RNA interference by osmotic lysis of pinosomes: liposome-independent transfection of siRNAs into mammalian cells

Abstract

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 µM, 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.

Delivering siRNAs

Implementation of the phenomenon of RNA interference as an experimental tool is fraught with technical challenges, including transfection of cells with biologically active RNA. Gruber et al. (p. 96) have adapted the process of osmotic lysis of pinosomes, traditionally used primarily for introduction of proteins and dyes, to deliver siRNA (short interfering RNA) into mammalian cells. Using fluorescently labeled siRNA and confocal microscopy, the authors show that siRNA in hypertonic medium is taken up by a variety of cell types by the process of pinocytosis and that the siRNAs are released from pinosomes into the cytoplasm when cells are subsequently “shocked” by treatment with hypotonic medium. They obtain silencing efficiencies of 60%–90% using the process that is dependent on uptake time and siRNA concentration. This novel approach provides a valuable alternative to existing techniques for transfecting cells with siRNA.

Acknowledgments

J.G. and G.B. contributed equally to this study. G.B. thanks Professor Petra Schwille for helpful discussions and technical support. The ConfoCor2 LCM510 microscope system was kindly provided by Carl Zeiss (Jena, Germany). This study was supported in part by the Max Planck Society and in part by the Bio-Future program (German Ministry of Research and Education).