i-Fect and Silencing of Gene Involved in Alcohol-Induced Anxiety

We are grateful to see our siRNA Transfection Kit (https://www.neuromics.com/transfection-kits) used in important research. Here, our kit is used to knock out a gene involved in alcohol-induced adolescent anxiety- EZH2-dependent epigenetic reprogramming in the central nucleus of amygdala regulates adult anxiety in both sexes after adolescent alcohol exposure (https://www.nature.com/articles/s41398-024-02906-y).

This research shows the impact of alcohol on mental health and potential treatments.

Mechanisms of Alcohol-Induced and Ameliorated Anxiety

High Fat and Diet Induced Obesity

i-Fect^TM Delivers Again!

Research shows that rats and humans on a high-fat diet (HFD) are less sensitive to satiety signals known to act via vagal afferent pathways. Impaired vagal afferent responsiveness to both gastric satiety hormones (CCK and leptin) and mechanical stimulation raises the possibility that changes in electrophysiological properties may be the underlying mechanism responsible for impaired vagal responsiveness to a wide variety of satiety signals.

Potassium channels play a central role. To demonstrate this researchers used our i-Fect siRNA Transfection Kit to silence TRESK and TASK1 to understand their impact on HFD and vagal responsiveness. Gintautas Grabauskas, Xiaoyin Wu, ShiYi Zhou, JiYao Li, Jun Gao, and Chung Owyang. (2019). High-fat diet–induced vagal afferent dysfunction via upregulation of 2-pore domain potassium TRESK channel. JCI Insight. https://doi.org/10.1172/jci.insight.130402.

Images: (A) Representative recordings of NG neuron responses to intra–superior pancreaticoduodenal artery infusions of CCK-8 (60 pmol/kg) and leptin (60 pmol/kg) in LFD-fed or HFD-fed rats and transfected with control siRNA or TRESK siRNA. Note that CCK-8 generated significantly fewer action potentials in HFD-fed rats compared with those fed an LFD. (B) Summary histograms showing single-unit discharges in response to CCK-8 in rats given an LFD and transfected with control siRNA (n = 11) or TRESK siRNA (n = 6), HFD + control siRNA (n = 12), and HFD treated with TRESK siRNA (n = 10). Data are represented as mean ± SEM. One-way ANOVA with Bonferroni’s test, *P < 0.05 vs. LFD + control siRNA; #P < 0.05 vs. HFD + control siRNA. (C) Summary histogram showing single-unit discharges in response to leptin in rats given an LFD and transfected with control siRNA (n = 11) and TRESK siRNA (n = 5), HFD (n = 12), and HFD treated with TRESK siRNA (n = 10). Data are represented as mean ± SEM. One-way ANOVA with Bonferroni’s test, *P less than 0.05 vs. LFD + control siRNA; #P less than 0.05 vs. HFD + control siRNA. (D) Summary histogram showing CCK-AR and ObR expression in vagal sensory ganglia from LFD- and HFD-fed rats were not significantly different. HPRT was used as a loading control. Data are represented as mean ± SEM. CCK-8, cholecystokinin-8.

Following 2 weeks of high-fat feeding, there was a significant upregulation of TRESK and a modest increase in TASK1 channels in the NG. Silencing studies indicate that the upregulation by TRESK channels is mainly responsible for a global decrease in excitability of vagal sensory neurons, which in turn dampens the response to satiety signals, such as CCK and leptin. 

This make TRESK a potential therapeutic target for treating Obesity.

i-Fect in Action

Knock-down of HIF-1a Attenuates Chemo Induced Pain
i-Fect ^TM is one of our original products. It has enjoyed 14 years of upping transfection percentages both in-vitro and in-vivo.

Here is a new study showing successful use of i-Fect to knock down HIF-1a in-vivo-Taylor Ludman and Ohannes K. Melemedjian. (2019). Bortezomib-induced aerobic glycolysis contributes to chemotherapy-induced painful peripheral neuropathy. Molecular Pain. https://doi.org/10.1177/1744806919837429.

