Abstract
For optimal use of antinociceptive gene therapy, it may be important to have extrinsic control of the expression of the transfected gene. To achieve this goal, we used a tetracycline-inducible system (Tet-On) composed of three plasmids coding for beta-endorphin, the tetracycline transcriptional activator rtTA, and the silencer tTS. The regulation of beta-endorphin expression was first assessed in cultures of dorsal root ganglion neurons. The three plasmids were then electrotransfected into the spinal cord of mononeuropathic rats and the analgesic potential of this therapy in vivo was evaluated by thermal-withdrawal latency and the mechanical-withdrawal threshold. Intraperitoneal injections of doxycycline were made to evaluate the possibility of exogenous upregulation of transfected beta-endorphin gene expression in vivo. The levels of beta-endorphin were analyzed by intrathecal microdialysis and radioimmunoassay. We found that, after doxycycline administration, the expression of beta-endorphin was rapid, stable, and tightly regulated (low background and high induction level) both in vitro and in vivo. The beta-endorphin protein was secreted into cerebrospinal. fluid at a peak level of 53 pmol/L in dialysate, which was sufficient to inhibit neuropathic pain. In conclusion, tightly controlled expression of beta-endorphin can be obtained following intrathecal electrotransfer of a tetracycline-inducible, three-plasmid-based system, and doxycycline-dependent beta-endorphin protein expression in this system alleviates sciatic nerve constriction-induced limb pain.
| Original language | English |
|---|---|
| Pages (from-to) | 208-216 |
| Number of pages | 9 |
| Journal | Journal of Gene Medicine |
| Volume | 10 |
| Issue number | 2 |
| DOIs | |
| Publication status | Published - 2008 Feb 1 |
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All Science Journal Classification (ASJC) codes
- Molecular Medicine
- Molecular Biology
- Genetics
- Drug Discovery
- Genetics(clinical)
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Intrathecal coelectrotransfer of a tetracycline-inducible, three-plasmid-based system to achieve tightly regulated antinociceptive gene therapy for mononeuropathic rats. / Chen, Kuan Hung; Wu, Chih Hsien; Tseng, Chia Chih; Shiau, Jieh Min; Lee, Chien Te; Lin, Chung Ren.
In: Journal of Gene Medicine, Vol. 10, No. 2, 01.02.2008, p. 208-216.Research output: Contribution to journal › Article
TY - JOUR
T1 - Intrathecal coelectrotransfer of a tetracycline-inducible, three-plasmid-based system to achieve tightly regulated antinociceptive gene therapy for mononeuropathic rats
AU - Chen, Kuan Hung
AU - Wu, Chih Hsien
AU - Tseng, Chia Chih
AU - Shiau, Jieh Min
AU - Lee, Chien Te
AU - Lin, Chung Ren
PY - 2008/2/1
Y1 - 2008/2/1
N2 - For optimal use of antinociceptive gene therapy, it may be important to have extrinsic control of the expression of the transfected gene. To achieve this goal, we used a tetracycline-inducible system (Tet-On) composed of three plasmids coding for beta-endorphin, the tetracycline transcriptional activator rtTA, and the silencer tTS. The regulation of beta-endorphin expression was first assessed in cultures of dorsal root ganglion neurons. The three plasmids were then electrotransfected into the spinal cord of mononeuropathic rats and the analgesic potential of this therapy in vivo was evaluated by thermal-withdrawal latency and the mechanical-withdrawal threshold. Intraperitoneal injections of doxycycline were made to evaluate the possibility of exogenous upregulation of transfected beta-endorphin gene expression in vivo. The levels of beta-endorphin were analyzed by intrathecal microdialysis and radioimmunoassay. We found that, after doxycycline administration, the expression of beta-endorphin was rapid, stable, and tightly regulated (low background and high induction level) both in vitro and in vivo. The beta-endorphin protein was secreted into cerebrospinal. fluid at a peak level of 53 pmol/L in dialysate, which was sufficient to inhibit neuropathic pain. In conclusion, tightly controlled expression of beta-endorphin can be obtained following intrathecal electrotransfer of a tetracycline-inducible, three-plasmid-based system, and doxycycline-dependent beta-endorphin protein expression in this system alleviates sciatic nerve constriction-induced limb pain.
AB - For optimal use of antinociceptive gene therapy, it may be important to have extrinsic control of the expression of the transfected gene. To achieve this goal, we used a tetracycline-inducible system (Tet-On) composed of three plasmids coding for beta-endorphin, the tetracycline transcriptional activator rtTA, and the silencer tTS. The regulation of beta-endorphin expression was first assessed in cultures of dorsal root ganglion neurons. The three plasmids were then electrotransfected into the spinal cord of mononeuropathic rats and the analgesic potential of this therapy in vivo was evaluated by thermal-withdrawal latency and the mechanical-withdrawal threshold. Intraperitoneal injections of doxycycline were made to evaluate the possibility of exogenous upregulation of transfected beta-endorphin gene expression in vivo. The levels of beta-endorphin were analyzed by intrathecal microdialysis and radioimmunoassay. We found that, after doxycycline administration, the expression of beta-endorphin was rapid, stable, and tightly regulated (low background and high induction level) both in vitro and in vivo. The beta-endorphin protein was secreted into cerebrospinal. fluid at a peak level of 53 pmol/L in dialysate, which was sufficient to inhibit neuropathic pain. In conclusion, tightly controlled expression of beta-endorphin can be obtained following intrathecal electrotransfer of a tetracycline-inducible, three-plasmid-based system, and doxycycline-dependent beta-endorphin protein expression in this system alleviates sciatic nerve constriction-induced limb pain.
UR - http://www.scopus.com/inward/record.url?scp=39449103344&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=39449103344&partnerID=8YFLogxK
U2 - 10.1002/jgm.1132
DO - 10.1002/jgm.1132
M3 - Article
C2 - 18064731
AN - SCOPUS:39449103344
VL - 10
SP - 208
EP - 216
JO - Journal of Gene Medicine
JF - Journal of Gene Medicine
SN - 1099-498X
IS - 2
ER -