Cytoplasmic expression of EGFP in dendritic cells transfected with in vitro transcribed mRNA or cellular total RNA extracted from EGFP expressing leukemia cells.
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-alpha, 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 micros. 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.
Takahashi
Masuhiro
M
School of Health Sciences, Faculty of Medicine, Niigata University Graduate School of Medical and Dental Sciences, 2-746 Asahimachi, Niigata 951-8518, Japan. matak@clg.niigata-u.ac.jp
Narita
Miwako
M
Ayres
Flavio
F
Satoh
Naoko
N
Abe
Takashi
T
Yanao
Toshio
T
Furukawa
Tatsuo
T
Toba
Ken
K
Hirohashi
Takeshi
T
Aizawa
Yoshifusa
Y
D003713:Dendritic Cells / Q000276:immunology / Q000378:metabolism*; D018274:Electroporation / Q000379:methods; D049452:Green Fluorescent Proteins; D006801:Humans; D007167:Immunotherapy / Q000379:methods; D008164:Luminescent Proteins / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics* / Q000302:isolation & purification; D014162:Transfection / Q000379:methods*; D014407:Tumor Cells, Cultured / Q000378:metabolism*
D008164:Luminescent Proteins; D012333:RNA, Messenger; D049452:Green Fluorescent Proteins