Figure 1. (a) Treatment of mice with bortezomib (Bor) for five days augmented HIF1A expression in L4-6 DRGs (*P ¼ 0.0412, five mice/ group) relative to the vehicle-treated group. (b) A schematic depicting the site of the intrathecal (IT) siRNA injection. The siRNA was administered between the L4 and L5 vertebrae which is around 17 mm rostral to the spinal cord (SC) section innervated by the L4-6 DRGs. (c) IT injection of siRNA (1 mg in 5 ml) that targets HIF1A (siRNA) but not control siRNA (Cont), for two consecutive days, significantly reduced the levels of HIF1A in L4-6 DRGs. (***P=0.0006, five mice/group). (d) IT siRNA did not affect HIF1A levels in L4-6 spinal cord (five mice/group). (e) After determining baseline withdrawal thresholds using von Frey filaments, male ICR mice received IP injection of vehicle or bortezomib (black arrows) and IT siRNA (blue arrows). The withdrawal thresholds were measured on days 7 to 14. IT HIF1A siRNA prevented the development of bortezomib-induced neuropathic pain. (****P less than 0.0001, five mice/group). DRG: dorsal rootganglia; HIF1A: hypoxia-inducible factor 1 alpha; IT: intrathecal; IP: intraperitoneal; siRNA: small interfering RNA.

This study is the first to demonstrate that the stabilization of HIF1A expression underpins the development of bortezomib-induced neuropathic pain. Crucially, these findings reveal that the initiation and maintenance of bortezomib-induced neuropathic pain are regulated by distinct mechanisms.

Looking to up your odds for high percentage siRNA Transfection? Try i-Fect.

i-Fect used to Study Angiogenesis in Brain Injury

Silencing Lactate Dehydrogenase A in vivo

Pathologic CNS is characterized by neuronal damage that leads to the release of intracellular components. However, the effect of damaged cells on angiogenesis has not been clarified. This study revealed that LDHA, which is a known damage marker, promotes CNS-specific angiogenesis. LDHA-mediated angiogenesis depends on vimentin on the surface of vascular endothelial cells. The work described here proposes a novel mechanism by which neurodegeneration drives angiogenesis in the CNS.

A mixture of our i-Fect^TM and LDHA siRNA, in this study, were directly injected into mice cortexes: Hsiaoyun Lin, Rieko Muramatsu, Noriko Maedera, Hiroto Tsunematsu, Machika Hamaguchi, Yoshihisa Koyama, Mariko Kuroda, Kenji Ono, Makoto Sawada, Toshihide Yamashita. Extracellular Lactate Dehydrogenase A Release From Damaged Neurons Drives Central Nervous System Angiogenesis. doi.org/10.1016/j.ebiom.2017.10.033.

Images: LDHA is sufficient to evoke CNS angiogenesis. (a) Representative images of CD105-labeled spinal cord sections obtained 7 days after LDHA administration. (b) Length of CD105+ neovessels around the LDHA administration site as indicated in a, n = 5 each. (c) Representative image of a Nissl-stained brain section after controlled cortical impact (CCI). (d) Representative image of the CD105-immunolabelled cerebral cortex obtained 7 days after CCI. (e) Length of CD105+ neovessels around CCI lesions as indicated in d; n = 5 each, all error bars represent the s.e.m. **P < 0.01, Student's t-tests. Scale bars, 200 μm.

The findings reveal unexpected neurovascular interactions in the injured adult CNS that may be relevant to our understanding of neuronal damage, which is a hallmark of many CNS disorders.

i-Fect Delivers Again!

Knocks Down Suspected Stress/Anxiety Receptor


The molecular pathogenesis underlying anxiety disorders is still unclear. Here, the authors demonstrate that myristoylated alanine-rich C-kinase substrate like 1 (MARCKSL1) overexpression in mice increases spine formation in the amygdala and induces stress hormone upregulation and anxiety-like behaviors. Suppression of MARCKSL1 in the amygdala ameliorates both the increase in stress hormones and the elevated anxiety-like behaviors. Our results indicate that MARCKSL1 expression in the amygdala plays an important role in anxiety-like behaviors.

