Kv Channels and Acute to Chronic Pain Transistion

i-Fect ^TMis a Proven Tool for Gene Manipulation in Studying All Types of Pain

I previously posted on use of our i-Fect Transfection Kit to silence Kv Channels Receptors. This has enabled researchers to study the role of these receptors in vitro and in vivo (see:i-Fect™ Delivers Your siRNA Payload).

Sample Data

Figure: Figures. siRNA-mediated knockdown of Kv1.1 expression in thoracic DRG significantly increased gastric sensitivity in naive adult rats. (A) Western blots showed a significant decrease in Kv1.1 protein in thoracic DRG (T8–T12) after intrathecal treatment with Kv1.1 siRNA but not with control siRNA. siRNA treatment did not alter TrpV1 expression (n = 5 rats each; *P < .01 vs control siRNA). (B) Naive rats treated with Kv1.1 siRNA showed a significant increase in VMR to gastric distention (n = 5 rats each, compared with pretreatment baseline; *P < .05). (C) Treatment with control siRNA had no significant effect on gastric hypersensitivity. (D) Patch clamp recordings from freshly dissociated gastric DRG neurons from FD-like and PND 10 saline-treated littermate controls showed a significant decrease in rheobase in FD-like rats (*P < .05), and (E) a significant increase in the number of action potentials elicited by current injection at 3× the rheobase in gastric DRG neurons from FD-like rats (*P < .05). (F) Sample voltage vs time traces showing action potentials evoked at ×1, ×2, and ×3 rheobase. The patch clamp data were obtained from 16 cells from 5 PND 10 saline control rats and 19 cells from 5 FD-like rats

I am pleased to share with you a new reference detailing how research use i-Fect to optimize and deliver euchromatic histone-lysine N-methyltransferase-2 (G9a) siRNA. This brings the number of publications referencing use of our Transfection Kits to over 45: Geoffroy Laumet, Judit Garriga, Shao-Rui Chen, Yuhao Zhang, De-Pei Li, Trevor M Smith, Yingchun Dong, Jaroslav Jelinek, Matteo Cesaroni, Jean-Pierre Issa & Hui-Lin Pan G9a is essential for epigenetic silencing of K+channel genes in acute-to-chronic pain transition. Nature Neuroscience (2015) doi:10.1038/nn.4165.

The authors report: "Selective knockout of the gene encoding G9a in DRG neurons completely blocked K+ channel silencing and chronic pain development after nerve injury. Remarkably, RNA sequencing analysis revealed that G9a inhibition not only reactivated 40 of 42 silenced genes associated with K+ channels but also normalized 638 genes down- or upregulated by nerve injury."

I will continue to post here new and unique solutions and related referencing for our Gene Expression Analysis Tools.

Introducing Sleeping Beauty

New Technology for Gene Transfer From Delivery to Stable Expression
Neuromics has a successful track record of helping our clients delivery siRNA, miRNA, Plasmids and other oligos in vitro and in vivo with our Transfection Kits...But my vision with our cell based assay solutions has always been to provide engineered cells and plasmids modified to study your genes of interest. I am pleased to announce we are working with Smart Cell /B-MoGen Technologies to make this happen. We now can provide:

Gene Transfer and Expression Products Leveraging the Sleeping Beauty Technology

Images: B-MoGen Transposon exhibiting stable expression of five fluorescent genes. 

Advantages of Sleeping Beauty Transposon System:
· Delivery method is time and cost effective compared to lentiviral delivery.
· Increased cargo-capacity when compared to lentiviral delivery.
· Safest insertion profile of all gene transfer methods.
· Commonly integrated as a single copy.
Custom vector design and assembly, including multi-gene (up to 6) vectors.
We are in the process of formulating standard offerings. In the meantime, I am positioned to offer favorable pricing and terms to early adopters of our Sleeping Beauty Solutions. Please contact me directly pshuster@neuromics.com or 612-801-1007. We can together determine your needs and desired outcomes and provide a statement of work with pricing, project milestones and delivery.

