siRNA, DsiRNA and Plasmid Transfection Efficiency

Delivering DNA to Cell Lines and Primary Cells

2011-12-15T03:18:00.000-08:00

Neuromics is pleased to introduce DNA-Fect^TMand DNA-Fect^TM293 in vivo transfection reagents.

These reagents deliver genes to various established cell lines as well as primary cells, which include HEK293, 293T, 293E, CHO, COS1, HeLa, NIH 3T3, insect cell lines (Sf9 and Sf21) and a variety of other eucaryotic cell lines with low cytotoxicity. GeneExpresso™ MAX reagent, 1.0 ml, is sufficient for 300 to 600 transfections in 24 well plates, or 150 to 300 transfections in 6 well plates.

Capabilities include:
• Proven to deliver DNA to difficult-to-transfect cells
•Stable and easy to use
•Suitable for high-thoroughput (HTS) applications

Image: Comparing DNA-Fect vs Lipofectamine 2000 by FACS Analysis

DNA Delivery Protocol:

β-arrestin siRNA Delivery in vivo and Increased Analgesia

2011-09-29T04:33:00.000-07:00

I have reported use of our i-FectTM siRNA delivery kit for gene expression analysis studies of Cav1.2, DOR, hTERT, The β3 subunit of the Na+,K+-ATPase, rSNSR1, NTS1. NAV1.8, , TRPV1, Survivin, Flaviviruses and more.

I would like to congratulate Dr. C.R. Lin and his team at National Taiwan University College of Medicine for silencing β-arrestin expression in vivo and the impact on opioid based analgesia. The results could be good news for improving opioid based pain therapies: C.-H.Yang, H.-W. Huang, K.-H.Chen, Y.-S.Chen, S.-M.Sheen-Chen and C.-R.Lin. Antinociceptive potentiation and attenuation of tolerance by intrathecal β-arrestin 2 small interfering RNA in rats. Br. J. Anaesth. (2011) doi: 10.1093/bja/aer291.

Background: Tolerance to the analgesic effect of opioids complicates the management of persistent pain states. We tested whether the intrathecal infusion of small interfering RNA (siRNA) against β-arrestin 2 would reduce tolerance to chronic morphine use and the severity of precipitated morphine withdrawal.

Methods: Intrathecal β-arrestin 2 (2 μg siRNA per 10 μl per rat) was injected once daily for 3 days. Rats then received a continuous intrathecal infusion of morphine (2 nmol h−1) or saline for 7 days. Daily tail-flick (TF) and intrathecal morphine challenge tests were performed to assess the effect of intrathecal β-arrestin 2 siRNA on antinociception and tolerance to morphine. Naloxone withdrawal (2 mg kg−1) was performed to assess morphine dependence.
Results: In the daily TF test, the antinociception of intrathecal morphine was increased and maintained in rats receiving β-arrestin 2 siRNA compared with the control group (morphine alone). In the probe response test, rats receiving morphine infusion with β-arrestin 2 siRNA treatment showed a significant left shift in their dose–response curve, as measured by per cent maximal possible effect (MPE), such that the AD50 was significantly decreased by a factor of 5.6 when compared with that of morphine-infused rats. In the naloxone-induced withdrawal tests, rats receiving β-arrestin 2 siRNA injection with morphine infusion showed a significant reduction in four of the six signs of withdrawal.
Conclusions: We show here that intrathecal β-arrestin 2 siRNA in rats enhances analgesia and attenuates naloxone-induced withdrawal symptoms. This may warrant further investigation in the context of long-term use of intrathecal opioids for controlling chronic pain.

Enhanced Transfection Efficiency of Human Embryonic Stem Cells

2011-09-26T17:41:00.000-07:00

Stem cell based drug discovery and cell based therapies hold great promise. Researchers are faced with many hurdles in developing and launching meaningful therapies. Key will be a thorough understanding of the genetically stability of the stem cells involved in these therapies. Transfection tools will help foward this understanding. Here's a representative publication: Luis G. Villa-Diaz, Jose L. Garcia-Perez and Paul H. Krebsbach. Stem Cells and Development. December 2010, 19(12): 1949-1957. doi:10.1089/scd.2009.0505.

Overview: Because human embryonic stem (hES) cells can differentiate into virtually any cell type in the human body, these cells hold promise for regenerative medicine. The genetic manipulation of hES cells will enhance our understanding of genes involved in early development and will accelerate their potential use and application for regenerative medicine. The objective of this study was to increase the transfection efficiency of plasmid DNA into hES cells by modifying a standard reverse transfection (RT) protocol of lipofection. We hypothesized that immobilization of plasmid DNA in extracellular matrix would be a more efficient method for plasmid transfer due to the affinity of hES cells for substrates such as Matrigel and to the prolonged exposure of cells to plasmid DNA. Our results demonstrate that this modification doubled the transfection efficiency of hES cells and the generation of clonal cell lines containing a piece of foreign DNA stably inserted in their genomes compared to results obtained with standard forward transfection. In addition, treatment with dimethyl sulfoxide further increased the transfection efficiency of hES cells. In conclusion, modifications to the RT protocol of lipofection result in a significant and robust increase in the transfection efficiency of hES cells.

Related Neuromics' Reagents for Testing and Gene Expression Analysis of Stem Cells:
LumiSTEM™ in vitro Cell Based Assays-Designed for Primary Stem and Explanted Cells, Stem and Other Cell Lines.
LUMENESC™ in vitro Cell Based Assays-Designed for Mesenchymal Stem/Stromal Cells (MSCs) and Cells derived from MSCs.
HALO® in vitro Cell Based Assays-Designed for Lympho-Hematopoietic Stem and Progenitor Cells.
Transfection Reagents

Bidirectional integrative regulation of Cav1.2 calcium channel by microRNA miR-103: role in pain

2011-08-20T11:15:00.000-07:00

I have reported use of our i-FectTM siRNA delivery kit for gene expression analysis studies of DOR, hTERT, The β3 subunit of the Na+,K+-ATPase, rSNSR1, NTS1. NAV1.8, , TRPV1, Survivin, Flaviviruses and more.

I am pleased to add the Cav1.2 calcium channel to this growing list. Congratulations to Dr. Marc Landry for discovering the interplay between microRNA-miR-103 and this calcium channel: Alexandre Favereaux, Olivier Thoumine, Rabia Bouali-Benazzouz, Virginie Roques, Marie-Amélie Papon, Sherine Abdel Salam, Guillaume Drutel, Claire Léger, André Calas, Frédéric Nagy and Marc Landry. Bidirectional integrative regulation of Cav1.2 calcium channel by microRNA miR-103: role in pain. The EMBO Journal , (29 July 2011) | doi:10.1038/emboj.2011.249.

Abstract: Chronic pain states are characterized by long-term sensitization of spinal cord neurons that relay nociceptive information to the brain. Among the mechanisms involved, up-regulation of Cav1.2-comprising L-type calcium channel (Cav1.2-LTC) in spinal dorsal horn have a crucial role in chronic neuropathic pain. Here, we address a mechanism of translational regulation of this calcium channel. Translational regulation by microRNAs is a key factor in the expression and function of eukaryotic genomes. Because perfect matching to target sequence is not required for inhibition, theoretically, microRNAs could regulate simultaneously multiple mRNAs. We show here that a single microRNA, miR-103, simultaneously regulates the expression of the three subunits forming Cav1.2-LTC in a novel integrative regulation. This regulation is bidirectional since knocking-down or over-expressing miR-103, respectively, up- or down-regulate the level of Cav1.2-LTC translation. Functionally, we show that miR-103 knockdown in naive rats results in hypersensitivity to pain. Moreover, we demonstrate that miR-103 is down-regulated in neuropathic animals and that miR-103 intrathecal applications successfully relieve pain, identifying miR-103 as a novel possible therapeutic target in neuropathic chronic pain.

MicroRNAs as targets for pain therapies are gathering momentum. This defned miR-103 is a compelling possibilty. I will be following the story closely as it unfolds.

PEI transfection and Implications for a Mechanism of Cytotoxicity

2011-08-10T13:44:00.000-07:00

This is a great article for undertanding the root causes of cytotoxicity caused by PEI-DNA Polyplexes.

Giovanna Grandinetti, Nilesh P. Ingle, and Theresa M. Reineke. Interaction of Poly(ethylenimine)–DNA Polyplexes with Mitochondria: Implications for a Mechanism of Cytotoxicity. Mol. Pharmaceutics, Article ASAP. Publication Date (Web): June 23, 2011. Copyright © 2011 American Chemical Society.

Poly(ethylenimine) (PEI) and PEI-based systems have been widely studied for use as nucleic acid delivery vehicles. However, many of these vehicles display high cytotoxicity, rendering them unfit for therapeutic use. By exploring the mechanisms that cause cytotoxicity, and through understanding structure–function relationships between polymers and intracellular interactions, nucleic acid delivery vehicles with precise intracellular properties can be tailored for specific function. Previous research has shown that PEI is able to depolarize mitochondria, but the exact mechanism as to how depolarization is induced remains elusive and therefore is the focus of the current study. Potential mechanisms for mitochondrial depolarization include direct mitochondrial membrane permeabilization by PEI or PEI polyplexes, activation of the mitochondrial permeability transition pore, and interference with mitochondrial membrane proton pumps, specifically Complex I of the electron transport chain and F0F1-ATPase. Herein, confocal microscopy and live cell imaging showed that PEI polyplexes do colocalize to some degree with mitochondria early in transfection, and the degree of colocalization increases over time. Cyclosporin a was used to prevent activation of the mitochondrial membrane permeability transition pore, and it was found that early in transfection cyclosporin a was unable to prevent the loss of mitochondrial membrane potential. Further studies done using rotenone and oligomycin to inhibit Complex I of the electron transport chain and F0F1-ATPase, respectively, indicate that both of these mitochondrial proton pumps are functioning during PEI transfection. Overall, we conclude that direct interaction between polyplexes and mitochondria may be the reason why mitochondrial function is impaired during PEI transfection.