This was proved, in part, by the knockdown of MARCKSL1 in vivo in mice using our i-Fect^TM. Tanaka, Takashi; Shimizu, Shoko; Ueno, Masaki; Fujihara, Yoshitaka; Ikawa, Masahito; Miyata, Shingo. MARCKSL1 Regulates Spine Formation in the Amygdala and Controls the Hypothalamic-Pituitary-Adrenal Axis and Anxiety-Like Behaviors. https://doi.org/10.1016/j.ebiom.2018.03.018

Figure: Knockdown of MARCKSL1 ameliorates anxiety-like behavior in MARCKSL1 Tg mice. (A and B) For the in vivo experiment, siRNA (blue) was injected into the CeA (total 4 sites) with i-Fect siRNA transfection reagents 5 days prior to behavioral tests. (C) In situ hybridization for Marcksl1 mRNA (blue) in the amygdala after injection of Marcksl1 siRNA into the CeA of Tg/Tg mice. Scale bar, 200 μm. (D and E) Light/dark transition test and elevated plus maze performance in 

MARCKSL1 knockdown mice (WT + control siRNA, n = 7; Tg/Tg + control siRNA, n = 7; Tg/Tg + Marcksl1 siRNA, n = 8).

We will continue to post new i-Fect results here.

i-Fect Delivers siRNA to Study Angiogenesis in Brain Injury

Silencing Lactate Dehydrogenase A in vivo

Pathologic CNS is characterized by neuronal damage that leads to the release of intracellular components. However, the effect of damaged cells on angiogenesis has not been clarified. This study revealed that LDHA, which is a known damage marker, promotes CNS-specific angiogenesis. LDHA-mediated angiogenesis depends on vimentin on the surface of vascular endothelial cells. The work described here proposes a novel mechanism by which neurodegeneration drives angiogenesis in the CNS.

A mixture of our i-Fect^TM and LDHA siRNA, in this study, were directly injected into mice cortexes: Hsiaoyun Lin, Rieko Muramatsu, Noriko Maedera, Hiroto Tsunematsu, Machika Hamaguchi, Yoshihisa Koyama, Mariko Kuroda, Kenji Ono, Makoto Sawada, Toshihide Yamashita. Extracellular Lactate Dehydrogenase A Release From Damaged Neurons Drives Central Nervous System Angiogenesis. doi.org/10.1016/j.ebiom.2017.10.033.

Images: LDHA is sufficient to evoke CNS angiogenesis. (a) Representative images of CD105-labeled spinal cord sections obtained 7 days after LDHA administration. (b) Length of CD105+ neovessels around the LDHA administration site as indicated in a, n = 5 each. (c) Representative image of a Nissl-stained brain section after controlled cortical impact (CCI). (d) Representative image of the CD105-immunolabelled cerebral cortex obtained 7 days after CCI. (e) Length of CD105+ neovessels around CCI lesions as indicated in d; n = 5 each, all error bars represent the s.e.m. **P < 0.01, Student's t-tests. Scale bars, 200 μm.

The findings reveal unexpected neurovascular interactions in the injured adult CNS that may be relevant to our understanding of neuronal damage, which is a hallmark of many CNS disorders

Delivering siRNA in vivo

New Publications

Our i-Fect transfection kit was one of the first products. This has resulted in it being widely used and frequently published.

We kick off 2018 with some new publications. Here we feature a study where researchers use our kit to deliver G-protein-coupled receptor C (MrgprC) siRNA in vivo. This receptor is part of the TRPV1 pathway.

• Ying-jun Liu, Xiao-xi Lin, Jian-qiao Fang, and Fang Fang. Involvement of MrgprC in electroacupuncture analgesia for attenuating CFA-induced thermal hyperalgesia by suppressing the TRPV1 pathway. Pre-pub-full text PDF

Image: Expression of TRPV1 in rats treated with MrgrprC si RNA vs controls (C).

