Transfection of "naked" siRNA results in endosomal uptake and metabolic impairment in cultured neurons.
RNA interference is rapidly becoming a powerful tool for gene silencing in mammalian cells. Introduction of siRNA into primary cells, however, remains one of the major difficulties of this novel technique. Using cationic lipid-based transfection reagents satisfactory transfection results are observed in cell lines, but low transfection efficiency and cytotoxicity limit applications in primary cells, especially primary neurons. The application of "naked" siRNA has been previously used successfully in nematodes and mammals in vivo. We therefore evaluated the effects of non-cationic-lipid-based siRNA application to primary hippocampal neuron cultures. "Naked" siRNA was bound to the cell surface and was taken up into endosomes. No significant silencing effect of endogenous or reporter genes was observed, rather application of "naked" siRNA was accompanied by a moderate downregulation of metabolic activity in culture. We postulate that endosomal degradation of "naked" siRNA in neurons prevents the induction of significant RNAi-mediated mRNA-downregulation and is accompanied by a global impairment of the cell metabolism. Transfection methods circumventing the endosomal pathway therefore might prove useful for siRNA transduction of primary neurons.
Lingor
Paul
P
Department of Neurology, University of Göttingen, Faculty of Medicine, S2-Laboratory, Waldweg 33, 37073 Göttingen, Germany. plingor@gwdg.de
Michel
Uwe
U
Schöll
Ulrike
U
Bähr
Mathias
M
Kügler
Sebastian
S
D000818:Animals; D002232:Carbocyanines / Q000737:chemistry / Q000378:metabolism; D002478:Cells, Cultured; D011992:Endosomes / Q000378:metabolism*; D005260:Female; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins; D006624:Hippocampus / Q000166:cytology; D007202:Indicators and Reagents; D008081:Liposomes; D008164:Luminescent Proteins / Q000096:biosynthesis / Q000235:genetics; D008247:Lysosomes / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008869:Microtubule-Associated Proteins / Q000096:biosynthesis / Q000235:genetics; D009474:Neurons / Q000378:metabolism*; D011247:Pregnancy; D034741:RNA, Small Interfering / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D014161:Transduction, Genetic; D014162:Transfection / Q000379:methods*
D002232:Carbocyanines; D007202:Indicators and Reagents; D008081:Liposomes; D008164:Luminescent Proteins; D008869:Microtubule-Associated Proteins; D034741:RNA, Small Interfering; C089187:cyanine dye 3; D049452:Green Fluorescent Proteins