Neuroscience-Best Transfection Practices

2011-05-21T08:54:00.000-07:00

I have multiple posting of customer success in transfecting siRNA and plasmids into neurons using Neuromics' i-Fect^TM and pn-Fect^TM...related publications.

Here I post excellent data, images and testimonials provided by researchers using our Magnetic Assisted Transfection (MATraTM). Our goal is to continue our journey towards having the best practices for gene expression analysis studies in the CNS.

With Magnet Assisted Transfection, IBA/Neuromics offers a very gentle and potent tool for the transfection of many kinds of neuronal cells. Magnet Assisted Transfection is the ideal solution to overcome problems related to the study of complex and easily interrupted systems.

Transfection of primary cortical neurons

Example 1

Embryonic cortical neurons were transfected with human NCAM.

Embryonic cortical neurons were transfected with human NCAM. After transfection membrane-localized NCAM (not endocytosed) was detected using a Cy3-coupled secondary antibody (red). Afterwards, the internalised, endocytosed NCAM was stained by a Cy2-coupled secondary antibody (green, see arrows) in the cell soma (left) and in axonal vesicles (right).

Example 2

pPrimary cortical neurons from mice embryonic day 15.5 (E15.5) were grown on poly-L-lysine coated coverslips at a density of 800.000 cells/well in a 24-well plate. The neurons were transfected after 1 day in vitro (DIV 1) with pCX-EGFP-N1 plasmid

Primary cortical neurons from mice embryonic day 15.5 (E15.5) were grown on poly-L-lysine coated coverslips at a density of 800.000 cells/well in a 24-well plate. The neurons were transfected after 1 day in vitro (DIV 1) with pCX-EGFP-N1 plasmid. Transfection was carried out as recommended by the manufacturer
(0.6 µg DNA, 0.6 µL Matra-A reagent). Cells were fixed 24 h later (DIV 2) and GFP fluorescence was visualized using a confocal laser scanning microscope.

"With MATra we achieved a higher transfection efficiency than with different liposomal transfection methods and no toxicity to the cells was observed." Dr. Simone Diestel, Institute of Animal Science, University Bonn, Germany

Cerebellar granular cells from CD1 mice

Cultured cerebellar granular cells from CD1 mice were transfected by below 4 constructs

Cultured cerebellar granular cells from CD1 mice were transfected by below 4 constructs (A-D) using MATra-A.
(A) MyrPalm-mCFP, cyan (provided by Dr. R. Tsien, UCLA)
(B) Actin-DsRed, red
(C) Flotillin-2-mVenus, yellow (B and C provided by Dr. R. Tikkanen, University of Giessen)
(D) Battenin-myc, detected by using GAM-Alexa647, dark green
(E) Surface: Crop of the whole image with 3D surface rendered fluorescence signals overlayed on phase contrast image.
(F) PhaCo: Phase contrast image

Primary hippocampal neurons (E14)

Primary hippocampal neurons (E14) were grown on 15 mm glass coverslips on a 12 well at density of 150.000/cm². The neurons were transfected 4 d.i.v. with pSyn-eGFP

Primary hippocampal neurons (E14) were grown on 15 mm glass coverslips on a 12 well at density of 150.000/cm². The neurons were transfected 4 d.i.v. with pSyn-eGFP using 25 µl MATra complex per well (prepared by adding a MATra-A Reagent-DNA complex mixture (2.8 µg cDNA; 2.8 µl beads) into 175 µl neuronal medium without serum). The cells were fixed 6 d.i.v. with 4% PFA and imaged
"With MATra we can transfect and modulate the expression levels of exogenous proteins in highly sensitive primary neurons without any toxicity. Once optimized, double and even triple transfections with different DNA ratios are easily achieved", said Dr. Mika Ruonala, Center for Membrane Proteomics, University of Frankfurt.
Neurosciences are a vast and expanding field of research focussing on highly sophisticated and enthralling questions. With Magnet Assisted Transfection IBA/Neuromics offers a very gentle and potent tool for the transfection of many kinds of neuronal cells. Magnet Assisted Transfection is the ideal solution to overcome problems related to the study of complex and easily interrupted systems.

PKA+siRNA Block Hyperalgesia

2011-05-13T11:00:00.000-07:00

I have reported use of our i-Fect^TM siRNA delivery kit for gene expression analysis studies of DOR, hTERT, The β3 subunit of the Na+,K+-ATPase, rSNSR1, NTS1. NAV1.8, , TRPV1, Survivin, Flaviviruses and more.

The data in this pub indicates that selective knock-down of spinal PKA activity by intrathecal (i.th.) pretreatment of rats with a PKA-selective small interference RNA (siRNA) mixture significantly attenuates sustained morphine-mediated augmentation of spinal CGRP immunoreactivity, thermal hyperalgesia, mechanical allodynia and antinociceptive tolerance. The present findings indicate that sustained morphine-mediated activation of spinal cAMP/PKA-dependent signaling may play an important role in opioid induced hyperalgesia: S. Tumati, W.R. Roeskea, T.M. Largent-Milnesa, T.W. Vanderaha, and EV Varga. Intrathecal PKA-selective siRNA treatment blocks sustained morphine-mediated pain sensitization and antinociceptive tolerance in rats. doi:10.1016/j.jneumeth.2011.04.036.

Figure: Intrathecal PKA-selective siRNA treatment blocks the development of morphine antinociceptive tolerance.

Male Sprague Dawley rats were pretreated i.th. with vehicle (inverted triangle) or PKA-selective siRNA (circle) for 3 days. After the pretreatments, the animals received continuous saline (open symbols, error bars within the symbol) or morphine (45 nmol/μl/h) (closed symbols, error bars within the symbol) infusion for 6 days, with continued i.th. siRNA or vehicle injections on alternate days. Sustained (6 days) systemic morphine (45 nmol/μl/h) infusion caused a rightward shift in the dose-response curve, with the previous A90 dose causing only 20±1% MPE (**p < 0.01 relative to control, one-way ANOVA, n=5). Intrathecal PKAselective siRNA pre-treatment greatly attenuated sustained morphine-mediated rightward shift in the morphine dose-response curve. Thus, re-challenge with the naive A90 dose (10 μg/5μl) produced 93±2% antinociception in the PKA-selective siRNA pre-treated rat**p < 0.01 relative to vehicle pre-treated morphine-infused rats, one-way ANOVA, n=5).

Transfection Kits and Related Reagents:

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.
n-Fect™ -A cationic lipid that has been specifically formulated for nervous system applications. n-Fect provides higher transfection efficiency than other commercially available broad-spectrum transfection reagents for glial cells, neuronal cell lines, and certain primary neuronal cultures.
n-Blast™ -A broad-spectrum transfection reagent successfully used in many cell types commonly used by neuroscientist.
pn-Fect™ -The latest advance in transfection technology for primary neuronal cells. This unique reagent provides ultra-high plasmid DNA delivery efficiencies and low cytotoxicity compared to competitive reagents.
p-Fect™ -Designed to delivery plasmids, DNA or RNA to hard to transfect Cell Lines.
pro-Fect™ -Is a unique lipid-based formulation that allows the delivery of proteins, peptides or other bioactive molecules into a broad range of cell types.
Penatratin-1™ -A peptide for delivering small molecules into Neurons and other cells. MP Biomedical is the manufacturer of Penetratin-1
MATra™ Products-Provides a system for Magnetically Driving the transfection process enhancing
the performance of transfectants.
Other Cells-Competent mammalian cells by Category
Primary Neurons and Astrocytes

I'll be posting more soon.

ASICs and Surgical Pain

2011-04-25T07:01:00.000-07:00

Dr. Eric Lingueglia, an INSERM group leader, and his team at the CNRS IPMC/IN2M have been doing impressive research using our i-Fect ™ siRNA Transfection Kits to study the role of Acid-Sensing Ion Channels in Postoperative Pain.

The etiology and pathophysiology of this pain is poorly understood. Their work is shedding light on potential root causes:
Emmanuel Deval, Jacques Noël, Xavier Gasull1, Anne Delaunay, Abdelkrim Alloui, Valérie Friend, Alain Eschalier, Michel Lazdunski, and Eric Lingueglia. Acid-Sensing Ion Channels in Postoperative Pain. The Journal of Neuroscience, 20 April 2011, 31(16): 6059-6066; doi: 10.1523/JNEUROSCI.5266-10.2011.

...Ten microliters of a siRNA (2 μg)/i-Fect (Neuromics) mix was injected intrathecally between the L4 and L5 vertebrae of rats using a Hamilton syringe and a 25 gauge needle. Animals received one injection per day for 4 d (Fig. 4A, protocol). ASIC3 (CUACACGCUAUGCCAAGGAdtdt) and the corresponding scramble (GCUCACACUACGCAGAGAUdtdt) siRNAs have been previously described (Deval et al., 2008)...

Highlights: Pharmacological inhibition of ASIC3 channels with the specific toxin APETx2 or in vivo knockdown of ASIC3 subunit by small interfering RNA led to a significant reduction of postoperative spontaneous, thermal, and postural pain behaviors (spontaneous flinching, heat hyperalgesia, and weight bearing). ASIC3 appears to have an important role in deep tissue but also affects prolonged pain evoked by skin incision alone.