The others conclude that MrgprC expression is impacted by electroacupuncture and downregulates  TRPV1.  This is the mechanism that results in pain relief.

i-Fect Deliver Plasmids to the CNS

Important for Gene Expression Studies.
I have posted many examples of how our customers use i-Fect^TM  and other Transfection Solutions for Gene Manipulation Studies. There are also many publications.

Here we feature how i-Fect was used to delivery plasmids to the CNS: Sara Elramah, María José López-González, Matthieu Bastide, Florence Dixmérias, Olivier Roca-Lapirot, Anne-Cécile Wielanek-Bachelet, Anne Vital, Thierry Leste-Lasserre, Alexandre Brochard, Marc Landry & Alexandre Favereaux. Spinal miRNA-124 regulates synaptopodin and nociception in an animal model of bone cancer pain. Scientific Reports 7, Article number: 10949 (2017) doi:10.1038/s41598-017-10224-1...Intrathecal administration of miRNAs and ShRNA To over-express miR-124, we cloned the pre-miRNA sequence of miR-124 into a plasmid. To determine cells expressing this miR-124 encoding plasmid, we added a GFP-coding sequence to the construct under the control of an IRES. Thus, miR-124 over-expressing cells also express GFP. To inhibit synaptopodin expression, we cloned a ShRNA sequence directed against synaptopodin into a plasmid. To determine cells expressing this ShRNA, we added a GFP-coding sequence to the construct under the control of an IRES. Thus, ShRNA expressing cells also expressed GFP. Two micrograms of these plasmids or the corresponding controls, were solubilized in 10 µl of i-Fect reagent (Neuromics, Edina, USA), and injected intrathecally between the L5 and L6 lumbar vertebrae every two days for a total of 3 injections, according to the manufacturer’s instructions and previously published experiments...

Figures: (C and D) Immunostaining of synpo in spinal cord after miR-124 intrathecal injections: only the dorsal horn which receive nociceptive information was quantified (white dash area). Measurement of synaptopodin stained area reveals ability of miR-124 to inhibit endogenous Synpo expression (20/3 and 17/3 denotes number of sections/animals for control and miR-124-injected mice, respectively.

I am confident there will be many more positive reports regarding our Transfection Reagents.

Desperately Seeking Data

Answering the Bell
We continue to seek data using our cells. We offer a reward of 25 USD Starbucks' Gift Card.

We were pleased to receive a recently published study from Dr. Mahendran Subramanian of Keele University. In this study, researchers showed that oscillating nanomagnetic gene transfection could be used to successfully transfect SH‐SY5Y cells as well as our primary hippocampal and cortical neurons on different days in vitro. This novel technique was used to effectively deliver genetic material into various cell types, resulting in high transfection efficiency and viability. Mahendran Subramanian, Aimee‐Jayne Tyler, Eva Maria Luther, Elena Di Daniel, Jenson Lim and Jon Dobson. Oscillating Magnet Array−Based Nanomagnetic Gene Transfection: A Valuable Tool for Molecular Neurobiology Studies. Nanomaterials 2017, 7, 28; doi:10.3390/nano7020028...Primary rat hippocampal and cortical neurons were obtained from Neuromics (Edina, MN, USA) and disassociated using papain disassociation kit (Worthington, NJ, USA) according to the manufacturer’s instructions. Isolated neurons were maintained using neurobasal medium supplemented with 5% FBS, 0.5 mM Glutamax, 2% B27 supplement, 25 μM L‐glutamine and seeded onto poly‐D‐lysine–coated cells culture plates...

Figure 1. Oscillating magnet array−based nanomagnetic gene transfection experimental setup. (A) Representation of a 96‐well oscillating magnet array–based nanomagnetic transfection setup using NdFeB magnetic array (nanotherics); (B) Dimensions of the permanent magnets and magnetostatic (vectorpotential) algorithm based magnetic field density |B| distribution (T) contour plot for the NdFeB magnetic array.