Silencing Cytokines in-vivo with i-Fect

Knocking Down Cytokines to Study Pain Response

We have many unique applications published by researchers using our Transfection Kits in vitro and in vivo. Here researchers simultaneously silence 3 immune/inflammatory response cytokines in vivo: Byung Moon Choi, Soo Han Lee, Sang Mee An, Do Yang Park, Gwan Woo Lee, and Gyu-Jeong Noh. The time-course and RNA interference of TNF-α, IL-6, and IL-1β expression on neuropathic pain induced by L5 spinal nerve transection in rats. Korean J Anesthesiol. 2015 Apr;68(2):159-169. English. Published online March 30, 2015. http://dx.doi.org/10.4097/kjae.2015.68.2.159.

Protocol: RNAs were administered as described, with modifications [11]. A cocktail of siRNA simultaneously targeting TNF-α (Silencer® Select siRNA; s128522, Ambion, Austin, TX, USA), IL-6 (Silencer® Select siRNA; s217844, Ambion, Austin, TX, USA) and IL-1b (Silencer® Select siRNA; s127941, Ambion, Austin, TX, USA), as well as a control siRNA (Silencer® Negative Control #1 siRNA; Cat #4635, Ambion, Austin, TX, USA), were prepared immediately prior to administration by mixing the RNA (200 µM) with the transfection reagent, i-Fect™ (Neuromics, Minneapolis, MN, USA), in a ratio of 1 : 5 (w : v). At this ratio, the final RNA/lipid complex concentration was 2 µg in 5 µl for each cytokine siRNA and 6 µg in 15 µl for the control siRNA. The cytokine siRNAs were combined and they and the control siRNA (15 µl each) were delivered to the lumbar region of the spinal cord via the intrathecal catheters. Injections were given daily on 5 consecutive days (-1, 0, 1, 2, 3 d after L5 SNT.

The changes in mechanically induced allodynia and hyperalgesia in the rats surviving for 6 d after SNT are shown in figure. Allodynia and hyperalgesia were lower in the COCK group than in the CON group by 2 d after SNT (P < 0.05) and the difference was maintained for the duration of the experiment.

Figure: The time course of mechanical allodynia (A) and hyperalgesia (B) in the ipsilateral hind paw of rats undergoing L5 spinal nerve transection (SNT) after the administration of control siRNA (CON group) or a cocktail of small interfering RNAs (siRNA) targeting TNF-α, IL-6 and IL1-β (COCK group). The data on the rats surviving for 6 d after SNT are expressed as mean ± SE. -1: 1 d prior to SNT, 0: the day of L5 SNT, 1, 2, 4 and 6: 1, 2, 4 and 6 d after L5 SNT. *P < 0.05 vs. CON group at each time point. MPE: maximal possible effect. The cut-off values for mechanical allodynia and hyperalgesia are 30 g and 250 g, respectively.

We will continue to post new applications and methods published by researchers using our Transfection Kits.

HDAC2 and Anxiety in Alcoholism

The Impact of HDAC2 Gene Expression on Anxiety

Our i-Fect Transfection Kit continues to be a potent tool for testing the impact of altered gene expression on behavior. see: SACHIN MOONAT. The Role of Amygdaloid Chromatin and Synaptic Remodeling in Anxiety and Alcoholism. THESIS Submitted as partial fulfillment of the requirements for the degree of Doctor of Philosophy in Neuroscience in the Graduate College of the University of Illinois at Chicago, 2014.

The author hypothesized that increased HDAC2 would have a positive impact on anxiety in alchohol preferring (P) rats. Specifically, HDAC2-induced histone modifications in the amygdala may play a role in the regulation of synaptic plasticity that may underlie the behavioral phenotypes of P rats. Furthermore, it could be possible that exogenous manipulation of HDAC2 levels in the amygdala may have an effect on anxiety-like behaviors and alcohol preference in P rats.

