Pushparaj, P.N., H'ng, S.C. and Melendez Romero, A.J. (2008) Refining siRNA in vivo transfection: silencing SPHK1reveals its key role in C5a-induced inflammation in vivo. International Journal of Biochemistry and Cell Biology, 40(9), pp. 1817-1825. (doi: 10.1016/j.biocel.2008.01.015)
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Publisher's URL: http://dx.doi.org/10.1016/j.biocel.2008.01.015
Abstract
The transfection of siRNA in vivo is essential for the study of gene functions, target validation, and for gene therapy. However, the successful delivery of siRNA in whole organisms is still very difficult to achieve. A high-pressure delivery technique, called the “hydrodynamics” method, has been used for siRNA transfection in mice. However, it is a method based on a high-speed and high-volume of i.v. injection, which makes it very difficult to implement in vivo, due to vascular breakage. Here, we systematically investigated ways to optimize the siRNA delivery, in order to avoid strong side effects, while achieving a high-efficiency siRNA-gene knockdown. We show here that the amount of siRNA delivered is crucial, as using too little or too much siRNA minimizes the knockdown effect. We demonstrate that by carefully identifying an optimal-minimal volume, and an optimal amount of siRNA, we achieve a high knockdown effect, with a 100% survival rate. We have previously shown that SphK1 plays a key role in anaphylatoxin (C5a) signaling in neutrophils and macrophages. Our approach, optimizing the dosage of siRNA, allowed us to successfully silence our target gene-product (SphK1) in vivo, and enabled us to validate SphK1 as a key player in our in vivo model of C5a-induced acute peritonitis and systemic inflammation including multi-organ damage, demonstrating that this improved siRNA-silencing method not only allowed us to identify SphK1 as a key therapeutic target, but brings us a step closer to the usage of siRNA for therapeutic intervention.
Item Type: | Articles |
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Status: | Published |
Refereed: | Yes |
Glasgow Author(s) Enlighten ID: | Pushparaj, Dr Peter and Melendez Romero, Dr Alirio |
Authors: | Pushparaj, P.N., H'ng, S.C., and Melendez Romero, A.J. |
College/School: | College of Medical Veterinary and Life Sciences > School of Infection & Immunity |
Journal Name: | International Journal of Biochemistry and Cell Biology |
Publisher: | Elsevier |
ISSN: | 1357-2725 |
ISSN (Online): | 1878-5875 |
Published Online: | 20 January 2008 |
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