ASIC3s are excitatory ion channels directly activated by extracellular protons that detect the painful drops in pH at incision points. Several factors may participate in the drop of extracellular pH, such as release of the acidic content of lyzed cells, degranulation of mast cells, organic acids released by metabolism..etc This makes makes the Ion Channel a great marker for the studying activation of pain and a potential therapeutic target for mitigating surgical pain.

I will continue to track and report progress.

Delivering TRPV1 shRNA to DRG of T8-L3 Segments of the Spinal Cord

2011-04-17T10:50:00.000-07:00

I have reported use of our i-FectTM siRNA delivery kit for gene expression analysis studies of DOR, hTERT, The β3 subunit of the Na+,K+-ATPase, rSNSR1, NTS1. NAV1.8, Survivin, Flaviviruses and more.

This is the first publication referencing the use of i-Fect to delivery shRNA intrathecally. In this study, researchers knockdown TRPV1 Channels in DRGs to study their role in regulation of blood pressure.

Shuang-Quan Yu, Donna H. Wang. Intrathecal injection of TRPV1 shRNA leads to increases in blood pressure in rats. DOI: 10.1111/j.1748-1716.2011.02285.x. Copyright © 2011 Scandinavian Physiological Society.

Aim: The transient receptor potential vanilloid type 1 (TRPV1) channels have been implicated to play a role in blood pressure regulation. However, contribution of tissue specific TRPV1 to blood pressure regulation is largely unknown. Here we test the hypothesis that TRPV1 expressed in dorsal root ganglia (DRG) of lower thoracic and upper lumbar segments (T8-L3) of the spinal cord and their central and peripheral terminals constitutes a counter regulatory mechanism preventing the increases in blood pressure.

Methods: TRPV1 was knocked down by intrathecal injection of TRPV1 shRNA in rats. Systolic blood pressure and mean arterial pressure (MAP) were recorded. The level of TRPV1 and tyrosine hydroxylase was measured by Western blot.

Results: Intrathecal injection of TRPV1 shRNA (6 μg kg−1 per day) for 3 days increased systolic blood pressure and MAP when compared to rats that received control shRNA (control shRNA: 112±2 vs TRPV1 shRNA: 123±2 mmHg). TRPV1 expression was suppressed in T8-L3 segments of dorsal horn and DRG as well as mesenteric arteries of rats given TRPV1 shRNA. Contents of tyrosine hydroxylase, a marker of sympathetic nerves, were increased in mesenteric arteries of rats treated with TRPV1 shRNA. Pretreatment with the 1-adrenoceptor blocker, prazosin (1 mg/kg/day, p.o.), abolished the TRPV1 shRNA-induced pressor effects.

Conclusion: Our data show that selective knockdown of TRPV1 expressed in DRG of T8-L3 segments of the spinal cord and their central and peripheral terminals increases blood pressure, suggesting that neuronal TRPV1 in these segments possesses a tonic anti-hypertensive effect possibly via suppression of the sympathetic nerve activity.

Transfecting Primary Cortical Neurons with a Plasmid for NCAM

2010-09-17T21:01:00.000-07:00

Harnessing the power of MATra^TM (Magnetic Assisted) Transfection Kits.

Background: The neural cell adhesion molecule (NCAM) plays a major role during development of the nervous system and in synapse plasticity in the adult brain (Diestel et al., 2007). Many studies provide evidence that NCAM can regulate processes like cell migration, axon growth and fasciculation. Endocytosis of NCAM might play a decisive role in these processes as it can potentially enable a quick change in cell adhesion between the cells or towards the extracellular matrix. Endocytosis of
NCAM might also influence these processes by activating specific signal transduction pathways.

Primary cortical neurons present a good in vitro system for these investigations since they allow analysis of molecules within growth cones. For analysis of NCAM, embryonic cortical neurons (E15.5) were transfected with human NCAM one day after isolation. Endocytosis of NCAM was induced 24 hours after transfection and detected by immunofluorescence analysis.

Results:

Images: Endocytosis of NCAM in cortical neurons. After transfection membrane-localized NCAM (not endocytosed) was detected using a Cy3-coupled secondary antibody (red). Afterwards, the internalised, endocytosed NCAM was stained by a Cy2-coupled secondary antibody (green, see arrows) in the cell soma (left) and in axonal vesicles (right).

The data presented here were provided by Simone Diestel, Institute for Animal Sciences, University
of Bonn, Germany. Published also in "Renker, B. et al. MATra - ein Trojanisches Pferd für eine zellschonende Transfektion. BIOSpektrum 04.10:441-442."
Literature: Diestel S, Schäfer D, Cremer H, Schmitz B. (2007) NCAM is ubiquitylated, endocytosed and
recycled in neurons. J Cell Sci. 120: 4035-49

Material and Methods: Primary cortical neurons from C57BL/6 mice embryonic day 15.5 (E15.5) were isolated and plated at a density of 800,000 cells per 24-well plate on poly-L-lysine-coated coverslips. The next day the neurons were transfected with an expression plasmid for human NCAM by Magnet Assisted
Transfection. To induce endocytosis, 24 hours after transfection cells were incubated 30 minutes at
37°C with an antibody which is specific for human NCAM. Subsequently the cells were fixed and
membrane-localized NCAM was visualized using a Cy3-coupled secondary antibody. After permeabilization of the cells internalised NCAM was stained by a Cy2-coupled secondary antibody. The cells were mounted on microscope slides and analysed using a Zeiss LSM510 MetaUV confocal microscope.

Magnet Assisted Transfection (MATra-A reagent): 0.6 μg DNA were dissolved in 50 μl Neurobasal medium. 0.6 μl MATra-A reagent were added, mixed well and incubated for 20 minutes at room temperature. During this incubation time the medium was exchanged with supplemented Neurobasal medium (containing B27 supplement and 2 mM L-glutamine). The transfection mixture was added drop by drop to the cells, dispersed evenly in the medium and immediately placed on the magnetic plate (37°C, 5% CO2, 15 minutes). After 6 hours half of the medium was exchanged with fresh, supplemented Neurobasal medium.

i-Fect and siRNA Delvery to Toll-like receptor 4

2010-08-25T04:32:00.000-07:00

I have reported use of our i-Fect^TM siRNA delivery kit for gene expression analysis studies of DOR, hTERT, The β3 subunit of the Na+,K+-ATPase, rSNSR1, NTS1. NAV1.8, Survivin, Flaviviruses and more.

These represent potential targets for Pain, Cancer and Infectious Disease Therapies.

The latest study involves successful knockdown of the Toll-like receptor 4 (TLR-4):

Wu Fx, Bian Jj, Miao Xr, Huang Sd, Xu Xw, Gong Dj, Sun Ym, Lu Zj, Yu Wf. Intrathecal siRNA against Toll-like receptor 4 reduces nociception in a rat model of neuropathic pain. Int J Med Sci 2010; 7:251-259.

Background: Neuropathic pain is characterized by hyperalgesia, allodynia and spontaneous pain. It often occurs as a result of injury to peripheral nerves, dorsal root ganglions (DRG), spinal cord, or brain. Recent studies have suggested that Toll-like receptor 4 (TLR4) might play a role in neuropathic pain. Methodology/Principal Findings: In this study, we investigated the role of TLR4 in a rat chronic constriction injury (CCI) model and explored the feasibility of treating neuropathic pain by inhibiting TLR4. Our results demonstrated that intrathecal siRNA-mediated suppression of TLR4 attenuated CCI-induced mechanical allodynia and thermal hyperalgesia through inhibiting the activation of NF-κB p65 and production of proinflammatory cytokines (e.g., TNF-α and IL-1β). Conclusions/Significance: These findings suggest that suppression of TLR4 mediated by intrathecally administered siRNA may be a new strategy for the treatment of neuropathic pain.

Images: Screening siRNA for an efficient suppression of TLR4 expression in vitro. HEK-293 cells were co-transfected with both pEGFRC1-TLR4 and either one of three independent siRNA oligonucleotides targeting TLR4 (TLR4-siRNA1-3) or a control siRNA (MM-siRNA). Two days after transfection, EGFP fluorescence was observed under microscope (A) or quantified by flow cytometry (B). (A) EGFP fluorescence under an inverted fluorescence microscope (×100) or cell density under an optical microscope (×100). A, control; B, siRNA1; C, siRNA2; D, siRNA3. (B) The quantification of TLR4-EGFP fluorescence intensity upon siRNA knockdown was evaluated by flow cytometry analysis. Immunofluorescence and flow cytometry results revealed that all 3 siRNAs had efficient inhibition on GFP fluorescence, and TLR4-siRNA2 was the most potent.

intra-i-Fect and intravenous delivery of siRNA

2010-08-16T10:57:00.000-07:00

Deliver siRNA in-vivo with stunning results! Introductory Special-200 to 600 USD (valid through 9/30/2010)
These intra-i-Fect kits are designed to deliver siRNA in vivo via intravenous injections with high efficiency to specific tissue in rats and mice. The protocol involves these simple steps: prep, mix, dry, hydrate and inject.

