Delivering DNA to Cell Lines and Primary Cells

Neuromics is pleased to introduce DNA-Fect^TM and 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

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

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

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

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

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

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

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

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.