Tuesday, February 28, 2012

pn-Fect and Transfection of DRG cultures

In Vitro Gene expression analysis assays are essential for understanding how up or down regulation of  related target proteins could result in pathologies. Dorsal Root Ganglion (DRG), Neuronal and Glial Cultures have proven hard to transfect as as many transfection reagents are toxic to these cells. It is important for the study of neuro-diseases that researchers have tools and methods that enable success.

In this study, researchers successfully transfect DRG cultures with IKAP-shRNA using our pn-Fect kit. The own regulation of IKAP in these cultures support findings that helped explain the potential pathology of Familial Dysautonomia (FD; Hereditary Sensory Autonomic Neuropathy; HSAN III): Hunnicutt BJ , Chaverra M , George L , Lefcort F , 2012 IKAP/Elp1 Is Required In Vivo for Neurogenesis and Neuronal Survival, but Not for Neural Crest Migration. PLoS ONE 7(2): e32050. doi:10.1371/journal.pone.0032050.

Cell culture: Dorsal root ganglia were dissected from Embryonic day 5–9.5 chick embryos (E5–9.5) and dissociated by incubation in 0.25% trypsin-EDTA (Gibco) for 7 min at 37 C followed by trituration through fire–pulled glass pipettes. The culture media consisted of Neurobasal medium (Invitrogen) supplemented with B27 (1X,Invitrogen), Glutamax (1X,Invitrogen), Hybrimax Antibiotic\Antimicotic (1:100, Sigma), NGF (10 ng/ml, gift from Dr. Thomas Large). Cells were plated on 8-well Nunc glass chamber slides that were coated with poly-D-lysine (1:100, Sigma) and laminin 20 ug/ml (Gibco). Approximately equal numbers of cells (52,500) were plated per well. Immediately after plating, cells were transfected with IKBKAP-7.4 shRNA or control shRNA via pn-Fect (Neuromics, PN3375). Several ratios of pnfect:DNA were tested with the optimum obtained 1.84:1. The cells were then cultured for approximately 29 h at 37 C, 5.5% C02. After incubation culture cells were fixed and inmunostained as previously described [24]. To determine whether IKBKAP shRNAs altered cell proliferation and/or neuronal differentiation in dissociated DRG cultures, Brdu was added to the cultures and the cells were fixed 24 hrs later (as described in [24]). The number of GFP+/BrdU+ or GFP+/Tuj-1+ cells were quantified for each experiment, and a ratio comparing control vs. IKBKAP shRNAs for each experiment determined. For the BrdU+ experiment, a total of 556 GFP+/control shRNA transfected cells were counted and 357 GFP+/IKBKAP shRNA transfected cells in 3 separate experiment. For determining neuronal differentiation, a total of 2866 GFP+/control shRNA transfected cells and 2913 IKBKAP shRNA transfected cells were counted, over 3 separate experiments.

Images: IKAP regulates neuronal differentiation in the DRG. Reduction in IKAP leads to increased numbers of neurons in the immature DRG (A–C). Embryos at St. 12 were transfected with either control shRNAs or IKBKAP shRNAs and analyzed at St 24/25. Embryos were sectioned, and immunolabled with the neuronal markers Tuj-1 or Ben and the percentage of GFP+ neurons determined. Significantly more IKBKAP shRNA transfected DRG precursor cells differentiated into neurons (arrows in B; IKBKAP shRNA 7.4; n = 3 embryos; p = 0.002; IKBKAP shRNA 1.6 & 4.5, n = 3 embryos; p = 0.001) than Control shRNA transfected DRG precursor cells (n = 5 embryos). (D–H) IKBKAP shRNA-transfected DRG precursor cells (n = 3 embryos; 252 cleaved-Caspase 3+ cells counted) were also more likely to die by apoptosis (compare D & E to F & G) than control shRNA-electroporated cells (n = 3 embryos; 117 cleaved caspase-3+ cells counted). (H) The number of cleaved Caspase 3+ cells was quantified in DRG on both the transfected side of the embryo and the non-transfected side of the embryo and a ratio determined. Significantly more cleaved-Caspase 3+ cells were present in the transfected DRG of IKBKAP shRNA transfected embryos than in the transfected DRG in embryos transfected with control shRNAs; p = 0.006. (I–N). Over-expression of the c-terminus of IKAP prevents neural crest cells from coalescing with the DRG (I, K, L; p = 0.004) but the few that do join, tend not to differentiate into neurons (p = 0.007; J, M, N). Embryos were transfected with either a construct driving expression of the c-terminus of chicken IKAP with a His tag (CT-IKAP-His; L,N) or a control, His-tagged construct (K, M) and analyzed at St. 21. The location and neuronal identity of transfected cells was determined in 3 embryos for each treatment; Control His-plasmid: n = 343 transfected cells counted; CT-IKAP-His: n = 278 transfected cells counted. Statistical analysis by Student t-test. doi:10.1371/journal.pone.0032050.g006. 

Check out our transfection reagents and capabilities. We will continue to post publications and data that support and add to our capabilities.

Sunday, February 26, 2012

Silencing NOV in vivo using i-Fect

Implications for Treating Neuropathic or Inflammatory Pain

It has been a awhile since I posted results here for researchers using Neuromics' i-Fect ™ siRNA Transfection Kits. Over the past months, we have enjoyed more growth in use of these kits so I anticipate more positive results to come.