Figure: siRNAs knock down profiles of the gene related to cancer, diabetes, obesity, steatosis hepatitis, cirrhosis and a gene specifically expressed in endothelial cells in liver

They are developed using a proprietary platform that uses nano-particles as the delivery vehicle. This platform enables:
•Effective delivery (60%+ knockdown) with no toxicity.
•Scalable to high throughput siRNA based gene screening.
•Consistent and reproducible results.

i-Fect, Survivin and Gliobastomas

2010-08-09T11:19:00.000-07:00

I would like to add Survivin to the list of genes successfully silenced in-vitro and in-vivo using our i-Fect^TMsiRNA delivery kit.

The list includes: DOR, hTERT, The β3 subunit of the Na+,K+-ATPase, rSNSR1, NTS1. NAV1.8 and more

Joseph George, Naren L. Banik and Swapan K. Ray. Survivin knockdown and concurrent 4-HPR treatment controlled human glioblastoma in vitro and in vivo. Neuro-Oncology, doi:10.1093/neuonc/noq079.

...survivin siRNA cDNA was suspended in RNAse free sterile water (25 μg DNA/10 μl) and mixed (1:4 v/v) with i-Fect transfection reagent (Neuromics)...

Delivery of the Surivivin siRNA resulted in significant decreases in Glioblatoma Tumor Size.

Using MATRa for siRNA Transfection of Carcinoma Cell Lines

2010-06-29T05:21:00.000-07:00

MATRa^TM-Magnet Assisted Transfection is an easy-to-handle, very fast and highly efficient technology to transfect cells in culture with siRNA. Multiple successes with the system includes Carcinoma Cell Lines.

Efficient transient transfection of siRNA in head and neck cancer cells. The cell line ANT-1 was transiently transfected with MATra-A (1 µl/1 µg DNA) in a 6 well format (5 x 105 cells/cavity) with siRNA against protein 1 (100 nM). After 24 hours total RNA was isolated and expression of protein 1-specific mRNA determined by RT-PCR (upper lane). SiRNA 13 are three different oligonucleotide sequences. Control for consistent loading and cDNA quality: expression of ubiquitary GAPDH mRNA (lower lane).
Protein 2 expression in head and neck cancer cells GHD-1. GHD-1 cells (5 x 105 cells/cavity of a 6 well plate) were transiently transfected with two different siRNAs against protein 2. Expression of protein 2 was detected with specific antibodies in an immunoblot 72 hours after transfection with MATra-A (1 µl / 1 µg DNA). As control ubiquitary β-actin was detected as well.
Treating the carcinoma cells with specific siRNA caused a clear inhibition of protein 1/protein 2 expression which indicates high transfection efficiencies.
(Data kindly provided by Rauch, Schaffrik, Ahlemann and Gires, LMU Munich and GSF, Munich, Germany).

"After having tested MATra in a variety of experimental set ups we can summarize the following advantages:

High transfection efficiency
Easier to handle
High reproducibility
Serum compatibility
Low sensibility against cell confluence"

Dr. Oliver Gires, LMU Munich, Germany

siRNA and i-Fect for the Study of Retinal Disease

2010-06-25T16:42:00.000-07:00

We continue to add new references to the many ways researchers are using i-Fect^TM to increase the potency of siRNA delivery.

This is a new reference from the book entitled Retinal Degenerative Diseases: Laboratory and Therapeutic Investigations By Robert E. Anderson, Joe G. Hollyfield, Matthew M. Lavail.

In this study, researchers used i-Fect to transfect and immortalized cell line from Mouse cones (661W) expressing ELOVL4. Using siRNA designed to silence the ELOVL4 gene, they used i-Fect + siRNA to transfect cells cultured at a density of 2X10⁵. Knockdown was achieved as confirmed by western blot analysis.

Raf-1- selective siRNA and Response to Pain

2010-05-23T18:03:00.000-07:00

I've reported Researchers' success in knocking down in vivo DOR, hTERT, The β3 subunit of the Na+,K+-ATPase, rSNSR1, NTS1. NAV1.8 and more using Neuromics' i-Fect^TM siRNA transfection reagent .

I am pleased to add Raf-1 to this growing list. Here's a recent publication by Dr. EV Varga, University of Arizona:

S Tumati, WR Roeske, T Largent-Milnes, R Wang, TW Vanderah and EV Varga. Sustained morphine-mediated pain sensitization and antinociceptive tolerance are blocked by intrathecal treatment with Raf-1- selective siRNA. This is an Accepted Article that has been peer-reviewed and approved for publication in the British Journal of Pharmacology, but has yet to undergo copy-editing and proof correction. Please cite this article as an "Accepted Article"; doi: 10.1111/j.1476-5381.2010.00869.x.

Background and purpose: Long-term morphine treatment enhances pain neurotransmitter (such as calcitonin gene-related peptide (CGRP)) levels in the spinal cord. It has been suggested previously that increased spinal CGRP may contribute to sustained morphine-mediated paradoxical pain sensitization and antinociceptive tolerance. Previous in vitro studies from our group indicated that Raf-1 kinase-mediated adenylyl cyclase superactivation played a crucial role in sustained morphine-mediated augmentation of basal and evoked CGRP release from cultured primary sensory neurons. The present study was aimed to evaluate the physiological significance of this molecular mechanism in vivo, in rats.

Experimental approach: Rats were intrathecally (i.th) injected with a Raf-1- selective small interfering RNA (siRNA) mixture for 3 days, and were subsequently infused with saline or morphine, s.c. for seven days. Thermal and mechanical sensory thresholds of the animals were assessed by daily behavioural tests. After final behavioural testing (day 6), spinal cords were isolated from each animal group and spinal CGRP and Raf-1 protein levels were measured using ELISA and immunohistochemistry.

Key results: Selective knockdown of spinal Raf-1 protein levels by i.th Raf-1- selective siRNA pre-treatment significantly attenuated sustained morphine-mediated upregulation of CGRP immunoreactivity in the spinal cord of rats and prevented the development of thermal hyperalgesia, mechanical allodynia and antinociceptive tolerance.

Conclusions and implications: Raf-1 played a significant role in sustained morphine-mediated paradoxical pain sensitization and antinociceptive tolerance in vivo. These findings suggest novel pharmacological approaches to improve the long-term utility of opioids in the treatment of chronic pain.

Andy Miller and Enabling RNAi Based Therapies

2010-05-07T19:46:00.000-07:00

Deliverying siRNA in vivo is faced with may challenges. Non specific and immune responses are at the top of the list. These hurdles stand in the way of optimally deliverying siRNA in vivo.

That said, I just came across an excellent presentation outlining methods to improve delivery. Creative chemistry indeed! Here's the link: http://www.labtube.tv/avc-interest.aspx?i=5&c=1&v=327.

Andy Miller talking at RNAi Europe 2009
Andy Miller from Imperial College London giving his keynote lecture at RNAi Europe in Berlin. The talk was entitled, 'Enabling RNAi Therapeutics with Safe, Synthetic, Self Assembling Nanoparticles'.
Published : 2009/10/27

RNAi.net-Check it Out!

2010-05-04T02:35:00.000-07:00

RNAi.net is a portal that has done an excellent job at providing a gateway to many resources to help researchers using siRNAs for gene expression analysis.

There webcast link is particulary useful. Included is a presentation by one of our collaborators: Dr. Mark Behlke. Here's the abstract:

Dicer-substrate siRNAs (DsiRNAs) are synthetic oligonucleotides that are processed by Dicer prior to RISC loading. DsiRNAs often show improved potency over traditional siRNAs in vitro and can have similar benefits in vivo. In collaboration with Dicerna Pharmaceuticals, systematic high throughput screening of DsiRNAs is in progress to identify ultra-potent sites in pharmaceutically relevant target genes. The results of a KRAS screening project will be discussed where over 400 synthetic siRNAs were tested in human and mouse cells. Chemical modification patterns have been defined that improve nuclease stability of the DsiRNA while retaining high potency and evade detection by the innate immune system. These improvements to DsiRNA design will be presented, which have particular utility for in vivo applications. In addition to work in RNAi, results will be presented relating to a new gene-knockdown technology that uses synthetic adaptor oligonucleotides to recruit the nuclear U1 snRNP complex to cleave nascent mRNAs prior to polyadenylation. RNAi and U1 adaptors work by different mechanisms at distinct sub-cellular locations and can be used together to improve knockdown of difficult targets.

Direct Application of siRNA for In Vivo Pain Research

2010-04-01T14:48:00.000-07:00

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

2010-02-18T10:14:00.000-08:00

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

2010-01-07T09:18:00.000-08:00

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...

Using i-Fect for treatment of Glioblastomas

2009-12-01T14:14:00.000-08:00

Dr. Swapan K. Ray, Department of Pathology, Microbiology, and Immunology, University of South Carolina School of Medicine and his team should positive results in reducing growth of Glioblastomas by knocking down hTERT expression using Neuromics' i-Fect ™ siRNA Transfection Kit. Here's the related pub:

Joseph George, Naren L. Banik, Swapan K. Ray. Combination of hTERT Knockdown and IFN-γ Treatment Inhibited Angiogenesis and Tumor Progression in Glioblastoma. Clin Cancer Res 2009;15(23):7186–95

...with i-Fect transfection reagent (Neuromics) to obtain 5 μg DNA/10 μL of injection volume...

Results: In vitro and in vivo angiogenesis assays showed inhibition of capillary-like network formation of microvascular endothelial cells and neovascularization under dorsal skin of nude mice, respectively. We observed inhibition of intracerebral tumorigenesis and s.c. solid tumor formation in nude mice after treatment with combination of hTERT siRNA and IFN-γ. Western blotting of solid tumor samples showed significant downregulation of the molecules that regulate cell invasion, angiogenesis, and tumor progression.

