Friday, April 14, 2006
Neuromics and AlphaGenix work closely with Dr. Mark Behlke, Vice President of Molecular Genetics at Integrated DNA Technologies to develop optimum strategies for the design of RNAi molecules.
This is followed by developing in vitro studies on primary cells, which are required in order to design in vivo strategies to inhibit targeted gene expression. While transfection studies enhance our understanding of RNAi technology, we believe that it is vital to demonstrate validation by including sufficent controls as well as demonstrating knockdown of endogenous message.
We will be routinely publishing new publications to this blog. In this way, researchers are able to get leading papers and reviews in these fields of research as well as having the ability to bring their own expertise to the table. Indeed, science is a collective effort and we encourage you as colleagues to help all of us keep on the cutting edge.
Stephen Hall, Ph.D.
Thursday, April 13, 2006
Neuromics and Alphagenix would like to announce our RNAi Transfection Alliance. We will be partnering with IDT to develop and commercialize better ways to deliver RNAi to Glia, Neurons and the Nervous System.
Here are some related publications:
Progress Towards in vivo Use of siRNAs
siRNAi: Applications in Functional Genomics and Potential as Therapeutics
Assembly and Function of RNAi Silencing Complexes
Synthetic dsRNA Substrates Enhance RNAi Potency and Efficacy
In vivo Publications:
Luo MC, Zhang DQ, Ma SW, Huang YY, Shuster SJ, Porreca F, Lai J (2005). An efficient intrathecal delivery of small interfering RNA to the spinal cord and peripheral neurons. Molecular Pain 2005, 1:29.
Priti Kumar, Sang Kyung Lee, Premlata Shankar, N. Manjunath (2006) A Single siRNA Suppresses Fatal Encephalitis Induced by Two Different Flaviviruses. PLOS Medicine, Volume 3 Issue 4 APRIL 2006
Monday, April 03, 2006
Priti Kumar1, Sang Kyung Lee2, Premlata Shankar1*, N. Manjunath1*
1 The CBR Institute for Biomedical Research and Department of Pediatrics, Harvard Medical School, Boston, Massachusetts, United States of America, 2 Department of Bioengineering, Hanyang University, Seoul, Korea
Japanese encephalitis virus (JEV) and West Nile virus (WNV) are neurotropic flaviviruses that can cause acute encephalitis with a high fatality rate. Currently there is no effective treatment for these infections.
Methods and Findings
We tested RNA interference (RNAi)-based intervention to suppress lethal JE and WN encephalitis in mice. To induce RNAi, we used either lentivirally expressed short hairpin RNA (shRNA) or synthetic short interfering RNA (siRNA). As target, we selected the cd loop-coding sequence in domain II of the viral Envelope protein, which is highly conserved among all flaviviruses because of its essential role in membrane fusion. Using as a target a species-specific sequence in the cd loop that is conserved only among the different strains of either JEV or WNV, we could achieve specific protection against the corresponding virus. However, by targeting a cross-species conserved sequence within the cd loop, we were able to protect mice against encephalitis induced by both viruses. A single intracranial administration of lentivirally delivered shRNA or lipid-complexed siRNA before viral challenge or siRNA treatment after viral challenge was sufficient for protection against lethal encephalitis.
RNAi-based intervention affords near complete protection from both JEV- and WNV- induced encephalitis in mice. Our results show, to our knowledge for the first time, that siRNA can be used as a broad-spectrum antiviral agent for treating encephalitis caused by multiple related viruses.
Funding: This work was supported by NIH grant U19 AI 056900 to NM and PS.
Competing Interests: The authors have declared that no competing interests exist.
Academic Editor: Clifford Lane, National Institutes of Health, United States of America
Received: September 20, 2005; Accepted: December 16, 2005; Published: February 14, 2006
Copyright: © 2006 Kumar et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Relevant to i-Fect (TM)
"Although there is a report of the successful use of naked siRNA targeting the pain-related cation channel P2X3 to treat chronic neuropathic pain in a rat model , other studies suggest that naked siRNA is poorly taken up by brain parenchymal cells [29,30]. Recently, a cationic lipid formulation, i-Fect (Neuromics) has been found to deliver siRNA into neuronal cells without toxicity (Dr. Josephine Lai, University of Arizona Health Sciences Center, personal communication). We tested if synthetic FvEJ siRNA (siFvEJ) complexed with i-Fect can protect mice against viral encephalitis. After confirming that i-Fect can transduse siFvEJ siRNA as efficiently as lipofectamine to inhibit JEV infection in Neuro 2a cells (Figure 3A), we infected mice by IC injection with JEV and, after allowing 30 min for viral adsorption, injected the synthetic siFvEJ or control luciferase siRNA (siLuc) complexed with i-Fect at the same site. All mice injected with siLuc died by day 5, whereas all of the siFvEJ-injected mice survived indefinitely (Figure 3B), suggesting that i-Fect can deliver siRNA into neuronal cells and result in protection that is similar to the lentivirally delivered shRNA."
Saturday, April 01, 2006
Thank you Kim Carnes, UIUC, for the image and protocol for the Ret staining of spermatagonial stem cells.
Depraffinize and hydration
Peroxidase blocking - 30 minutes in 0.06% H2O2 in methanol.
Antigen retrieval - 0.01M Citrate buffer (pH6.0) in microwave (10 minutes, power level 7)
Serum blocking - horse serum (unfiltered normal, diluted1:100) for 30 minutes.
Primary antibody - Neuromics Ret antibody (1:200), overnight at 4º C in a moisture chamber.
Secondary Ab - this was a horse universal biotinylated ab that came in a kit (with the above horse serum) diluted 1:50 with PBS and horse serum (also 1:50) for 60 minutes at room temp.
ABC incubation (Vector ABC Elite kit) - 30 minutes.
Colorization with DAB, counterstain with hematoxylin followed by dehydration.
We use PBS rinses between steps.
Thank you Alex Vila, UCLA, for your image and protocol for the SHANK 1a C-terminus staining of retinol tissue
Species: Mouse C57B6
Fixation: 15 minute 4% paraformaldehyde fixation
Antibody dilution: 1:800 overnight.
Secondary Alexa 568 1:500 for 45 minutes
Tissue preparation: Blocked for 2 hrs at room temp in 10% normal goat serum and
Triton X-100 (0.5%) in 0.1 M Phosphate buffered saline (PBS)
Washed: 3X 0.1 M PBS between primary and secondary step.
Mounted tissue with Paramount.
Confocal: Images are 2048 X 2048, 12 bit, collected with a 40X water objective, 10 stacks 0.55 um per stack