New BDNF and NT-3 IHC Images

Images from Dr. Matt Ramer's Lab, University of British Columbia, Courtesy of Dr. Lowell McPhail.















NT-3 Staining in Rat Dorsal Horn















BDNF Staining in Rat Cerebellum

You've Been Patient-Now We Have It!

We have finally produced a new and robust P2X3 Guinea Pig Antibody. It tested out beautifully in IHC.

The Neuromics-AlphaGenix alliance

We are thrilled to be working with Neuromics in the area of RNAi and believe it has enormous potential for genetic-based therapies for neurological disorders, neuro-oncology, viral infections and inherited genetic disorders.

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.

Best regards,

Stephen Hall, Ph.D.

RNAi Transfection Alliance

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

New Article with RNAi and i-Fect in vivo Results

A Single siRNA Suppresses Fatal Encephalitis Induced by Two Different Flaviviruses

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

ABSTRACT
Background
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.
Conclusions
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
DOI: 10.1371/journal.pmed.0030096
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 [28], 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."

More Customer Data-Ret and SHANK 1a-C-terminus

Ret-(Cat#GT15002)

Thank you Kim Carnes, UIUC, for the image and protocol for the Ret staining of spermatagonial stem cells.

Protocol

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.

SHANK 1a C-terminus

Thank you Alex Vila, UCLA, for your image and protocol for the SHANK 1a C-terminus staining of retinol tissue

Protocol

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

New Park7 Data-Thank you Amanda Ashley

We have received extremely positive feedback on the quality of our PARK7 (DJ-1) antibody. In addition to feedback, Amanda Ashley, in Dr. Legare's Laboratory, Colorado State University, resently sent us a very clean Western Blot using the PARK7 (DJ-1) antibody comparing wildtype to PARK7/DJ-1 knockout animals





Click to see full sized image.


WB: Wild type and PARK7 (DJ-1) KO animals
Data courtesy of Amanda Ashley and Dr. Marie Legare, Colorado State University.

Western conditions:
– 25mg protein from mouse brain lysate
– 1:2000 Park7 (DJ-1) antibody (Cat # RA19006)
– 12% gel
– Electrotransferred onto PVDF
– Blocked with 5% BSA in TBS-Tween
– Detected with ImmunStar HRP (BioRad)
– Dually probed for beta-actin (1:1000, Sigma)

News Release from Neuromics RNAi Collaborators

Alnylam - Developing Technology With Gene Silencing Potential
... of Dr. Frank Porreca and Dr. Josephine Lai at the University of Arizona
evaluated siRNAs that target the gene for NPY, a pain-associated peptide. ...

Annual SFN Chicago Meeting-March 3rd 2006

The meeting will be held at the Harold Washington Library in downtown Chicago.

We are pleased that Dr Tomas Hokfelt, a friend and key Neuromics' collaborator, will be presenting:

Three Decades of with Three Swedish Peptides: Substance P, NPY and Galanin

“Working with Neuromics has truly been a collaborative effort, from the initial antibody generation strategy to its development and characterization. This interaction between our groups was an important factor in our successful generation of a novel NPY Y2 antibody.” Dr. Tomas Hokfelt, Karolinska Institute

More specifics to follow...

PDF to Walter Low's Stem Cell Data

Here's the Link

Walt Low's PDF Report on Neuromics' Rat NSCs

Neural Stem Cell Data Was Generated Using Neuromics' Neural Stem Cell Kit (Cat#: NP37100)

New Antibody from The David Holtzman Lab

ABCA1

By Western blot mouse anti-ABCA1 (MO13101; clone HJ1) recognizes rat (left image), mouse (right image) and to a lesser extent human (left image) ABCA1.

Dr. David Holtman Lab-Washington University School of Medicine

http://www.neuro.wustl.edu/people/holtzman.html

Finding a Better Way

FACS/Phenotyping-A constant challenge working with neural stem and progenitors cells is to determine the percentage of cells of a given phenotype within the culture. Phenotypic characterization of the entire cell population is of the utmost importance in determining the efficacy of your cell culture conditions to generate the neural or glial cell of interest. Immunocytochemistry can get you a long way to understanding the types of cells you obtained in your culture, but it is both time consuming and qualitative in nature.
A far better approach is to analyze your cells by FACS. Although routinely employed within many branches of biology and medical technology, FACS is not a common technique used in Neuroscience. It is our hope that by offering the IntraCtyeTM kits, intracellular FACS analysis will be more accessible to and used more often by neuroscientists. We are currently offering several basic kits as well as a kit designed for rat neural progenitor cell phenotyping (IntraCyte-rNSC). The kit has been tested with our rat Neural Stem Cell Kit and is a powerful analytic tool to analyze the neural and glial cell within your culture.

The IntraCyte kit technology and the precision and accuracy of flow cytometry, enables you to get statistically significant, multi-parameter data at the single-cell level, on thousands of cells per sample in seconds. Even though we enjoy nothing more than looking at beautifully labeled neuronal cultures, we can do without the tedious and inaccurate counting of these cells in the dark!