Inflammation plays and evil role in Neuropathic Pain. Sustained neuroinflammation cased by  release of pro-inflammatory cytokines and chemokines (including TNF-α, IL-1β, IL-6 and CCL2). Emerging studies have established that the extracellular matrix (ECM) components, particularly matrix metalloproteinases (MMPs) actively participate in the generation and maintenance of pain.
In this study, investigators show how modulating expression of nephroblastoma overexpressed gene (NOV) can mitigate expression of the MMPs and thus regulate Pain: Lara Kular, Cyril Rivat, Brigitte Lelongt, Claire Calmel, Maryvonne Laurent, Michel Pohl, Patrick Kitabgi, Stephane Melik-Parsadaniantz and Cecile Martinerie. NOV/CCN3 attenuates inflammatory pain through regulation of matrix metalloproteinases-2 and -9. Journal of Neuroinflammation 2012, 9:36 doi:10.1186/1742-2094-9-36.
Results:  NOV was expressed in neurons of both dorsal root ganglia (DRG) and dorsal horn of the spinal cord (DHSC). After intraplantar CFA injection, NOV levels were transiently and persistently down-regulated in the DRG and DHSC, respectively, occurring at the maintenance phase of pain (15 days). NOV-reduced expression was restored after treatment of CFA rats with dexamethasone. In vitro, results based on cultured DRG neurons showed that siRNA-mediated inhibition of NOV enhanced IL-1beta- and TNF-alpha-induced MMP-2, MMP-9 and CCL2 expression whereas NOV addition inhibited TNF-alpha-induced MMP-9 expression through beta1 integrin engagement. In vivo, the intrathecal delivery of MMP-9 inhibitor attenuated mechanical allodynia of CFA rats. Importantly, intrathecal administration of NOV siRNA specifically led to an up-regulation of MMP-9 in the DRG and MMP-2 in the DHSC concomitant with increased mechanical allodynia. Finally, NOV intrathecal treatment specifically abolished the induction of MMP-9 in the DRG and, MMP-9 and MMP-2 in the DHSC of CFA rats. This inhibitory effect on MMP is associated with reduced mechanical allodynia.
Conclusions:  This study identifies NOV as a new actor against inflammatory pain through regulation of MMPs thus uncovering NOV as an attractive candidate for therapeutic improvement in pain relief.

Figure 9. Effect of in vivo endogenous NOV inhibition on MMP-2/-9 expression and mechanical allodynia. In CFA rats, NOV-specific siRNA (2 μg) or control non-silencing siRNA (Ctr) were delivered intrathecally (i.t) daily for 3 consecutive days. (A) NOV protein levels in DHSC. Representative western blot (left panel) and quantification of protein levels normalized to GAPDH (right panel) (**P <0.01, siNOV versus Ctr, n = 6) (B, C) Levels of MMP-9 and MMP-2 mRNA in DRG (B) and DHSC. (C) Transcript levels were quantified by RT-qPCR and values were normalized to rat S26 mRNA level. Data represent the mean value ± SEM of two independent experiments realized with three rats per condition (*P <0.05 siNOV versus Ctr). (D) Representative gelatin zymograph showing MMP-9 and MMP-2 activities in DRG (left panel) and quantification of MMP-2 and MMP-9 gelatinolytic bands (right panel). Data represent the mean ± SEM of six rats per group (**P <0.01 siNOV versus Ctr). (E) Paw withdrawal threshold (g) of CFA rats intrathecally injected with NOV-specific siRNA or control siRNA evaluated using the von Frey test. Data represent the mean ± SEM of eight rats per group (*P <0.05 siNOV- versus Ctr-treated rats), BL: baseline In order to test whether endogenously produced NOV could modulate inflammatory pain, we evaluated the mechanical allodynia of CFA rats treated with NOV. As shown in Figure 9E, intrathecal delivery of siNOV resulted in a significant increase of mechanical allodynia compared to rats injected with control siRNA (*P <0.05, n = 8). These data strongly suggest that endogenously produced NOV influences pain intensity and further support the hypothesis that NOV downregulation could participate in pain processes through
upregulation of MMP-2 and MMP-9.

Learn more about Neuromics' Transfection Reagents!

Monday, February 13, 2012

In Vivo application of RNAi to study pain

This overview is from 2010. I am posting a link because it undercores the need to have transfection reagents that have the ability to deliver small doses of siRNA in vivo.
"One of the biggest challenges in using RNAi in pain research is delivery of siRNA to the CNS in sufficient concentrations. This obstacle exists because siRNA by itself does not cross the blood brain barrier (BBB) and is degraded in the blood by endonucleases. Intravenous or oral administration is, therefore, inadequate to achieve desired protein knockdown. The use of transfection agents and intrathecal delivery has enhanced siRNA uptake by target tissues in recent studies." Zachary J Clark, Gurwattan S. Miranpuri, Daniel K Resnick. In Vivo application of RNAi to study pain. Annals of Neurosciences, Volume 17, Number 3, July 2010.

Please click through the link and you will learn of techniques currently used to delivery siRNA in vivo for pain research.

Saturday, February 4, 2012

siRNA Delivery Group on Linkedin

I wanted to make readers aware of an excellent discussion group on Linkedin named "siRNA Delivery". Included are tip, updates on commercialization and key publications.

Here're some examples:
Happy reading.