent reporter to assess the location and persistence of gene expression. A functional β-glucuronidase (βG) was stably expressed on the surface of murine CT26 colon adenocarcinoma cells where it selectively hydrolyzed the cell-impermeable FDGlcU probe. FDGlcU was also preferentially converted to fluorescent probe by (βG) on CT26 tumors. The fluorescent intensity in βG-expressing CT26 tumors was 240 times greater than the intensity in control tumors. Selective imaging of gene expression was also observed after intratumoral injection of adenoviral βG vector into carcinoma xenografts. Importantly, mβG did not induce an antibody response after hydrodynamic plasmid immunization of Balb/c mice, indicating that the reporter gene product displayed low immunogenicity. A membrane-anchored form of human βG also allowed in vivo imaging, demonstrating that human βG can be employed for imaging. This imaging system therefore, displays good selectivity with low immunogenicity and may help assess the location, magnitude and duration of gene expression in living animals and humans.

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Acknowledgements

This work was supported by the National Research Program for Genomic Medicine (NRPGM), National Science Council, Taipei, Taiwan (NSC95-3112-B-037-001 and NSC94-2745-B-037-010-URD) and the Genomic and Proteomic Program, Academia Sinica, Taipei, Taiwan (94M007-2). The Molecular-Genetic Imaging Core-Cell and tissue-imaging core of National Yang-Ming University is also gratefully acknowledged.

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Authors and Affiliations

  1. Faculty of Biomedical Science and Environmental Biology, Kaohsiung Medical University, Kaohsiung, Taiwan

    Y-C Su & T-L Cheng

  2. Graduate Institute of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan

    K-H Chuang & C-M Cheng

  3. Faculty of Medicinal and Applied Chemistry, Kaohsiung Medical University, Kaohsiung, Taiwan

    Y-M Wang

  4. Graduate Institute of Medical Genetics, Kaohsiung Medical University, Kaohsiung, Taiwan

    S-R Lin

  5. Faculty of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan

    J-Y Wang

  6. Faculty of Biomedical Imaging and Radiological Sciences, National Yang-Ming University, Taipei, Taiwan

    J-J Hwang

  7. Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan

    B-M Chen, K-C Chen & S Roffler

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Correspondence to S Roffler or T-L Cheng.

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Su, YC., Chuang, KH., Wang, YM. et al. Gene expression imaging by enzymatic catalysis of a fluorescent probe via membrane-anchored β-glucuronidase. Gene Ther 14, 565–574 (2007). https://doi.org/10.1038/sj.gt.3302896

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