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[Transfection of GFP mRNA in dendritic cells and analysis of some factors involved].

AIM
To investigate the transfection of green fluorescent protein (GFP) mRNA in dendritic cells (DC) and analyze some factors which influence the transfection efficiency.


METHODS
GFP (as a report gene) mRNA with cap was synthesized, in vitro, with mMESSAGE RNA Transcription Kit containing T7 RNA polymerase, and then the poly(A) was added to the GFP caped-mRNA by yeast poly(A) polymerase. DC were generated from the monocytes isolated from human peripheral blood by stimulation of GM-CSF and IL-4. The GFP mRNA was transfected into DC mediated by transfection reagent. The transfection efficiency and the expression levels were measured by flow cytometry.


RESULTS
GFP expression in DC has been obtained by transfecting its mRNA synthesized in vitro. The transfection reagents, mRNA concentrations and cell densities have the significant effects on the transfection. The high level of transfection efficiency (up to 27%) was obtained using Transmessenger Transfection Kit with 1 microg gfp mRNA in 200 microL X-VIVO-15 serum-free medium at the cell density of 2.5x10(9)/L.


CONCLUSION
The high-level transfection efficiency of gfp gene in DC could been achieved by using GFP mRNA in the optimum transfection conditions.