Figure 2. Gene delivery by oscillating nanomagnetic gene transfection in primary cortical neurons. Images of pmaxGFP plasmid expressed in primary neurons using fluorescence microscopy and its corresponding Hoechst 33,342 stained counterpart of transfected DIV 1 (A,C) and DIV 5 (B,D) mature neurons were taken 48 h post transfection.

If you have data to share email it to me, pshuster@neuromics.com and we'll email you a 25 USD gift card. Thank you. Pete Shuster, CEO & Owner.

More iFect in-vivo

The parade of publications continues to grow.

Here researchers use our i-Fect^TM Transfection Kit for delivering sh-IRF3 in vivo: Rui Li, Li-guo Wang, Qi Wang, Zhi-hua Li, Ya-li Ma, Qing-Duo Guo. Silencing of IRF3 alleviates chronic neuropathic pain following chronic constriction injury. doi.org/10.1016/j.biopha.2017.01.085... The oligonucleotides for sh-IRF3 were: 5′-CACCGCGTCTAGGCTGGTGGTTATTCGAAAATAACCACCAGCCTAGACGC-3′ −3′. Then, 10 μg sh-IRF3 dissolved in 30 μl i-Fect transfection reagent (Neuromics, Edina, MN, USA) was administered intrathecally once daily for 7...

Fig. Down-regulation of IRF3 attenuated mechanical allodynia and thermal hyperalgesia in CCI rats. (A) The mRNA expression level of IRF3 in the DRG at postoperative day 7. (B) The protein expression level of IRF3 in the DRG at postoperative day 7. (C and D) PWT and PWL were measured 1 day before CCI and 1, 3, 7 and 14 days after intrathecal injection of sh-IRF3 or scramble.

Down-regulation of IRF3 inhibited the production of pro-inflammatory cytokines in the DRG of CCI rats.

These results indicated that IRF3 was involved in the development of neuropathic pain. Down-regulation of IRF3 attenuated neuropathic pain in CCI rats by inhibiting the activation of NF-κB signaling pathway, suggesting that IRF3 may be a novel and potential target for the treatment of neuropathic pain.

Delivering miRNA in vivo

More Transfection Success! Great Research Tools!

Our i-Fect^TM  Transfection Kit is used to study Epigenetics and pain. Here's yet another example: M. Leinders, b, N. Üçeyler, R.A. Pritchard, C. Sommer, L.S. Sorkin. Increased miR-132-3p expression is associated with chronic neuropathic pain. Experimental Neurology. Volume 283, Part A, September 2016, Pages 276–286...The inhibitor and mimetic were administered to awake rats via the it catheters. Prior to injection, active or mismatch inhibitors were mixed with (1:5 w/v) i-Fect™ in vivo transfection reagent(Neuromics, Edina, USA) to final doses of 5, 2 and 1 μg in 10 μl...

.

Spinal administration of miR-132-3p antagonists via intrathecal (i.t.) catheters dose dependently reversed mechanical allodyina  and eliminated pain behavior in the place escape avoidance paradigm (p < 0.001). Intrathecal administration of miR-132-3p mimetic dose-dependently induced pain behavior in naïve rats (p < 0.001). Taken together these results indicate a pro-nociceptive effect of miR-132-3p in chronic neuropathic pain.

Finding like these could pave the way for an miRNA like therapy for pain.

i-Fect used to Study Epigenetics in Nerve Injury

i-Fect used In Vivo for Study

Researchers use our i-Fect^TM to effectively deliver miR-126 in vivo to modulate Methyl-CpG-binding protein 2 (MeCP2).

