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Journal of Biomedicine and Biotechnology
Volume 2009 (2009), Article ID 410260, 7 pages
http://dx.doi.org/10.1155/2009/410260
Methodology Report

Efficient siRNA Delivery by the Cationic Liposome DOTAP in Human Hematopoietic Stem Cells Differentiating into Dendritic Cells

Dipartimento di Medicina Sperimentale e Scienze Biochimiche, Sezione di Biochimica e Biologia Molecolare, Università di Perugia, Via del Giochetto, 06100 Perugia, Italy

Received 11 December 2008; Accepted 20 March 2009

Academic Editor: Bibekanand Mallick

Copyright © 2009 Sabata Martino et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

RNA interference technology is an ideal strategy to elucidate the mechanisms associated with human hematopoietic stem cell differentiation into dendritic cells. Simple manipulations in vitro can unequivocally yield alloreactive or tolerogenic populations, suggesting key implications of biochemical players that might emerge as therapeutic targets for cancer or graft-versus-host disease. To knockdown proteins typically involved in the biology of dendritic cells, we employed an siRNA delivery system based on the cationic liposome DOTAP as the carrier. Freshly-isolated cells were transfected with siRNA for cathepsin S with negligible cytotoxicity and transfection rates (>60%) comparable to the efficiency shown by lentiviral vectors. Further, cathepsin S knockdown was performed during both cell commitment and through the entire 14-day differentiation process with repeated transfection rounds that had no effect per se on cell development. Tested in parallel, other commercially-available chemical reagents failed to meet acceptable standards. In addition to safe and practical handling, a direct advantage of DOTAP over viral-mediated techniques is that transient silencing effects can be dynamically appraised through the recovery of targeted proteins. Thus, our findings identify DOTAP as an excellent reagent for gene silencing in resting and differentiating cells, suggesting a potential for applications in related preclinical models.