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.

EPO Protects Against Cerebral Ischemia

Neuromics' i-Fect ^TM used to Modulate phospho-Connexin 43

In this study data suggest the protective effects of EPO on NUV injuries are highly associated with the increase of p-Cx43, which improves GJIC to reduce neurotoxic substances: Ziyi Zhoua, Xiaobai Weib, Jun Xiang, Junpeng Gao, Lixin Wang, Jinsong You, Yefeng Cai , Dingfang Caid. Protection of erythropoietin against ischemic neurovascular unit injuries through the effects of connexin43. Biochemical and Biophysical Research Communications. doi:10.1016/j.bbrc.2015.02...The strands were incubated at 90°C for 5 min and then at 37°C for 1 h. SiRNA was prepared immediately before administration by mixing the RNA solution (1 μg/μl in annealing buffer) with the transfection reagent i-Fect (v/v: 1/3; Neuromics, Edina, MN, USA) to a final siRNA ...

Highlights
•EPO has protective effects on ischemic NVU injuries.
•EPO up-regulates phosphorylation of Cx43, not total Cx43.
•EPO's protective effects on NUV injuries are p-Cx43-GJIC dependent.