MeCP2 regulates gene expression through activation, repression and chromatin remodeling. Mutations in MeCP2 cause Rett syndrome, and these patients display impaired nociception. The researchers observed an increase in MeCP2 expression in mouse dorsal root ganglia (DRG) after peripheral nerve injury: Melissa T. Manners, Adam Ertel, Yuzhen Tian and Seena K. Ajit. Genome-wide redistribution of MeCP2 in dorsal root ganglia after peripheral nerve injury. Epigenetics & Chromatin 20169:23. DOI: 10.1186/s13072-016-0073-5© The Author(s) 2016 Received: 11 March 2016. Accepted: 27 May 2016Published: 7 June 2016...miRNA administration protocol was adapted from previous report of intrathecal miRNA delivery. To administer miRNA mimics, a polyurethane catheter (25G, 5.5 cm long, SAI infusion) was placed into the intrathecal space of the lumber L4–L5 vertebrae under isoflurane anesthesia. The catheter was stereotactically secured under the skin and occluded between injections. A custom miRCURY (Exiqon) miR-126 mimic containing a 5′ cholesterol tag and 3′ fluorescein label was injected at 2 nmol concentration with 4 µl iFECT transfection reagent (Neuromics). A total of 6 µl was delivered into the catheter connection juncture using a 25G blunt end needle on a Hamilton syringe. The catheter was then flushed with 7 µl sterile PBS to ensure miRNA reached the intrathecal space...

Figure: Expression of miR-126 and its target genes Dnmt1 and Vegfa in the DRG after nerve injury. a Relative expression of miR-126 determined by qPCR shows a reduction in miR-126 in SNI model compared to DRG from sham control. U6 was used for normalization (n = 8 sham, n = 7 SNI). b Relative expression of Dnmt1 mRNA and c Vegfa transcripts showed an increase in the DRG after nerve injury compared to control (n = 3). Gapdh was used as a normalizer. d Representative Western blot and quantification showed an increase of Dnmt1 protein in the DRG after nerve injury. e Western blot and quantification showed Vegfa protein was not significantly different in DRG after nerve injury (n = 3 from pooled samples, three DRG were pooled for each sample).

Conclusions: The study shows a regulatory role for MeCP2 in that changes in global redistribution can result in direct and indirect modulation of gene expression in the DRG. Alterations in genome-wide binding of MeCP2 therefore provide a molecular basis for a better understanding of epigenetic regulation-induced molecular changes underlying nerve injury.

Epigenetics and Pain Research

i-Fect Used to Study Impacts

Our i-Fect siRNA, miRNA and shRNA Trasfection Kit was recently used to study the impact of G9a-specific siRNA (AGUAACGGGCAUCAAUGC) on Mu Opioid Receptors: Yuhao Zhang, Shao-Rui Chen, Geoffroy Laumet, Hong Chen and Hui-Lin Pan. Nerve Injury Diminishes Opioid Analgesia through Lysine Methyltransferase-Mediated Transcriptional Repression of µ-Opioid Receptors in Primary Sensory Neurons. First Published on February 25, 2016, doi: 10.1074/jbc.M115.711812... In some SNL rats, G9a-specific siRNA (4 µg) or the negative control siRNA was administered intrathecally. G9a-specific siRNA(AGUAACGGGCAUCAAUGC) or universal negative control siRNA (#SIC001, Sigma-Aldrich) was mixed with i-Fect (Neuromics, Edina, MN) to a final concentration of 400 mg/L for the intrathecal injections...

Figures: G9a knockdown with siRNA reverses the MOR expression in the DRG and the morphine analgesic effect diminished by nerve injury. (A,B) Quantitative PCR (A) and Western blotting (B) analyses show the mRNA and protein levels of MORs in the DRGs of sham and SNL rats treated with control or G9a-specific siRNA (n = 10 rats in each group). The ipsilateral L5 and L6 DRG tissues were removed 24 h after the last siRNA injection. The amount of MOR mRNA and protein was normalized to GAPDH in the same samples, and the mean value of MOR levels in sham control rats was considered to be 1. (C) Time course of the intrathecal morphine effects on the tactile and pressure withdrawal thresholds in sham and SNL rats treated with G9a-specific siRNA or negative control siRNA (n = 9 rats in each group). The withdrawal thresholds after the last siRNA injection were plotted as the baseline control (BL).


Summary: The findings provide new insight into the epigenetic mechanism regulating MOR expression in primary sensory neurons in neuropathic pain. This multidisciplinary approach provides conclusive evidence for G9a as a key chromatin regulator responsible for MOR downregulation in the DRG and the analgesic efficacy of opioids reduced by nerve injury. A better understanding of the epigenetic mechanisms underlying nerve injury-induced downregulation of MORs in primary sensory neurons could help improve the analgesic efficacy of opioids for treating chronic neuropathic pain. G9a inhibitors could be used to enhance the opioid analgesic effect and reduce opioid consumption in patients with chronic neuropathic pain.

