Investigation of TRPV1 loss-of-function phenotypes in transgenic shRNA expressing and knockout mice Thomas Christoph, a, ⁎ Gregor Bahrenberg, b Jean De Vry, a Werner Englberger, b Volker A. Erdmann, c Moritz Frech, a Babette Kögel, a Thomas Röhl, c Klaus Schiene, a Wolfgang Schröder, a Jost Seibler, d and Jens Kurreck c,e a Preclinical Research and Development, Department of Pharmacology, Grünenthal, Zieglerstrasse 6, 52078 Aachen, Germany b Preclinical Research and Development, Department of Molecular Pharmacology, Grünenthal, Zieglerstrasse 6, 52078 Aachen, Germany c Institute for Chemistry and Biochemistry, Free University Berlin, Thielallee 63, 14195 Berlin, Germany d ARTEMIS Pharmaceuticals, Neurather Ring 1, 51063 Cologne, Germany e Institute of Industrial Genetics, University of Stuttgart, Allmandring 31, 70569 Stuttgart, Germany Received 23 July 2007; revised 28 November 2007; accepted 6 December 2007 Available online 15 December 2007 The function of the transient receptor potential vanilloid 1 (TRPV1) cation channel was analyzed with RNA interference technologies and compared to TRPV1 knockout mice. Expression of shRNAs targeting TRPV1 in transgenic (tg) mice was proven by RNase protection assays, and TRPV1 downregulation was confirmed by reduced expression of TRPV1 mRNA and lack of receptor agonist binding in spinal cord membranes. Unexpectedly, TRPV3 mRNA expression was upregulated in shRNAtg but downregulated in knockout mice. Capsaicin-induced [Ca 2+ ] i changes in small diameter DRG neurons were significantly diminished in TRPV1 shRNAtg mice, and administration of capsaicin hardly induced hypothermia or nocifensive behaviour in vivo. Likewise, sensitivity towards noxious heat was reduced. Interestingly, spinal nerve injured TRPV1 knockout but not shRNAtg animals developed mechanical allodynia and hypersensitivity. The present study provides further evidence for the relevance of TRPV1 in neuropathic pain and characterizes RNA interference as valuable technique for drug target validation in pain research. © 2008 Elsevier Inc. All rights reserved. Introduction TRPV1 is a cation channel that is predominantly expressed by nociceptive sensory neurons. TRPV1 is activated by capsaicin, nox- ious heat and protons (Caterina et al., 1997; Tominaga et al., 1998). A variety of intracellular pain-related pathways and molecules regulate nociception and signal transmission by TRPV1 (Szallasi et al., 2007; Tominaga, 2007). As TRPV1 appears to be a central molecular integrator of noxious stimuli, it is considered to be an attractive target for new analgesic drugs. Knockout mice lacking TRPV1 exhibit diminished sensitivity to heat and perturbed micturition (Birder et al., 2002; Caterina et al., 2000; Davis et al., 2000). Interestingly, TRPV1 knockout mice do not reveal a significantly altered phenotype in the partial sciatic nerve ligation model of mononeuropathic pain as compared to wild- type animals (Caterina et al., 2000) and even show increased me- chanical hyperalgesia in polyneuropathic pain models (Bolcskei et al., 2005). In contrast, intrathecal administration of the TRPV1 antagonist capsazepine was reported to block A-fiber-evoked res- ponses in dorsal horn neurons of rats after spinal nerve ligation (Kelly and Chapman, 2002), and more selective TRPV1 antagonists attenuated mechanical allodynia and hyperalgesia in rat models of mononeuropathic pain (Christoph et al., 2006; Honore et al., 2005; Kanai et al., 2005; Pomonis et al., 2003). The precise role of TRPV1 in neuropathic pain is therefore still controversial. We decided to use an RNA interference (RNAi) approach to investigate further the role of TRPV1 in an animal model of neu- ropathic pain. RNAi is an evolutionary conserved mechanism of posttranscriptional gene silencing mediated by double stranded RNA molecules (Grünweller and Hartmann, 2005; Kim and Rossi, 2007). For applications in mammalian cells, 21 nucleotides long small interfering RNAs (siRNAs) can be employed to specifically silence a particular gene. To achieve long-term inhibition of a target gene, self-complementary short hairpin (shRNAs) can be expressed intracellularly (Shi, 2003). The shRNAs are processed by the RNase Dicer to give siRNA-type molecules. For in vivo experiments, trans- genic animals have been generated which continuously express the shRNA, thereby permanently knocking down the targeted gene (Seibler et al., 2005). Several studies have already demonstrated the potential of RNAi to validate new targets in pain research (for reviews, see Ganju and www.elsevier.com/locate/ymcne Mol. Cell. Neurosci. 37 (2008) 579 – 589 ⁎ Corresponding author. E-mail address: thomas.christoph@grunenthal.com (T. Christoph). Available online on ScienceDirect (www.sciencedirect.com). 1044-7431/$ - see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.mcn.2007.12.006