Conclusions: Our study showed that the combination of hTERT siRNA and IFN-γ effectively inhibited angiogenesis and tumor progression through the downregulation of molecules involved in these processes. Therefore, the combination of hTERT siRNA and IFN-γ is a promising therapeutic strategy for controlling the growth of human glioblastoma.

Delivering 27mer DsiRNAs to Mice DRGs

2009-06-23T08:39:00.000-07:00

I have been a proponent of using 27mer DsiRNAs (Dicer Substrate Small Interfering RNAs) with our i-Fect kits to deliver siRNA to the CNS for gene expression analysis. The potency of this platform was highlighted in my profile of Dr. Mark Behlke.

It was further confirmed by in Studies conducted by Dr. Philippe Serrat and his team at University of Sherbrooke.

Louis Doré-Savard, Geneviève Roussy, Marc-André Dansereau, Michael A Collingwood, Kim A Lennox, Scott D Rose, Nicolas Beaudet, Mark A Behlke and Philippe Sarret. Central Delivery of Dicer-substrate siRNA: A Direct Application for Pain Research. Molecular Therapy (2008); Jul;16(7):1331-9. Epub 2008 Jun 3 doi:10.1038/mt.2008.98.

Using ultra low dose of DsiRNAs complexed with Neuromics’ i-Fect , they were able to successfully reduce NTS2 gene expression by up to 86% in rat lumbar Dorsal Root Ganglia after only two intrathecal injections. This was confirmed by Western Blot and qPCR analysis.

We now have further confirmation of the capabilities of this delivery platform in a just released publication by Dr. Jeffrey Mogil and team:

Michael L. LaCroix-Fralish, Gary Mo, Shad B. Smith, Susana G. Sotocinal, Jennifer Ritchie, Jean-Sebastien Austin, Kara Melmed, Ara Schorscher-Petcu, Audrey C. Laferriere, Tae Hoon Lee, Dmitry Romanovsky, Guochun Liao, Mark A. Behlke, David J. Clark, Gary Peltz, Philippe Séguéla, Maxim Dobretsov and Jeffrey S. Mogil. The β3 subunit of the Na+,K+-ATPase mediates variable nociceptive sensitivity in the formalin test. doi:10.1016/j.pain.2009.04.028.

IT Delivery of siRNA in vivo supplement

Knockdown of rSNSR1 in vivo

2009-04-20T08:12:00.000-07:00

The parade of success with use our i-FectTM in vivo grows. Here's the most recent study:

Christian Ndong, Amynah Pradhan, Carole Puma, Jean-Pierre Morello, Cyrla Hoffert, Thierry Groblewski , Dajan O’Donnell , Jennifer M.A. Laird. Role of rat sensory neuron-specific receptor (rSNSR1) in inflammatory pain: Contribution of TRPV1 to SNSR signaling in the pain pathway. PAIN 143 (2009) 130–137.
...For experiments in which siRNA was delivered by bolus injections, 10 ul of siRNA or vehicle was injected directly into the intrathecal catheter once daily for 4 days. In this case, siRNAs were prepared immediately prior to administration by mixing the RNA solution (200 uM in annealing buffer) with the transfection reagent i-FectTM (Neuromics) at a ratio of 1:4 (w:v) for a final siRNA/ lipid complex concentration of 2 ug/10 ul...

Related Data:

Images: in vivo characterization of knockdown produced by rSNSR1 siRNA. (A) A dose-dependent decrease in rSNSR1 mRNA levels measured in lumbar L3/L4/L5 DRGs was
observed when rSNSR1 siRNA (n = 7–14/group) or MM siRNA (n = 6/group) was delivered by four daily bolus injections. *p < 0.05; **p < 0.01; ***p < 0.001 as determined by oneway analysis of variance followed by sequential testing. (B) rSNSR1 immunoreactivity in dorsal horn of the spinal cord was visibly reduced in rSNSR1 siRNA-treated animals (5 lg/day, left panel). Immunoreactivity with neuron-specific isolectin B4 (IB4; right panel) did not change between treatment groups, showing the integrity of each dorsal horn analyzed (n = 6/group). (C) A semi-quantitative score of rSNSR1 immunoreactivity showed that siRNA treatment greatly decreased rSNSR1 protein levels compared to MM and control groups. A blinded observer scored 9–12 individual sections taken from a 1 cm segment of the spinal cord.

Did Your RNAi Experiment Work?

2009-04-06T07:37:00.000-07:00

This is a good methods publication on RNAi transfection: Reliably Validating RNA Interference with qRT-PCR.

Bill Wang, Song Tian, Qiong Zhou, and Xiao Zeng. SA Biosciences.

Intrathecal Delivery of siRNA

2008-12-16T11:44:00.000-08:00

We wanted to present yet another publication referencing successful delivery of siRNA using i-Fect^TM:

Suneeta Tumati, Tally Largent Milnes, Henry I. Yamamura, Todd W. Vanderah, William R. Roeske and Eva V. Varga. Intrathecal Raf-1-selective siRNA attenuates sustained morphine-mediated thermal hyperalgesia. doi:10.1016/j.ejphar.2008.10.033

...siRNAs stock solutions (100 μM) were prepared in double distilled RNAse free water and stored in aliquots at −80 °C. For intrathecal treatment, aliquots of the stock solution (2 μg of the appropriate siRNA) were mixed (1:5 v/v)with i-Fect transfection reagent (Neuromics, Edina, MN). After recovery from the surgery (5–7 days), the animals received intrathecal injections (2 ug siRNA/10 ul/rat) of either a lipid encapsulated Raf-1-selective siRNA mixture (Smart pool siRNA, Dharmacon Inc; Chicago, IL, Cat # L-087699-00) (Raf-1 siRNA groups) or i-Fect encapsulated non-targeting dsRNA (Dharmacon, #D-001810-01-20) (control mismatch siRNA groups) or the transfection lipid alone, once daily, for 3 days, as described earlier (Gardell et al., 2002). Intrathecal injections of the siRNAs or the transfection agent alone did not cause any sign of behavioral toxicity. Western blots, using a Raf-1-selective antibody, indicated that intrathecal treatment with the Raf-1-selective siRNA mixture for 3 days significantly reduced Raf-1 protein levels in the dorsal root ganglion and in the dorsal horn of the spinal cord...

Delivering Naked siRNA by Direct Injection

2008-11-02T07:00:00.000-08:00

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).

ACIC3 Receptors Knockdown in vivo

2008-10-19T12:10:00.000-07:00

Researchers using siRNA complexed with our i-Fect ™ transfection regent have successfully knocked down ASIC3 Receptors in vivo. This publication joins the growing parade (starting with Luo et al, 2005) that refererence successuful modulation of receptors involved in pain using siRNA complexes.

These studies all share animal behavior studies showing a marked change in response to pain stimuli after treatment.

In this study, Dr. Eric Lingueglia and his team found Peripheral ASIC3 channels are thus essential sensors of acidic pain and integrators of molecular signals produced during inflammation where they contribute to primary hyperalgesia.

Emmanuel Deval, Jacques Noël, Nadège Lay, Abdelkrim Alloui, Sylvie Diochot, Valérie Friend, Martine Jodar, Michel Lazdunski and Eric Lingueglia. ASIC3, a sensor of acidic and primary inflammatory pain. The EMBO Journal advance online publication 16 October 2008; doi: 10.1038/emboj.2008.213

Cy3-labelled siRNA no. 1121 and its corresponding scramble (no. 1121S; GCTCACACTACGCAGAGAT) synthesized by MWG Biotech (Germany) were injected in rats by intrathecal bolus to the lumbar region of the spinal cord once a day for 3 days before the induction of inflammation with CFA. Each 10-ml injection corresponded to 2 mg of siRNA complexed with i-Fect siRNA transfection reagent (Neuromics) at a ratio of 1:4 (w:v) (Luo et al, 2005), following the supplier’s suggested protocol. siRNA uptake in lumbar DRGswas monitored by fluorescence microscopy on cryostat sections 24 h after a single intrathecal injection.

Here’s a synopsis of results:
Inflammation was produced by CFA injection, which led to primary heat hyperalgesia, and this hyperalgesia was drastically reduced by the ASIC3 blocker APETx2 injected subcutaneously, which only access cutaneous nociceptors. It was also drastically reduced when, before triggering the inflammation state, intrathecalinjections of an siRNA against ASIC3 had induced a knockdown of ASIC3 expression in lumbar DRGs.

siRNA-mediated gene silencing

2008-10-04T07:08:00.000-07:00

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.

Transfecting Sympathetic Neurons

2008-08-28T14:50:00.000-07:00

GDNF and Ret are important to the growth, maintenance and survival of Neurons. The GDNF ligands act via activation of Ret. Every step towards understanding the intricacies of this pathway, brings researchers closer towards unlocking the code for Neurodegenerative Disease therapies.

siRNA is an important tool for studying the Neurotrophic pathways as researchers can use it to modulate the expression of related receptors. The tricky part is getting sufficient siRNA into neurons to do the appropriate studies of how modulating targeted genes results in changes in protein expression.

Here Drs. Cynthia Tsui and Brian Pierchala have published results from there studies of C2AP and Cbl-3/Cbl-c and Ret Transduction. One of the keys to this study was using siRNA to silence CD2AP and Cbl-3 expression. By turning these off they were able to identify a critical checkpoint in the Ret pathway.

Cynthia C. Tsui and Brian A. Pierchala CD2AP and Cbl-3/Cbl-c Constitute a Critical Checkpoint in the Regulation of Ret Signal TransductionJ. Neurosci., Aug 2008; 28: 8789 - 8800 ; doi:10.1523/JNEUROSCI.2738-08.2008.