Figure 1. Chromatin remodeling via histone acetylation and DNA methylation regulates gene transcription associated with changes in synaptic plasticity. During gene transcriptional processes, the chromatin structure associated with DNA to be transcribed is in a relaxed chromatin conformation due to hyperacetylation of histone proteins and hypomethylation of DNA, which allows access to transcriptional machinery. This relaxed chromatin structure results in increased gene transcription, which in neurons may cause increased expression of synaptically active proteins that result in the positive modulation of synaptic plasticity, such as increased dendritic spine density (DSD). DNA methyltransferase (DNMT) methylates DNA at CpG islands, leading to hypermethylated DNA and recruiting of methyl-CpG binding domain protein (MBD) complexes which block binding of transcriptional machinery. The MBD complex can in turn recruit histone deactylases (HDAC) which remove acetyl groups from histone proteins resulting in chromatin condensation thereby decreasing gene transcription. HDACs and histone acetyltransferases (HAT) control the histone acetylation profile, such that HDACs remove acetyl groups and HATs add acetyl groups to histone proteins. In this manner, increased HDAC expression results in hypoacetylation of histones leading to a condensed chromatin structure. Chromatin condensation resulting from HDAC-induced histone deacetylation or DNMT-induced DNA methylation causes reduced gene transcription. In neuronal cells, the reduction in gene transcription may be associated with decreased expression of synaptically active proteins and negative modulation of synaptic plasticity, such as reduced DSD. Treatment with DNMT inhibitors or HDAC inhibitors may block these enzymatic processes and return chromatin to a relaxed state, resulting in increased gene transcription and synaptic plasticity (Moonat and Pandey, 2012).

Methods: P rats that had been previously cannulated for delivery of solutions directly into the CeA were infused with either HDAC2 siRNA, control siRNA or vehicle. The siRNAs were dissolved in iFect solution (Neuromics, Edina, MN), a cationic lipid-based transfection solution, such that the final concentration of the solution was 2 µg/µL. The sequence of the HDAC2 siRNA was as follows: 5’-CAAGUUUCUACGAUCAACATT-3’; 5’- UAUUGAUCGUAGAAACUUGAT-3’. Some of the HDAC2 siRNA (Qiagen, Valencia, CA) had been modified to include a 5’ Alexa Fluor-488 fluorescent probe in order to determine the transfection efficiency and cellular localization of transfection. The control siRNA used was the AllStars Negative Control siRNA (Qiagen), which shows no homology to any known mammalian gene. To prepare the vehicle, RNase-free water was dissolved in the iFect solution in place of any siRNA. The solutions (0.5 µL) were infused bilaterally into the CeA of P rats using an automatic infusion pump which resulted in a dose of 1 µg of siRNA per side. The automatic pump was attached to a microdialysis probe which seated in the guide cannula and extended 3 mm past the tip of the cannula into the CeA.

For the experiments which looked at the anxiolytic effect of HDAC2 siRNA infusion, P rats were infused with either HDAC2 siRNA, control siRNA or vehicle at the end of the light cycle. 16 hours after the infusion, the rats were tested for anxiety-like behaviors. Immediately following behavioral testing, rats were anaesthetized and brains were collected for further analysis. For the voluntary drinking experiment, P rats were infused with either HDAC2 siRNA or vehicle when the bottles were changed following the third day of 9% ethanol exposure. The rats continued to be monitored for the intake of 9% ethanol for 7 days following the infusion. After the final day of voluntary drinking, the rats were anaesthetized for collection of brains and blood to confirm the cannula position and the blood alcohol levels, respectively.

Figure. The effects of HDAC2 siRNA Infusion into the CeA of P rats on voluntary ethanol consumption as measured by the two-bottle free choice paradigm. Monitoring the voluntary ethanol consumption of alcohol-preferring (P) rats via the two bottle free choice paradigm following infusion of vehicle or histone deacetylase isoform 2 (HDAC2) siRNA into the central amygdala (CeA) demonstrates that high HDAC2 levels may mediate the high alcohol drinking behaviors of P rats. P rats were given access to water and 7% ethanol followed by water and 9% ethanol. On the sixth day of ethanol access P rats received infusion of vehicle or HDAC2 siRNA and consumption of water and 9% ethanol were monitored for sevnfusion. Total fluid intake did not significantly differ between the groups. Values are represented as the mean ± SEM of the ethanol consumption (g / kg / day) and total fluid intake (mL) plotted daily for n=6 rats per treatment group. *Significantly different between the groups.

