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Immunogenicity of enhanced green fluorescent protein (EGFP) in BALB/c mice: identification of an H2-Kd-restricted CTL epitope

Gene Therapy volume 7, pages 2036–2040 (2000)Cite this article

Abstract

Enhanced green fluorescent protein (EGFP) is a novel marker gene product, which is readily detectable using techniques of fluorescence microscopy, flow cytometry, or macroscopic imaging. In the present studies, we have examined the immunogenicity of EGFP in murine models. A stable transfectant of the transplantable CMS4 sarcoma of BALB/c origin expressing EGFP, CMS4-EGFP-Zeo, was generated. Splenocytes harvested from mice immunized with a recombinant adenovirus expressing EGFP (Ad-EGFP) were restimulated in vitro with CMS4-EGFP-Zeo. Effector lymphocytes displayed strong cytotoxicity against CMS4-EGFP-Zeo, but not against mock-transfected CMS4-Zeo tumor cells. A number of candidate H2-Kd-binding peptides derived from the EGFP protein were chosen according to an epitope prediction program and synthesized. These peptides were tested for their ability to bind to H2-Kd molecules and stimulate IFNγ-production by splenocytes harvested from Ad-EGFP-immunized mice. Using this methodology, the peptide, HYLSTQSAL (corresponding to EGFP200–208) which strongly binds to H2-Kd molecules, was identified as a naturally occurring epitope of EGFP. These results should facilitate the use of EGFP as a model tumor antigen in BALB/c mice.

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Acknowledgements

This work was supported by National Institute of Health (NIH) Grants CA 64623 (ABD), CA59371 (PDR), a Telethon Grant (AG), and grant Tu 90/2–1 from the Deutsche Forschungsgemeinschaft (TT).

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Authors and Affiliations

  1. Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA

    A Gambotto, T Kenniston & PD Robbins

  2. Department of Surgery, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA

    A Gambotto

  3. Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA

    G Dworacki, V Cicinnati & AB DeLeo

  4. Department of Dermatology, University of Mainz, Germany

    J Steitz & T Tüting

  5. University of Pittsburgh Cancer Institute, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA

    AB DeLeo

Authors
  1. A Gambotto
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  2. G Dworacki
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Gambotto, A., Dworacki, G., Cicinnati, V. et al. Immunogenicity of enhanced green fluorescent protein (EGFP) in BALB/c mice: identification of an H2-Kd-restricted CTL epitope. Gene Ther 7, 2036–2040 (2000). https://doi.org/10.1038/sj.gt.3301335

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  • DOI: https://doi.org/10.1038/sj.gt.3301335

Keywords

  • EGFP
  • BALB/c
  • epitope
  • H2-Kd

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