...Control, CD2AP, and Cbl-3 siRNAs (Applied Biosystems/Ambion) were transfected into 4 DIV sympathetic neurons using the i-Fect ™ reagent according to the manufacturer’s instructions (Neuromics). Transfection efficiency was determined by the cotransfection of a fluorescently labeled nontargeting, control siRNA (siGLO RISC-free siRNA; Dharmacon RNA Technologies). Immunoblotting of the targeted proteins determined that the maximal knockdown of protein expression was observed 72 h after siRNA transfection. Greater than 90% of SCG neurons were transfected, as ascertained by the level of intracellular fluorescence of the siGLO siRNA.

Down Regulating the Smad and Neuro-regeneration

2008-08-24T22:51:00.000-07:00

The inventors down regulated Smad 2/3 (an inhibitor of neuro-regeneration) in vivo via delivery of siRNA to the spinal cord using catheters.

Inhibiting smad signaling promotes neuron regeneration.
Inventors: Fan Wang, Zhigang He
USPTO Application #: 20080031911
Inhibition of Smad2/3 Signaling Promotes Axonal Regeneration after Spinal Injury in Rats-Gene Expression Knockdown in vivo!
One hour after the spinal cord is lesioned, the rats in the SB-505124 group receive a bolus injection of SB-505124 (30 mg/kg) in 0.9% saline administered via a tail vein. The treatment is repeated every 24 hours on days 1 through 7 post-lesion. Vehicle only control rats undergo the same treatment but are injected with an equal volume of 0.09% saline in a tail vein. At the same treatment time points as the SB-505124 group, the Smad2/3 siRNA group and corresponding controls receive 10 .mu.l rat Smad2/3 siRNA (Dharmacon, Lafayette, Colo.), mismatch siRNA, or transfection reagent only delivered to the spinal cord via the catheters. The siRNA (or mismatch siRNA control) complexes are prepared immediately prior to administration by mixing the RNA solution (200 .mu.M in annealing buffer) with a transfection reagent, i-Fect ™ . (Neuromics, Edina, Minn.), in a ratio of 1:4 (w:v). At this ratio, the final concentration of RNA as an RNA/lipid complex is 2 .mu.g in 10 .mu.l.

siRNA and Diabetes

2008-07-31T18:19:00.000-07:00

I'll be keeping my eyes on this and post updates.

Quark Pharmaceuticals Announces First Patient Dosing by Pfizer in Phase II Trial of RNAi Therapy in Diabetic M
Clinical Program Leverages Quark's RNAi Technology FREMONT, Calif., July 30 /PRNewswire/ -- Quark Pharmaceuticals, Inc., a development-stage pharmaceutical company discovering and developing novel RNA interference (RNAi)-based therapeutics,...

Improving 27mer DsiRNA Performance

2008-07-20T19:53:00.000-07:00

The DsiRNA story marches forward with yet another important publication. We will give you an opportunity to upload the fulltext article at the end of this posting.

Dr. Mark Behlke, Dr John Rossi and team have been gaining deeper understanding of the Mechanism of Dicer-substrate small-interfering RNA (DsiRNA) processing. This understanding is leading to better and better designs of the RNA duplexes. These designs or chemical modifications are necessary steps in the drug design and development process.

This publication looks at design from the perspective of:

Nuclease Stability
Pharmacokinectics
immune response

I believe this is an important publication for researchers wanting to better understand:

The mechanisms behind successful delivery of DsiRNA for gene expression studies.
Variations in potency.

upload article: oligo-18-p187-2008-collingwood-dsirna-modifications1

Coming Soon

2008-07-08T07:20:00.001-07:00

Agenda Topics:
Target Discovery and Validation
Transfection
siRNA Therapeutics
in vivo RNAi-recent in vivo RNAi Pubs
Bioinformatics of small RNAs
siRNA library screens
microRNAs in
Disease Biology:

Stem Cell Biology

Diagnostics

Virology

Biogenesis

Development

RNAi Researchers Galvanized by Advances

2008-06-18T07:33:00.000-07:00

Technology's Viability in Drug Development Is Finally Established
Author: Elizabeth Lipp
Publication: Genetic Engineering & biotechnology News
Publisher: Mary Ann Liebert, Inc. publishers
Date: Jun 1, 2008
Copyright © 2008 GEN Publishing

Article Link: http://www.genengnews.com/articles/chitem.aspx?aid=2493

Notable Quotables:

“Long dsRNAs have been employed for many years as a means to modulate gene expression in plants, yeast, and C. elegans,” noted Mark Behlke, M.D., Ph.D., svp of molecular genetics and CSO at Integrated DNA Technologies (IDT; www.idtdna.com).
“Similar attempts in higher organisms failed due to interferon activation, however we now know that short RNA duplexes can be safely used in mammalian systems both in vitro and in vivo. The technology has rapidly matured, thanks in large part to all that was learned over the past 20 years using antisense oligonucleotides. RNAi is now routinely employed in vivo as an experimental tool and numerous groups are vigorously pursing the use of RNAi compounds as therapeutics. Several siRNA drugs are already in clinical trials and more are in preclinical development.”

Hi Thru Put Delivery of siRNA/DsiRNA

2008-06-16T07:58:00.000-07:00

NEW PRODUCT

i-Fect ™ Hi Put 96-New

Learn about transfection products @ neuromics

Central Delivery of DsiRNA

2008-06-04T12:53:00.000-07:00

Louis Doré-Savard, Geneviève Roussy, Marc-André Dansereau, Michael A Collingwood, Kim A Lennox, Scott D Rose, Nicolas Beaudet, Mark A Behlke and Philippe Sarret. Central Delivery of Dicer-substrate siRNA: A Direct Application for Pain Research. Molecular Therapy (2008); doi:10.1038/mt.2008.98.

Images: Cellular uptake of Texas Red–tagged Dicer-substrate small-interfering RNA (DsiRNA) by spinal nociceptive structures. (a,b) Distribution of fluorescence in lumbar dorsal root ganglia at 24 hours after intrathecal injection of a control siRNA conjugated with Texas Red (1 μg administered twice with a 24-hour interval; n = 3). As seen by confocal microscopy, the staining is not uniformly distributed among the cells. Higher-magnification images also show that the fluorescent signal is detected in the form of small intracytoplasmic hot spots, sparing the nucleus. (c,d) Expression of Texas Red–tagged DsiRNA in a dorsal spinal cord section taken from an L5 segment. Fluorescence clusters are present in the cytoplasm of the cells. Note that the labeling is also detected in neuronal processes. Scale bar: 60 μm in a, 30 μm in b,25 μm in c and 15 μm in d. Courtesy of Dr. Nicolas Beudeat. Published in Molecular Therapy (2008); doi:10.1038/mt.2008.98

Use of Dicer Substrate siRNAs

Dicer-substrate siRNAs (DsiRNAs) have recently been employed for in vivo studies using intraperitoneal and intrathecal routes of administration. “IDT got into RNAi research in collaboration with John Rossi at The City of Hope and the Beckman Research Institute five years ago,” explained Dr. Behlke. In vivo, long dsRNAs are cleaved by the RNase III class endoribonuclease dicer into 21–23 base duplexes having 2-base 3´-overhangs. These species, called small interfering RNAs (siRNAs), enter the RISC and serve as a sequence-specific guide to target degradation of complementary mRNA species.
Typically, siRNAs are chemically synthesized as 21 mers with a central 19 bp duplex region and symmetric 2-base 3´-overhangs on the termini, reported Dr. Behlke. These duplexes are transfected into cells lines, directly mimicking the products made by dicer in vivo. Most siRNA sequences can be administered to cultured cells or to animals without eliciting an interferon response.

“We observed,” added Dr. Behlke, “that the use of slightly longer sequences that were substrates for dicer showed improved potency, which we theorize relates to participation of dicer in RISC loading. We are now focusing on the use of these compounds in vivo.”

IDT recently completed a collaborative study with the laboratory of professor Phillipe Sarret at the Université de Sherbrooke in Quebec. The collaboration studied the use of DsiRNA to knockdown the GPCR NTS2 (neurotensin type 2 receptor) in rat spinal cord and dorsal root ganglia. The RNA duplexes were administered by intrathecal injection in a cationic lipid slurry. Stimulation of NTS2 with a chemical agonist resulted in analgesia. Pain responses were monitored in treated animals by dipping their tails in hot water with and without the chemical agonist.

“The anti-NTS2 DsiRNA treated animals showed a marked difference of response to the test stimulus,” said Dr. Behlke. “We recorded differences of up to five seconds, which is quite a long time for a rat to sit with its tail in hot water. While interesting, this work mainly represents a pilot study to demonstrate the feasibility of using DsiRNA to study pain pathways in rats. We were amazed at the low dose it takes to get knockdown—we used 1 mcg/200 g rat, which is only a 0.005 mg/kg dose.” Modulating CNS disease and affecting brain processes is clearly possible, but better methods of delivery are going to be needed to move this approach from a research tool into the clinic, noted Dr. Behlke.

Ret Knockdown Using Small Interference RNA

2008-04-09T08:30:00.000-07:00

Nearly 100% Transfection Efficiency Reported in vitro with i-Fect ™.

