1066. Optimisation of the Tet-On System for In Vivo Electro Gene Transfer to Muscle Tissue in Mice

Abstract

Top of pageAbstract Aim: In vivo electric pulses delivery to muscle for gene transfer (electro gene transfer) can yield long term high levels of gene expression. The method holds great promise for safe and simple gene delivery for gene therapy. A system to regulate the expression of transferred genes is the Tet-On system, which modulates gene expression by administration of doxycycline. The transactivator rtTA induces gene expression in the presence of doxycycline while the repressor tTS inhibits gene expression in the non-induced state. We have optimised the rtTA/tTS system in vivo to minimise DNA use and improve silencing in the non-induced state. Methods: Control of gene expression was investigated by in vivo delivery of three plasmids, one encoding the transactivator (pTet-On), one encoding the repressor (pTet-tTS), and one encoding the green fluorescent protein (GFP) under a Tet-On transactivator (rtTA) responsive promoter (ptetO-GFP). Different amounts of plasmids were co-injected in the tibialis cranialis muscle and followed by transfer into the muscle fibres by electro gene transfer. Results & Conclusion: It was found that 1) the total amount of the injected DNA could be significantly reduced whilst maintaining local expression, 2) the non-induced basal expression of GFP could be significantly reduced by increasing the repressor/activator ratio. When using a mixture of 5 |[mu]|g of ptetO-GFP, 5 |[mu]|g of pTet-On, and 5 |[mu]|g of pTet-tTS transferred into the tibialis cranialis muscle in mice, it was possible both to limit the toxicity related to exposure of high concentrations of DNA in the muscle, improve the control of gene expression as well as reduce the consumption of DNA for experiments. Indeed the use of less DNA may lead to fewer physiological changes happening in the muscle, a particular advantage for physiological studies of the organ.