This data suggest reduction of HDAC2 levels in the CeA leads to reduced DSD associated with a reduction in anxiety-like behaviors and alcohol preference in P rats and could prove to have therapeutic value.

Neuromics' Transfection Kits-Genes Studied

Delivering siRNA, miRNA, Plasmids and Viral Vectors for Gene Expression Analysis.

I have shared the many genes researchers have studied using our Transfection Kits. These include: β-arrestin, ABCA, ASIC, β-arrestin, CAV1.2, CX3CR1, DOR, EHDAC2, LOVL4, IKBKAP, K+-ATPase, KV1.1, KV9.1 , neuroligin 2, The β3 subunit of the Na+,K+-ATPase, NTS1, NAV1.8, NTS1, NOV, Raf-1, RANK, SNSR1, hTert, NOV, Survivin, TLR4, Troy and TRPV1 and More!

We can now add GPNMB to this list: Lili Hou, Yanfeng Zhang, Yong Yang, Kai Xiang, Qindong Tan, Qulian Guo. Intrathecal siRNA Against GPNMB Attenuates Nociception in a Rat Model of Neuropathic Pain. Journal of Molecular Neuroscience. July 2014...Ten micrograms of siRNA1- GPNMB dissolved in 30 μl i-Fect transfection reagent (Neuromics, Edina, MN, USA) was administered intrathecally once daily for 7 days, starting from 1 day before CCI surgery...

Abstract: Neuropathic pain is characterized by hyperalgesia, allodynia, and spontaneous pain. Recent studies have shown that glycoprotein nonmetastatic melanoma B (GPNMB) plays a pivotal role in neuronal survival and neuroprotection. However, the role of GPNMB in neuropathic pain remains unknown. The aim of the present study was to assess the role of GPNMB in neuropathic pain. In cultured spinal cord neurons, we used two small interfering RNAs (siRNAs) targeting the complementary DNA (cDNA) sequence of rat GPNMB that had potent inhibitory effects on GPNMB, and siRNA1-GPNMB was selected for further in vivo study as it had the higher inhibitory effect. After sciatic nerve injury in rats, the endogenous level of GPNMB was increased in a time-dependent manner in the spinal cord. Furthermore, the intrathecal injection of siRNA1-GPNMB inhibited the expression of GPNMB and pro-inflammatory factors (TNF-α, IL-1β, and IL-6) and alleviated mechanical allodynia and thermal hyperalgesia in the chronic constriction injury (CCI) model of rats. Taken together, our findings suggest that siRNA against GPNMB can alleviate the chronic neuropathic pain caused by CCI, and this effect may be mediated by attenuated expression of TNF-α, IL-1β, and IL-6 in the spinal cord of CCI rats. Therefore, inhibition of GPNMB may provide a novel strategy for the treatment of neuropathic pain.

If you would like to learn how you can optimize your gene expression analysis studies, do not hesitate to e-mail: pshuster@neuromics.com or direct line: 612-801-1007.

i-Fect Delivers IGF1 siRNA to the Mouse Brain

Intraventricular Injections Used to Study Neuronal Survival During Post Natal Development

Neuromics' i-Fect^TM Transfection Kit continue to be successfully used to deliver siRNA, shRNA and miRNA to cell cultures and the CNS in vivo (via intrathecal, epidural and intraventricular injection). Genes studied include: ABCA, ASIC, β-arrestin, CAV_1.2, IGF1, CX3CR1, DOR, ELOVL4, IKBKAP, K+-ATPase, K_V1.1, K_V9.1 ,The β3 subunit of the Na+,K+-ATPase, NTS1, NAV_1.8, NTS1, NOV, Raf-1, RANK,SNSR1, hTertTRPV1 NOV, Survivin, TLR4, Troy and TRPV1. Related Publications.

