RNA interference by osmotic lysis of pinosomes: liposome-independent transfection of siRNAs into mammalian cells.
The osmotic lysis of pinosomes procedure has been adapted to deliver small interfering RNAs (siRNAs) into cells in culture. Under hypertonic conditions, siRNAs were internalized into pinosomes. A subsequent osmotic shock in hypotonic buffer disrupted the pinosomes and caused the release of siRNAs into the cell cytoplasm. Both steps could be demonstrated directly using fluorescein-labeled siRNAs and confocal laser-scanning microscopy. Uptake by the pinocytosis/osmotic lysis procedure is concentration- and time-dependent. At an siRNA concentration of 0.4 microM, treatment for 40 or 80 min results in silencing efficiencies of 60% and 90%, respectively, after 44 h. A double treatment resulted in approximately equal silencing efficiencies but in reduced viability. This method has been used on a variety of human and murine cell lines including HEK293, HeLa SS6, and SW3T3 cells. Targets such as lamin A/C and Eg5 were effectively silenced. Novel silencing data are provided for Ki67, one of the few reliable prognostic markers for tumor patients. The new procedure avoids certain technical problems encountered with commercial transfection reagents while yielding silencing efficiencies that are comparable to those obtained with liposome-mediated siRNA transfection.
Gruber
Jens
J
Max Planck Institute for Biophysical Chemistry, Goettingen, Germany.
Boese
Guido
G
Tuschl
Thomas
T
Osborn
Mary
M
Weber
Klaus
K
D016475:3T3 Cells; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D005456:Fluorescent Dyes; D020868:Gene Silencing; D006801:Humans; D008081:Liposomes; D051379:Mice; D008856:Microscopy, Fluorescence; D009995:Osmosis; D010873:Pinocytosis; D012313:RNA; D034622:RNA Interference; D014162:Transfection
D005456:Fluorescent Dyes; D008081:Liposomes; C034477:RNA primers; D012313:RNA