RNAi and Huntington's

From the Cover: RNA interference improves motor and neuropathological abnormalities in a Huntington's disease mouse model.Harper SQ, Staber PD, ..., Paulson HL, Davidson BLProc Natl Acad Sci U S A 2005 Apr 19 102(16):5820-5 [abstract on PubMed] [related articles] [order article]

Excellent Neural Stem Cell Data and images

We would like to thank Drs. Walter Low and Jing Xiao of the University of Minnesota for providing these excellent images from experiments using select Neuromics' Neural Stem Cell Reagents



















Figure1 and Figure2
Customer Data generated using Neuromics' Neural Stem Cell Kit Catalog #: NP37100. Data Courtesy of Walter Low and Jing Xiao, University of Minnesota
Neural progenitor cells were plated and maintained in Proliferation Media (Figure 1). For differentiation, cells were added to 4 well chamber slides, grown in proliferation media for 24 hours and then switched to Commitment Media with various growth factors added. Cells were cultured for 14 days and then processed by immunocytochemistry. Figure 2 shows representative data from 1 experiment using bFGF/EFG (figure 2B) for differentiation.

Post Comments

As a company always looking for ways to better serve our customers, we welcome any comments, information, input on products, etc. from the world of Neuroscience. We send a link out each month to 9,000 plus researchers so any information or knowledge posted will extend its reach. Thank you!

Sam Shuster
CEO and CSO

Stem Cell to Neurons

Stem Cells to Nerve Cells
Small synthetic molecule initiates differentiation of adult neuronal rat brain cells to neurons
12/28/2005 - Adult neuronal stem cells have the potential to mature into functional nerve cells (neurons) and neuron support cells (astroglia). The mechanisms for this process are thus far not well understood. Res...
http://www.bionity.com/news/e/51090/01065/137777

Important Nature Journal Article on 27 vs 21mer potency. Comments Welcome!

http://www.nature.com/nbt/journal/v23/n2/abs/nbt1051.html

Dong-Ho Kim, Mark A Behlke, Scott D Rose, Mi-Sook Chang, Sangdun Choi & John J Rossi. Synthetic dsRNA Dicer substrates enhance RNAi potency and efficacy. Nature Biotechnology 23, 222 - 226 (2004).

Neuro-transfection

27mers vs 21mers for Effective Gene Silencing in the Nervous System

In cells, small interfering RNAs (siRNAs) are produced by enzymatic cleavage of long dsRNAs by the RNase-III class endoribonuclease Dicer. The siRNAs associate with the RNA Induced Silencing Complex (RISC) in a process that is facilitated by Dicer. Dicer-Substrate RNAi methods take advantage of the link between Dicer and RISC loading that occurs when RNAs are processed by Dicer as a heterodimer with TRBP. Traditional 21-mer siRNAs are chemically synthesized RNA duplexes that mimic Dicer products and bypass the need for Dicer processing. Dicer-Substrate RNAs are chemically synthesized 27-mer RNA duplexes that are optimized for Dicer processing and show increased potency when compared with 21-mer duplexes.

An Efficient Intrathecal Delivery of Small Interfering RNA to the Spinal Cord and Peripheral Neurons.

Luo MC, Zhang DQ, Ma SW, Huang YY, Shuster SJ, Porreca F, Lai J.We have developed a highly effective method for in vivo gene silencing in the spinal cord and dorsal root ganglia (DRG) by a cationic lipid facilitated delivery of synthetic, small interfering RNA (siRNA). A siRNA to the delta opioid receptor (DOR), or a mismatch RNA, was mixed with the transfection reagent, i-FectTM (vehicle), and delivered as repeated daily bolus doses (0.5 microgram to 4 micrograms) via implanted intrathecal catheter to the lumbar spinal cord of rats. Twenty-four hours after the last injection, rats were tested for antinociception by the DOR selective agonist, [D-Ala2,Glu4]deltorphin II (DELT), or the mu opioid receptor (MOR) selective agonist, [D-Ala2, N-Me-Phe4, Gly-ol5]enkephalin (DAMGO). Pretreatment with the siRNA, but not the mismatch RNA or vehicle alone, blocked DELT antinociception dose-dependently. The latter was concomitant with a reduction in the spinal immunoreactivity and receptor density of DOR, and in DOR transcripts in the lumbar DRG and spinal dorsal horn. Neither siRNA nor mismatch RNA pretreatment altered spinal immunoreactivity of MOR or antinociception by spinal DAMGO, and had no effect on the baseline thermal nociceptive threshold. The inhibition of function and expression of DOR by siRNA was reversed by 72 hr after the last RNA injection. The uptake of fluorescence-tagged siRNA was detected in both DRG and spinal cord. The low effective dose of siRNA/i-FectTM complex reflects an efficient delivery of the siRNA to peripheral and spinal neurons, produces no behavioral signs of toxicity, and this delivery method may be optimized for other gene targets.
The full-text, Open Access, article can be found online at:http://www.molecularpain.com/content/1/1/29.