It is always an honor for one our products to be referenced in one of the Nature publications. Here researchers use i-Fect to study the impact of microglial derived IGF1 silencing on Neuronal Survival: Masaki Ueno,Yuki Fujita, Tatsuhide Tanaka, Yuka Nakamura, Junichi Kikuta, Masaru Ishii, Toshihide Yamashita. Layer V cortical neurons require microglial support for survival during postnatal development. Nature Neuroscience 16, 543–551 (2013) doi:10.1038/nn.3358. ...vehicle (PBS) was delivered intraventricularly through the cisterna magna with a glass pipette while P3 mice were cold anesthetized. Igf1 siRNA (stealth siRNA, Invitrogen) or Igfbp5 siRNA with i-Fect reagent (Neuromics) was delivered intraventricularly through the cisterna magna at P3 twice with a 12-h interval...

Images: (a) Igf1 expression (blue) and Iba1-positive microglia (brown) in P5 brain. Scale bar represents 400 μm. (b) Magnified view of dotted square in a. Scale bar represents 50 μm. (c,d) IGF1Ra expression (red) in CTIP2-positive (c) and SATB2-positive (d) layer V neurons at P5. Scale bar represents 50 μm. (e) IGF1 protein levels in medium from cultured cortical neurons, microglia and neurons with microglia in transwell systems detected by enzyme-linked immunosorbent assay (ELISA). **P < 0.01 (neuron, microglia + neuron, n = 5; microglia, n = 6 experiments; one-way ANOVA followed by Tukey-Kramer test). (f) The number of cleaved caspase-3–positive neurons in transwell systems treated with LY294002 or H-1356 or transfected to microglia with Igf1 siRNA. *P < 0.05, **P less than  0.01 (n = 3 experiments, one-way ANOVA followed by Tukey-Kramer test). (g) Neuronal phospho-AKT expression in cultured cortical neurons and those with microglia in transwell system. (h) TUNEL-positive cells in H-1356–treated cortex (36 h after treatment). Scale bar represents 100 μm. (i) The number of TUNEL-positive apoptotic cells in each layer in vehicle- (phosphate-buffered saline, PBS) or H-1356–treated mice (36 h after injection). *P < 0.05 (n = 4 brains, one-way ANOVA followed by Tukey-Kramer test). (j) Cleaved caspase-3–labeled cells (red) expressing CTIP2 (green, arrowheads). Scale bar represents 100 μm. (k,l) TUNEL-positive cells in the cortex treated with control or Igf1 siRNA (48 h after treatment). Scale bar represents 100 μm. (m) The number of TUNEL-positive apoptotic cells in each layer in control siRNA– and Igf1 siRNA–treated mice. **P less than 0.01 (n = 5 brains, one-way ANOVA followed by Tukey-Kramer test). Error bars represent s.e.m.

I will continue to post new developments and successes.

BDNF and Recovery of Motor Function After Brian Injury

Researchers use i-Fect^TM to Study BDNF Silencing in Mice

Brain injury that results in an initial behavioural deficit is frequently followed by spontaneous recovery. The intrinsic mechanism of this functional recovery has never been fully understood. Here, we show that reorganization of the corticospinal tract induced by target-derived brain-derived neurotrophic factor is crucial for spontaneous recovery of motor function following brain injury:  Masaki Ueno, Yasufumi Hayano1, Hiroshi Nakagawa and Toshihide Yamashita. Intraspinal rewiring of the corticospinal tract requires target-derived brain-derived neurotrophic factor and compensates lost function after brain injury. Brain (2012) doi: 10.1093/brain/aws053. ... motor cortex at 14 days after the injury, using i-Fect™ transfection reagents (Neuromics) according to the manufacture's instruction .

Findings Overview: After destruction of unilateral sensorimotor cortex, intact-side corticospinal tract formed sprouting fibres into the specific lamina of the denervated side of the cervical spinal cord, and made new contact with two types of spinal interneurons—segmental and propriospinal neurons. Anatomical and electrophysiological analyses revealed that this rewired corticospinal tract functionally linked to motor neurons and forelimb muscles. This newly formed corticospinal circuit was necessary for motor recovery, because transection of the circuit led to impairment of recovering forelimb function. Knockdown of brain-derived neurotrophic factor in the spinal neurons or its receptor in the intact corticospinal neurons diminished fibre sprouting of the corticospinal tract. Our findings establish the anatomical, functional and molecular basis for the intrinsic capacity of neurons to form compensatory neural network following injury.