Images: Ret receptor knockdown using small interference RNA (siRNA) in podocytes. (A) Transfection efficiency: mouse podocytes were transfected with 100 nM concentrations of Ret siRNA or vehicle alone. (a) When podocytes were exposed to i-Fect alone, there was no toxicity. (a and b) A transfection efficiency of nearly 100% was achieved with 100 nM concentration of Ret siRNA (b). Co-transfection with a fluorescently tagged control siRNA was used to determine the transfection efficiency, and fluorescence microscopy revealed a perinuclear localization of the tagged RNA (b, arrowhead). (B) Western blot analysis of Ret after transfection: Ret immunoblotting (top) of WCL 2, 3, or 4 d after transfection revealed that Ret was downregulated within 2 d after transfection with 100 nM Ret siRNA. Transfection of control siRNA at day 4 served as a negative control, and the maximal knockdown of Ret was observed 4 d after transfection. GAPDH immunoblotting confirmed equal protein loading (bottom). doi:10.1681/ASN.2005080835. (full text publication)

Methods: Ret small interference RNA (siRNA) knockdown was performed by using transient transfection of pooled functionally validated Ret siRNA (SMARTpool mouse RET siRNA; Dharmacon, Lafayette, CO). HSMP cells that were differentiated for 10 to 12 d were maintained at 10% FBS/RPMI as described above and transfected using the i-Fect siRNA
transfection reagent (Neuromics, Northfield, MN). For determination
of the transfection efficiency, a Texas Red–labeled siRNA (siGLO RISCFree
siRNA; Dharmacon) was co-transfected with Ret siRNA and visualized
using fluorescence microscopy. For control siRNA samples,
identical conditions were used with the substitution of siGLO-RISCFree
siRNA for Ret siRNA. For determination of the efficiency of Ret
knockdown, Western analysis for Ret was performed on WCL from
cells 24 to 96 h after the transfection. Several concentrations of Ret
siRNA (40, 60, and 100 nM) were tested to determine optimal knockdown
conditions. For apoptosis assays, podocytes were exposed to
UV-C or PA (40 g/ml) on days 3 to 4 after transfection of Ret siRNA
or control siRNA (100 nM). Apoptosis was measured by counting
podocytes with Hoechst-positive pyknotic nuclei 3 h after UV and 5 h
after exposure to PA.

RNAi therapeutics: a potential new class of pharmaceutical drugs

2007-11-26T09:27:00.000-08:00

Comprehensive and Cogent overview on delivery methods:

David Bumcrot1, Muthiah Manoharan1, Victor Koteliansky1 and Dinah W Y Sah1. RNAi therapeutics: a potential new class of pharmaceutical drugs

Nature Chemical Biology 2, 711-719 (2006) doi:10.1038/nchembio839

i-Fect and Posters @ SfN

2007-11-07T17:44:00.000-08:00

Program#/Poster#:
72.13/EE20
Title:
RNAi of neuropeptide Y for neuropathic pain
Location:
San Diego Convention Center: Halls B-H
Presentation Start/End Time:
Saturday, Nov 03, 2007, 1:00 PM - 2:00 PM
Authors:
*M.-C. LUO1, D. ZHANG1, E.-T. ZHANG1, Q. CHEN1, P. GE2, D. SAH2, T. VANDERAH1, F. PORRECA1, J. LAI1; 1Dept Pharmacol, Univ. Arizona Hlth. Sci. Ctr., Tucson, AZ; 2Alnylam Pharmaceuticals, Inc., Boston, MA
Neuropeptide Y (NPY) is upregulated after L5/L6 spinal nerve ligation (SNL) injury in large diameter dorsal root ganglion (DRG) neurons that project, via the ipsilateral dorsal column, to the brain stem gracile nucleus. Action of NPY in the gracile nucleus promotes hypersensitivity to innocuous touch, which mimics neuropathic pain in human (Ossipov et al., 2002). We hypothesize that a knock down of NPY in the injured DRG by small interfering RNA (siRNA) blocks the nerve injury induced tactile hypersensitivity.A number of synthetic siRNA that target preproNPY were screened by transfecting the cell line, F-11, that expresses endogenous NPY. A maximal knock down of 89% of preproNPY mRNA and 47% of the peptide was observed in vitro. The knock down of both mRNA and peptide lasted at least 72 hr following a single transfection. Sequence specificity of the siRNA-mediated knock down of NPY is confirmed by mismatch RNA control.A siRNA for preproNPY was delivered intrathecally to the lumbar spinal cord once daily at 2 μ g (with the vehicle i-Fect) in rats beginning one day prior to SNL for 7 days. SiRNA, but not mismatch RNA treatment, significantly attenuated tactile hypersensitivity in the injured paw. A moderate attenuation of NPY expression was confirmed by NPY-immunoreactivity in lumbar spinal cord and DRG in siRNA treated rats. Tactile hypersensitivity returned after cessation of siRNA treatment. siRNA treatment initiated after the tactile hypersensitivity was established was ineffective in reversing the abnormal pain behavior. Thus, early but not delayed intervention of NPY expression in the injured nerve significantly attenuated nerve injury induced tactile hypersensitivity, which is likely due to limited efficacy of the siRNA against a highly abundant gene target. Effect of siRNA may be further optimized in vivo by chemical stabilization and delivery. This study is supported by NIH grant R01NS046785.
Disclosures:
M. Luo , None; D. Zhang, None; E. Zhang, None; Q. Chen, None; P. Ge, None; D. Sah, None; T. Vanderah, None; F. Porreca, None; J. Lai, None.
Support:
NIH grant R01NS046785
[Authors]. [Abstract Title]. Program No. XXX.XX. 2007 Neuroscience Meeting Planner. San Diego, CA: Society for Neuroscience, 2007. Online.

Program#/Poster#:
509.6/PP9
Title:
Small interfering RNA-mediated selective knockdown of NTS2 receptors reverses neurotensin-induced analgesia in rats
Location:
San Diego Convention Center: Halls B-H
Presentation Start/End Time:
Monday, Nov 05, 2007, 2:00 PM - 3:00 PM
Authors:
*L. DORE-SAVARD1,2, G. ROUSSY1, M.-A. DANSEREAU1, K. BELLEVILLE1, N. BEAUDET1, M. BEHLKE2, P. SARRET1; 1Physiology and Biophysics, Univ. Sherbrooke, Sherbrooke, PQ, Canada; 2Integrated DNA Technologies Inc., Coralville, IA
We have previously shown that NTS2 receptors play an important role in the regulation of nociceptive functions at the spinal level. Indeed, intrathecal (i.t.) administration of NTS2-selective agonist, levocabastine and JMV-431, induced a dose-dependent antinociceptive responses in the tail-flick test. Recent discoveries revealed that the delivery of small interfering RNA (siRNA) in vivo resulted in the potent, long-lasting, post-transcriptional silencing of specific genes. Thus, we investigated the effect of i.t. injection of siRNA targeting NTS2 receptors for the modulation of pain. Using Real-time PCR analysis, we first identified several siRNA capable of a high-selective attenuation of NTS2 message in rNTS2 stably transfected CHO cells. Dicer-substrate siRNA (DsiRNA), which have been shown to have increased potency in vitro compared to 21-mers, were therefore administered i.t. at the lumbar spinal cord level on days 0 and 1 at a dose of 1 µg formulated in the cationic lipid i-Fect transfection agent. Twenty-four hours after the last dose of DsiRNA, NTS2 protein levels were markedly reduced when examined by Western blot in dorsal root ganglia (DRG, 43.4%) and spinal cord (27.4%), compared to rats receiving control DsiRNA. Rats were then tested for antinociception by the NTS2-selective agonist, JMV-431 in the tail-flick test. Pretreatment with the DsiRNA targeting NTS2, but not the mismatch RNA or vehicle alone reduced by 93.3% the analgesic effects of JMV-431. The functional inhibition of NTS2 by DsiRNA was progressively reversed within 4 days after the last RNA injection. Texas Red-labeled DsiRNA were clearly detected in the cytoplasm of both lumbar DRG and spinal cord neurons, indicating that DsiRNA were taken up and transported within spinal nociceptive structures. Taken together, these results demonstrate that silencing of NTS2 receptors using a DsiRNA approach abolishes NT-induced antinociception and further support a role for NTS2 in the management of acute pain.
Disclosures:
L. Dore-Savard, None; G. Roussy, None; M. Dansereau, None; K. Belleville, None; N. Beaudet, None; M. Behlke, None; P. Sarret, None.
Support:
CIHR, FRSQ and CRS
[Authors]. [Abstract Title]. Program No. XXX.XX. 2007 Neuroscience Meeting Planner. San Diego, CA: Society for Neuroscience, 2007. Online.2007 Copyright by the Society for Neuroscience all rights reserved. Permission to republish any abstract or part of any abstract in any form must be obtained in writing by SfN office prior to publication.

More on Delivering siRNA in vivo

2007-10-30T05:29:00.000-07:00

We are excited about this upcoming presentation at Society for Neuroscience comference presesentation. Neuromics' i-Fect ™ reagent was used to deliver siRNA in vivo to silence the NTS2 gene.

