Investigation of TRPV1 loss-of-function phenotypes in transgenic shRNA expressing and knockout mice
Introduction
TRPV1 is a cation channel that is predominantly expressed by nociceptive sensory neurons. TRPV1 is activated by capsaicin, noxious 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 mechanical 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 responses 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 neuropathic 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, transgenic 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 Hall, 2004, Röhl and Kurreck, 2006). The pain-related RNAi approaches published to date made use of repeated injections or continuous infusion of siRNA molecules. With this experimental design, however, the knockdown was intended to last only for a limited time of several days. We have also performed some initial functional investigations by transient inhibition of TRPV1 expression by intrathecal injection of antisense oligonucleotides (Christoph et al., 2007) or siRNA molecules (Christoph et al., 2006).
In order to perform a detailed comparison of different gene silencing strategies, we generated transgenic animals that express a TRPV1-directed shRNA and analyzed expression of the transgene, knockdown of TRPV1 and pain-related behaviour. Our study demonstrates that transgenic animals expressing shRNAs can be used for functional investigations in pain research and suggests a role of TRPV1 in neuropathic pain.
Section snippets
Generation of shRNA transgenic mice
In a previous study, we screened for efficient siRNAs targeting the mRNA of TRPV1. One siRNA, named VsiR1, was found to inhibit expression of a TRPV1–GFP fusion construct in the subnanomolar range in co-transfection experiments (Grünweller et al., 2003) and was efficacious upon intrathecal administration in rodent models of mononeuropathic and visceral pain (Christoph et al., 2006). In vitro and in vivo activities of this particular siRNA have recently been confirmed by an independent group (
Discussion
The present study successfully demonstrates that RNAi is a useful approach to validate potential novel targets for pain therapy. Previous studies have shown that the knockdown of the expression of several pain-related genes, such as the delta opioid receptor (Luo et al., 2005), the ionotropic nucleotide receptor P2X3 (Dorn et al., 2004), the NMDA receptor NR2B subunit (Tan et al., 2005) or TRPV1 (Christoph et al., 2006), by acute or sub-acute local administration of siRNAs resulted in analgesic
Construction and testing of the shRNA expression vector
For the generation of transgenic animals, an exchange vector, named pRMCE-U6-shTrpv1, was constructed that encodes an shRNA under control of the U6 promotor: The vector contains the F3 site and the FRT site in the same configuration as in the rosa26 targeting vector described by Seibler et al. (2005). The vector was generated using standard cloning procedures and has the following order in 5′ to 3′ direction: synthetic polyA signal, F3 site and a neomycin resistance gene lacking the start ATG.
Acknowledgments
The technical assistance of Birgit Bieber, Franz-Josef Butz, Elisabeth Haase, Elisabeth Krings, Thomas Krüger, Beatrice Petter, Simone Pfennings, Silke Rosenow, Stefanie Voß and the secretarial assistance of Ingrid Loeser is gratefully acknowledged. Furthermore, we want to thank Clemens Gillen, Kurexi Yunusi and Arnold Grünweller for initial contributions to the project and Oliver Bogen and Frank Nicolai Single for helpful discussion. The work was sponsored by Grünenthal GmbH, RiNA network for
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