We will continue to post new references to our Transfection Kits.

siRNA Delivery Group on Linkedin

I wanted to make readers aware of an excellent discussion group on Linkedin named "siRNA Delivery". Included are tip, updates on commercialization and key publications.

Here're some examples:

• Alnylam Announces Publication of Pre-clinical Results with ALN-HTT, an RNAi Therapeutic for the Treatment of Huntington’s Disease, in Experimental Neurology
• Delivering siRNA to Neurons
• Life Technologies develops new drug delivery technology
• Recent experiments demonstrate a novel method of liposomal encapsulation of siRNA/RNA/DNA at 4C, could be a promising method to minimize degradation.

Happy reading.

Direct Application of siRNA for In Vivo Pain Research

My friends at McGill University have recently published in depth methods for using siRNA to study pain. Dr. Philippe Sarret have done extensive work delivering siRNA + i-Fect^TM in vivo for gene expression analysis of specific pain receptors.

Here's a link to the book chapter from Springer Protocols:

25. Direct Application of siRNA for In Vivo Pain Research
By: Philippe Sarret , Louis Doré-Savard, Nicolas Beaudet
Affiliation(s): (1) Department of Physiology and Biophysics, Faculty of Medicine and Health Sciences, Université de Sherbrooke, Sherbrooke, QC, Canada
Book Title: RNA Interference: From Biology to Clinical Applications
Series: Methods in Molecular Biology Volume: 623 Pub. Date: May-01-2010 Page Range: 383-395 DOI: 10.1007/978-1-60761-588-0_25

Abstract: Pain is the new burden of the twenty-first century, raising enormous socio-economic costs to developed and underdeveloped countries. Chronic pain is a central nervous system (CNS) pathology, affecting a large proportion of the population. Morphine and its derivatives are still the golden clinical standards for treating pain although they induce severe side effects. To this day, we still have poor understanding of nociceptive pain and its underlying complex mechanisms; furthermore, novelty in clinical analgesics is lacking.

RNA interference technologies are promising both for pain research and treatment. This genetic approach will likely provide new insights into pain mechanisms and eventually offer nonpharmacological therapeutic approaches. In vivo research is thus crucial to reach this goal. Preclinical studies on rodents are necessary to validate small interfering RNA (siRNA) candidates and to target precise physiological pain modulators. Aiming treatment at the CNS is delicate work, and here we will describe how to perform adequate pain research using siRNA, including siRNA preparation and injection, animal behavioral models, and CNS tissue collection.

i-Fect and More Knockdown Success

Neuromics' Customers have published their success using i-Fect^TM for gene expression studies Genes studied include: DOR, hTERT, The β3 subunit of the Na+,K+-ATPase, rSNSR1, NTS1. NAV1.8 and more.

We are now pleased to add knockdown L Calcium Channel Subtypes to study differential effects of neuropathic pain.

In this study researchers showed specific knockdown of CaV1.2 in the spinal dorsal horn reversed the neuropathy-associated mechanical hypersensitivity and the hyperexcitability and increased responsiveness of dorsal horn neurons. Intrathecal application of anti-CaV1.2 siRNAs confirmed the preceding results.

Here's a link to the related pub: Pascal Fossat, Eric Dobremez, Rabia Bouali-Benazzouz, Alexandre Favereaux, Sandrine S. Bertrand, Kalle Kilk, Claire Léger, Jean-René Cazalets, Ülo Langel, Marc Landry and Frédéric Nagy. Knockdown of L Calcium Channel Subtypes: Differential Effects in Neuropathic Pain. The Journal of Neuroscience, January 20, 2010, 30(3):1073-1085; doi:10.1523/JNEUROSCI.3145-09.2010

We used siRNA targeting several splice variants of CaV1.2 ("Silencer Select Pre-designed and Validated siRNA", Ambion). They consisted of a pool of two 21 nt duplex. siRNAs were selected to target two distinct CaV1.2 mRNA regions to enhance silencing. The antisense sequences were as follows: UCUAUUGUCAUAUCGCAGG and UAUCCGAACAGGUAUAGAG.