Program#/Poster#:509.6/PP9
Title:Small interfering RNA-mediated selective knockdown of NTS2 receptors reverses neurotensin-induced analgesia in rats
Location:San Diego Convention Center: Halls B-H
Presentation Start/End Time:Monday, Nov 05, 2007, 2:00 PM - 3:00 PM
Authors:*L. DORE-SAVARD1,2, G. ROUSSY1, M.-A. DANSEREAU1, K. BELLEVILLE1, N. BEAUDET1, M. BEHLKE2, P. SARRET1;
1Physiology and Biophysics, Univ. Sherbrooke, Sherbrooke, PQ, Canada; 2Integrated DNA Technologies Inc., Coralville, IA2007

Copyright by the Society for Neuroscience all rights reserved. Permission to republish any abstract or part of any abstract in any form must be obtained in writing by SfN office prior to publication

i-Fect and Nav1.8 Gene Silencing in vivo

2007-05-22T17:07:00.000-07:00

Referenced in United States Patent 20070105806

Link to patent: Free Patents Online
[0241] The effect of siRNAs against Nav1.8, formulated with iFECT, on complete Freund's adjuvant-induced tactile hypersensitivity was evaluated in rats (FIG. 5). Adult male Sprague-Dawley rats received an injection of CFA (150 uL) into the hindpaw on day 0. siRNAs against Nav1.8 were then administered by intrathecal bolus to the lumbar region of the spinal cord on days 1, 2 and 3; specifically, for each bolus injection, 2 ug of siRNA was complexed with iFECT transfection reagent (Neuromics, Minneapolis Minn., USA) at a ratio of 1:4 (w:v) in a total volume of 10 uL. Five groups of rats (with 5 rats per group) were treated with either siRNA (AL-DP-6049, AL-DP-6209, AL-DP-6217 or AL-DP-6218; Table 1), or PBS, in the presence of iFECT. Tactile hypersensitivity was expressed as tactile withdrawal thresholds which were measured by probing the hindpaw with 8 calibrated von Frey filaments (Stoelting, Wood Dale Ill., USA) (0.41 g to 15 g). Each filament was applied to the plantar surface of the paw. Withdrawal threshold was determined by sequentially increasing and decreasing the stimulus strength and calculated with a Dixon non-parametric test (see Dixon, W. J. (1980) "Efficient analysis of experimental observations" Annu Rev Pharmacol Toxicol 20:441-462; Chaplan, S. R., F. W. Bach, et al.(1994) "Quantitative assessment of tactile allodynia in the rat paw" J Neurosci Methods 53:55-63). Tactile thresholds were measured before CFA injection to assess baseline thresholds, and then on day 4 after CFA and treatment with test articles. In rats treated with PBS, tactile hypersensitivity was pronounced on day 4, as evidenced by reduced paw withdrawal threshold, as expected. In rats treated with AL-DP-6209, tactile thresholds were nearly normalized on day 4, demonstrating that the Nav1.8 siRNA, AL-DP-6209, is efficacious in vivo against inflammation-induced hyperalgesia. Treatment with the Nav1.8 siRNA, AL-DP-6217, resulted in the average tactile threshold trending towards baseline, with one of five rats demonstrating a normal tactile response. AL-DP-6049 and AL-DP-6218 did not significantly alter tactile thresholds compared to PBS treatment, in this experimental paradigm.[0242] These results demonstrate that siRNAs targeting Nav1.8, formulated with transfection reagent and administered intrathecally, alleviate CFA-induced tactile hyperalgesia, and therefore represent a novel approach to providing effective treatment of clinical inflammatory pain.

Magnetically Enhanced siRNA Transfection

2007-04-02T16:44:00.000-07:00

We have added a new set of products that magnetically drive transfection.

MATra products are designed to enhance the transfection capabilties of our i-Fect and pn-Fect kits.

The technique requires lease or purchase of the Universal Magnetic Plate AND for i-Fect and other siRNA transfection reagents, please also purchase the MATra-siRNA Reagent. For pn-Fect and other Nucleic Acid transfectants, you would buy the MATra-A Regeant.
If your cells are in suspension, you will also need MATra-s-Immobilizer.
Manual for All MATra Products
MATra FAQs

Magnet Assisted Transfection Animation

References:

Publications:
Bertram, J. (2006) MATra - Magnet Assisted Transfection: Combining Nanotechnology and Magnetic Forces to Improve Intracellular Delivery of Nucleic Acids. Current Pharmaceutical Biotechnology 7, 277-285.

Goulimari, P., Kitzing, T.M., Knieling, H., Brandt, D. T., Offermanns, S.
and Grosse, R. (2005) G?12/13 Is Essential for Directed Cell Migration and Localized Rho-Dia1 Function. J. Biol. Chem., Vol. 280, Issue 51, 42242-42251.

Kumbrink, J., Gerlinger, M., and Johnson, J.P. (2005) Egr-1 Induces the Expression of Its Corepressor Nab2 by Activation of the Nab2 Promoter Thereby Establishing a Negative Feedback Loop. J. Biol. Chem., Vol. 280, Issue 52, 42785-42793.

Liman, J., Ganesan, S., Dohm, C.P., Krajewski, S., Reed, J.C., Bähr, M., Wouters, F.S. and Kermer, P. (2005) Interaction of BAG1 and Hsp70 Mediates Neuroprotectivity and Increases Chaperone Activity. Molecular and Cellular Biology, Vol. 25, Issue 9, 3715-3725.

Luo, W., Wang, Y., Hanck, T., Stricker, R. and Reiser, G. (2006) JAB1, A Novel Protease-Activated Receptor-2 (PAR-2) - Interacting Protein Is Involved in PAR-2-Induced Activation of AP-1. J. Biol. Chem. Papers in press. Published on January 12, 2006 as Manuscript M510784200.

Duration of siRNA studies-pain study example

2007-02-06T20:07:00.000-08:00

We are working with an investigator on using RNAi to relieve pain and his study prompted some discussion about the length of time before an effect is seen. It is important to note that these types of experiments take time to see an effect. In studying the pain response, the effect may take even longer.

Dorn et al (Nucleic Acids Res. 2004 Mar 16;32) examined the ability of siRNA to relieve chronic neuropathic pain targeting P2X3 in vivo. They showed a significant effect on the ability of the siRNA to relieve the pain; however there was no significant effect until around 6 days post.

Another study by Hemmings-Mieszczak et al (Nucleic Acids Research, 2003, Vol. 31, No. 8 2117-2126) showed that in an in vitro model, oligofectamine-mediated transfection of siRNA resulted in 60±90% downregulation of P2X3, but only after about 3 days.

So a key factor in any siRNA transfection study is to carry the experiment out long enough, which in in vivo work can be longer than for in vitro studies. As Mark points out, "Normally in cell culture we would look at mRNA levels at 24 h and protein levels at 48-72 h post transfection, depending upon the cell division rate and the half life of the protein. Long lived proteins in non-dividing cells could theoretically take multiple administrations of reagent over a week or more to knock down your targeted protein in vivo."

Best regards,

Steve

Transfecting Primary Cells

2007-01-15T11:18:00.000-08:00

We welcome any comments on you experiences with transfecting primary cell lines with cationic lipids and/or electroporation.

Here're are interesting comments of transfecting primary cells from Dr. Mark Behlke, CSO of IDT:

"Just wanted to give you a project update. We have good transfection conditions worked out for LTK cells and CHO cells now. Different reagents and different conditions proved to be optimal for each cell line.

The LTK cells proved to be a real nightmare. We were testing out different siRNAs and had a weird problem that it seemed that none of the siRNA worked even a little, with the weird observation that our controls were “very low”. After quite a bit of investigation, we finally figured out that the LTK cells were triggering an IFN pathway response to the siRNAs. Unfortunately, it was a bit odd and it took some time to figure out what was going on. The cells did not just “die” or show changes in the usually time frame expected for Type-I IFN responses, but clearly were activating new cytokine related transcription units. The promoter driving your transgene is regulated by the transcription factors induced, such that NTS1 mRNA levels increased upon transfection. The controls were “low”, showing basal levels, and the transfected cells were all “high”. It turns out that our dye-labeled transfection control sequence was triggering the pathway in a sequence-specific fashion. We are now leaving out the transfection control and things are OK. We can also totally evade the response using 2’OMe modified duplexes (we usually use these modified duplexes for siRNA intended for IV administration, since exposure to PBMCs usually triggers any/all possible IFN responses).

We can avoid these responses and now have the assay system working. So, we can finally start proper testing!"

Preliminary Results from Dr. Helen Hellmich of UTMB:

"It looks like the I-fect delivered the control labeled IDT oligos to approximately the right place in the rat hippocampus 48 hours post-injection. After my research assistant comes back from her vacation, we will try a longer time point, 72 hours and alter our stereotactic coordinates slightly to more directly target the CA3 subregion. After that we will try the real siRNA injections with I-fect, hopefully in the next few weeks. I will keep you all posted to whether this really works."

Protocols for Delivering siRNA to Neurons

2007-01-12T14:33:00.000-08:00

Here's a link to the protocol: Transfecting Schwann Cells with i-Fect and below are related data.

Figure:si RNA-mediated suppression of target gene expression in Schwann cells. A, Detergent extracts of siNeg- or siGly1- transfected Schwann cells were digested with heparitinase and subjected to immunoblot analysis with anti-glypican-1 antibodies (top);aliquots of undigested extracts wereimmunoblotted with anti-actin antibodies (bottom) to verify equal sample loading. B, Cell surface expression of glypican-1 was assessed by immunofluorescent staining of transfected cells 48 h after transfection using anti-glypican- 1 antibodies (green) and DAPI (4',6'-diamidino-2 phenylindole) to stain nuclei (red). C, Schwann cells were transfected with siNeg or si 4(V)and conditioned media and cell lysates were harvested 48 h later (left) or at the indicated times after transfection (right);aliquots of medium (top) or cell lysates (bottom) were subjected to immunoblot analysis and stained with anti- 4(V) collagen (top) or anti- -actin (b ottom) antibodies.

intrathecal in vivo delivery of siRNA (pdf - 57Kb)

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