Regulated delivery of glial cell line-derived neurotrophic factor into rat striatum, using a tetracycline-dependent lentiviral vector

In this study, a tetracycline-regulated lentiviral vector system, based on the tetracycline-dependent transactivator rtTA2(S)-M2, was developed for controlled expression of glial cell line-derived neurotrophic factor ( GDNF) in the rat brain. Expression of the marker gene green fluorescent protein (GFP) and GDNF was tightly regulated in a dose-dependent manner in neural cell lines in vitro. Injection of high-titer lentiviral vectors into the rat striatum resulted in a 7-fold induction of GDNF tissue levels ( 1060 pg/mg tissue), when doxycycline ( a tetracycline analog) was added to the drinking water. However, low levels of GDNF ( 150 pg/mg tissue) were also detected in animals that did not receive doxycycline, indicating a significant background leakage from the vector system in vivo. The level of basal expression was markedly reduced when a 10-fold lower dose of the tetracycline-regulated GDNF vector was injected into the striatum ( 3 - 11 pg/mg tissue), and doxycycline-induced GDNF tissue levels obtained in these animals were about 190 pg/mg tissue. Doxycycline-induced expression of GDNF resulted in a significant downregulation of the tyrosine hydroxylase (TH) protein in the intact striatum. Removal of doxycycline from the drinking water rapidly ( within 3 days) turned off transgenic GDNF mRNA expression and GDNF protein levels in the tissue were completely reduced by 2 weeks, demonstrating the dynamics of the system in vivo. Accordingly, TH protein expression returned to normal by 2 - 8 weeks after removal of doxycycline, indicating that GDNF-induced downregulation of TH is a reversible event.