Delivering siRNA in vivo

New Publications

Our i-Fect transfection kit was one of the first products. This has resulted in it being widely used and frequently published.

We kick off 2018 with some new publications. Here we feature a study where researchers use our kit to deliver G-protein-coupled receptor C (MrgprC) siRNA in vivo. This receptor is part of the TRPV1 pathway.

• Ying-jun Liu, Xiao-xi Lin, Jian-qiao Fang, and Fang Fang. Involvement of MrgprC in electroacupuncture analgesia for attenuating CFA-induced thermal hyperalgesia by suppressing the TRPV1 pathway. Pre-pub-full text PDF

Image: Expression of TRPV1 in rats treated with MrgrprC si RNA vs controls (C).

The others conclude that MrgprC expression is impacted by electroacupuncture and downregulates  TRPV1.  This is the mechanism that results in pain relief.

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.

Knockdown of rSNSR1 in vivo

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.