In contrast to PNA, these siRNAs targeted the 5'-coding region. Mismatch siRNA was a nontargeting 21 nt duplex designed as a negative control. The siRNAs (2 µg) were solubilized in 10 µl of reagent i-Fect (Neuromics) following Neuromics instructions and published protocol (Luo et al., 2005), and applied intrathecally according to the same protocol as for the PNA.

Delivering siRNA in Mice for Studying Opioid-Induced Hyperalgesia

Researchers have successfully delivered siRNA in-vitro and in-vivo using Neuromics' i-Fect ™ siRNA Transfection Reagent. Gene expression studies include: DOR, hTERT, The β3 subunit of the Na+,K+-ATPase, rSNSR1, NTS1. NAV1.8 and more.

Here's a link to all transfection publications: Transfection Kit Pubs

We are pleased to present yet another study and related publication. This includes one of the first successful delivery of siRNA in mice using i-Fect ™ :

Yan Chen, Cheng Yang, and Zaijie Jim Wang. Ca2+/Calmodulin-Dependent Protein Kinase II Is Required for the Initiation and Maintenance of Opioid-Induced Hyperalgesia. The Journal of Neuroscience, January 6, 2010, 30(1):38-46; doi:10.1523/JNEUROSCI.4346-09.2010.

...KN93 and KN92 were administered intrathecally by percutaneous puncture through the L5-L6 intervertebral space, as described previously (Hylden and Wilcox, 1980; Chen et al., 2009). A lateral tail flick was considered as success of the intrathecal injection. To inhibit CaMKII, CaMKII was targeted by small interfering RNA (siRNA). Four days after morphine pellet implantation, mice were treated with CaMKII siRNA (5'-CACCACCAUUGAGGACGAAdTdT-3', 3'-dTdTGUGGUGGUAACUCCUGCUU-5') (Zayzafoon et al., 2005) or Stealth RNAi negative control (Invitrogen) (2 µg, i.t., twice per day for 3 consecutive days). These oligos were mixed with the transfection reagent i-Fect (Neuromics), in a ratio of 1:5 (w/v) (Luo et al., 2005). Mechanical and thermal sensitivity tests were performed daily...

Delivering Naked siRNA by Direct Injection

This blog has featured methods for delivering siRNA in vivo by intrathecal injections. These methds all highlight conjugation with novel cationic lipid based carriers like i-Fect ™. Here we highlight delivery by direct injection:

Dr. Matthew Farrer and his team, including researchers from Alnylam, have successfully demontrated the delivery of naked siRNA by direct injection. In this study, they delivered chemically modified murine and human alpha-synuclein (SNCA) siRNAs to the hippocampus by direct injection resulting in silencing of gene expression.

To learn more access:

In vivo silencing of alpha-synuclein using naked siRNA Jada Lewis, Heather Melrose, David Bumcrot, Andrew Hope, Cynthia Zehr, Sarah Lincoln, Adam Braithwaite, Zhen He, Sina Ogholikhan, Kelly Hinkle, Caroline Kent, Ivanka Toudjarska, Klaus Charisse, Ravi Braich, Rajendra K. Pandey, Michael Heckman, Demetrius M Maraganore, Julia Crook, Matthew J Farrer. Molecular Neurodegeneration 2008, 3:19 (1 November 2008).

siRNA-mediated gene silencing

Dr. Josephine Lai (Professor of Pharmacology, University of Arizona) is a pioneer in developing experimental designs and methods for delivering siRNA to the CNS for gene expression analysis.She and her team have documented these in the publication:


Modulating Sensory Systems Using RNAi(pdf - 187Kb)© 2007 Lai


For researchers desiring to effectively deliver siRNA to the CNS for gene expression analysis of specific receptors, this publication offers proven methods. These include:

• The Choice of siRNA


• Choosing and Optimizing Transfection Reagents for siRNA Delivery to the Nervous System
  
• Delivery Systems-Microinjection and Infusion (using mini-osmotic pumps)
  
• Validation

We will continue to track advances by Dr. Lai and team.