Delivery of siRNA, miRNA, shRNA and Plasmids Guaranteed

Potent and Frequently Published Transfection Solutions


We have proven and frequently published transfection solutions. 
Powerful punch and versatility are now needed more than ever with the 
hyper growth in gene manipulation technologies like Sleeping Beauty^TM
and CRISPR-Cas9.

We have published examples of the use of our solutions for delivering siRNA, miRNA, 
shRNA and plasmids both in vitro and in vivo. here are some of your options.
i-Fect ™ -A novel cationic lipid formulation specifically 
designed for efficient delivery of 27mer DsiRNAs(dicer 
substrate small Interfering RNAs)& 21mer siRNAs (small interfering RNAs) in vitro and in vivo.
p-Fect™ -Designed to delivery plasmids, DNA or 
RNA to hard to transfect Cell Lines.

pn-Fect™ -The latest advance in transfection 
for primary neuronal cells. 
This unique reagent provides ultra-high plasmid DNA delivery 
efficiencies and low cytotoxicity compared to competitive reagents.
Here's a recent i-Fect Publication:
Liuming Jiang, Qun Wu , Tao Yang. Silencing of Id2 Alleviates 
Chronic Neuropathic Pain Following Chronic Constriction Injury.
Journal of Molecular Neuroscience\pp 1-7.First online: 15 January 2016
 

Figure: Knockdown of Id2 attenuated mechanical allodynia and thermal 

hyperalgesia in CCI rats. (a and b) PWT and PWL were measured 1 day before 

CCI and 1, 3, 7, and 14 days after intrathecal administration of shRNA-Id2.

If you are looking for transfection solutions, do not hesitate to contact me @ direct phone: 612-801-1007 or pshuster@neuromics.com. Thank you, Pete Shuster, CEO and Owner, Neuromics

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.

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.

miRNA, Inflammation and Coronary Heart Disease

i-Fect^TM Delivers miRNA for the Study of Cardiovascular Pathogenesis

We have posted over 35 publications that reference use of our i-Fect Transfection Kit to deliver siRNA, miRNA and shRNA in vitro and in vivo. Results documented in these publications prove that this kit is both non-toxic and delivers ultra-high transfection efficiency.

Here i-Fect is used to silence miR-21 microRNAs. This miRNA stimulates pro-inflammatory pathways that are at the root of Coronary Heart Disease: Guo Weizao, Liu Huichen, Li Lin, Yang Man and Du Aihua. Regulation of lovastatin on a key inflammation-related microRNA in myocardial cells. Chinese Medical Journal 2014;127(16):2977-2981:10.3760/cma.j.issn.0366-6999.20140780...... miRNA functional inhibition assay Anti-miR miRNA antagonist for miR-21 (Ambion/Life Technologies, Grand Island, NY, USA) was transfected into H9c2(2-1) cells using iFect transfection kit (Neuromics, Edina, MN, USA) according to the manufacturer's manual...

Results:Inhibition of miR-21 upregulates STAT-3 and exerts a critical role in the upregulation of cardioprotective and anti-apoptotic proteins.

Fig: Inhibition of miR-21 attenuated the up-regulation of phosphorylation of STAT3 in H9c2(2-1) cells by lovastatin (LST) in lipopolysaccharide (LPS) treated cardiomyocytes. Combination of treatments are indicated under the image, the basic comparison was 1 vs 3. 

This study demonstrates the relationship between miR-21 and the STAT3 pathway in Coronary Heart Disease. Delivering inhibitory miRNA into cardiomyocytes was key to establishing this relationship. Further study could enable discovery of STAT3 related targets for CV protective drug. In the spirit of helping researchers find the best solutions and protocols for Gene Expression Analysis Studies, we will continue to post new findings.