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.
Georgievska
Biljana
B
Division of Neurobiology, Department of Physiological Sciences, Wallenberg Neuroscience Center, Lund University, 221 84 Lund, Sweden.
Jakobsson
Johan
J
Persson
Elisabeth
E
Ericson
Cecilia
C
Kirik
Deniz
D
Lundberg
Cecilia
C
D000818:Animals; D003342:Corpus Striatum / Q000378:metabolism*; D018491:Dopamine Agonists / Q000378:metabolism; D015536:Down-Regulation; D004318:Doxycycline / Q000494:pharmacology; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D018014:Gene Transfer Techniques; D015316:Genetic Therapy / Q000379:methods; D005822:Genetic Vectors; D051100:Glial Cell Line-Derived Neurotrophic Factor; D049452:Green Fluorescent Proteins / Q000378:metabolism; D016086:Lentivirus / Q000235:genetics*; D018613:Microscopy, Confocal; D008957:Models, Genetic; D009414:Nerve Growth Factors / Q000235:genetics* / Q000378:metabolism; D009474:Neurons / Q000378:metabolism*; D010300:Parkinson Disease / Q000235:genetics; D016133:Polymerase Chain Reaction; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000737:chemistry; D013752:Tetracycline / Q000494:pharmacology; D013997:Time Factors; D019076:Transgenes / Q000235:genetics; D014446:Tyrosine 3-Monooxygenase / Q000096:biosynthesis
D018491:Dopamine Agonists; C495041:Gdnf protein, rat; D051100:Glial Cell Line-Derived Neurotrophic Factor; D009414:Nerve Growth Factors; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; D014446:Tyrosine 3-Monooxygenase; D013752:Tetracycline; D004318:Doxycycline