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Cytoplasmic expression of EGFP in dendritic cells transfected with in vitro transcribed mRNA or cellular total RNA extracted from EGFP expressing leukemia cells

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Abstract

The present study was designed for identifying the protein synthesis in cytoplasm of dendritic cells transfected with in vitro transcribed mRNA and cellular total RNA extracted from tumor cells. Dendritic cells were generated from cord blood-CD34+ cells by culture with GM-CSF, SCF, and TNF-α, or from peripheral blood adherent cells or CD14+ cells by culture with GM-CSF and IL-4. Dendritic cells were transfected with in vitro transcribed EGFP mRNA or cellular total RNA, which was isolated from EGFP expressing K562, by electroporation using a square-wave pulse. Optimal in vitro transcribed EGFP mRNA transfection efficiency (>90%) was observed in a single electroporation of 1.75 kV/cm (electric field strength) with a pulse width of 250 µs. Although the intensity of EGFP expression in dendritic cells transfected with cellular total RNA was less compared with that in dendritic cells transfected with in vitro transcribed EGFP mRNA, a definite cytoplasmic synthesis of EGFP was demonstrated in dendritic cells transfected with cellular total RNA. The visual identification of cytoplasmic expression of cellular total RNA in dendritic cells revealed that electroporation of tumor cell-derived RNA could be a useful tool to load dendritic cells with tumor antigens for establishing an efficient dendritic cell-based tumor immunotherapy.

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Authors and Affiliations

  1. Division of Hematology, Department of Regenerative and Transplant Medicine, Niigata University Graduate School of Medical and Dental Sciences, 951-8518, Niigata, Japan

    Flavio Ayres, Naoko Satoh, Takashi Abe, Toshio Yanao, Ken Toba & Yoshifusa Aizawa

  2. Division of Stem Cell Transplantation, Niigata University Hospital, 951-8518, Niigata, Japan

    Tatsuo Furukawa

  3. Hirohashi Gynecology and Obstetrics Clinic, 951-8518, Niigata, Japan

    Takeshi Hirohashi

  4. School of Health Sciences, Faculty of Medicine, Niigata University, 2-746, Asahimachi, 951-8515, Niigata, Japan

    Masuhiro Takahashi M.D. & Miwako Narita

Authors
  1. Masuhiro Takahashi M.D.
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  2. Miwako Narita
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  3. Flavio Ayres
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  4. Naoko Satoh
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  5. Takashi Abe
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  6. Toshio Yanao
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  7. Tatsuo Furukawa
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  8. Ken Toba
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  9. Takeshi Hirohashi
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  10. Yoshifusa Aizawa
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Correspondence to Masuhiro Takahashi M.D..

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Takahashi, M., Narita, M., Ayres, F. et al. Cytoplasmic expression of EGFP in dendritic cells transfected with in vitro transcribed mRNA or cellular total RNA extracted from EGFP expressing leukemia cells. Med Oncol 20, 335–348 (2003). https://doi.org/10.1385/MO:20:4:335

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  • DOI: https://doi.org/10.1385/MO:20:4:335

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