Intrathecal coelectrotransfer of a tetracycline-inducible, three-plasmid-based system to achieve tightly regulated antinociceptive gene therapy for mononeuropathic rats

Kuan Hung Chen, Chih Hsien Wu, Chia Chih Tseng, Jieh Min Shiau, Chien Te Lee, Chung Ren Lin

Research output: Contribution to journalArticle

12 Citations (Scopus)

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 languageEnglish
Pages (from-to)208-216
Number of pages9
JournalJournal of Gene Medicine
Volume10
Issue number2
DOIs
Publication statusPublished - 2008 Feb 1

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beta-Endorphin
Tetracycline
Genetic Therapy
Plasmids
Doxycycline
Gene Expression
Dialysis Solutions
Microdialysis
Spinal Ganglia
Neuralgia
Sciatic Nerve
Intraperitoneal Injections
Constriction
Radioimmunoassay
Analgesics
Spinal Cord
Proteins
Up-Regulation
Extremities
Hot Temperature

All Science Journal Classification (ASJC) codes

  • Molecular Medicine
  • Molecular Biology
  • Genetics
  • Drug Discovery
  • Genetics(clinical)

Cite this

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title = "Intrathecal coelectrotransfer of a tetracycline-inducible, three-plasmid-based system to achieve tightly regulated antinociceptive gene therapy for mononeuropathic rats",
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.",
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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 journalArticle

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AU - Chen, Kuan Hung

AU - Wu, Chih Hsien

AU - Tseng, Chia Chih

AU - Shiau, Jieh Min

AU - Lee, Chien Te

AU - Lin, Chung Ren

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