Transfecting Primary Cortical Neurons with a Plasmid for NCAM

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

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

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

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

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

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

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

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!

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

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

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

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

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

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

i-Fect and More Knockdown Success

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

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

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

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

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

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

Delivering siRNA in Mice for Studying Opioid-Induced Hyperalgesia

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

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

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

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

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