Thursday, September 21, 2017

i-Fect Deliver Plasmids to the CNS

Important for Gene Expression Studies.
I have posted many examples of how our customers use i-FectTM  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.

Tuesday, January 31, 2017

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.

Monday, January 30, 2017

More iFect in-vivo

The parade of publications continues to grow.

Here researchers use our i-FectTM 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.

Friday, August 19, 2016

Delivering miRNA in vivo

More Transfection Success! Great Research Tools!

Our i-FectTM  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...
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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.

Monday, June 20, 2016

i-Fect used to Study Epigenetics in Nerve Injury

i-Fect used In Vivo for Study

Researchers use our i-FectTM 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.

Thursday, March 24, 2016

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.

Wednesday, January 20, 2016

Delivery of siRNA, miRNA, shRNA and Plasmids Guaranteed

Potent and Frequently Published Transfection Solutions

Gene Tools
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 BeautyTM
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
 i-Fect
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