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.
Sunday, April 17, 2011
Friday, September 17, 2010
Transfecting Primary Cortical Neurons with a Plasmid for NCAM
Harnessing the power of MATraTM (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.
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:
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.
Wednesday, August 25, 2010
i-Fect and siRNA Delvery to Toll-like receptor 4
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.
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.
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.
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.
Monday, August 16, 2010
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 liverThey 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.
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•Effective delivery (60%+ knockdown) with no toxicity.
•Scalable to high throughput siRNA based gene screening.
•Consistent and reproducible results.
Monday, August 9, 2010
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-FectTMsiRNA 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.
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.
Tuesday, June 29, 2010
Using MATRa for siRNA Transfection of Carcinoma Cell Lines
MATRaTM -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).
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:
Dr. Oliver Gires, LMU Munich, Germany |
Friday, June 25, 2010
siRNA and i-Fect for the Study of Retinal Disease
We continue to add new references to the many ways researchers are using i-FectTM 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.
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.
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