Dual-promoter lentiviral vectors for constitutive and regulated gene expression in neurons

• Published in: Journal of neuroscience methods 2008-02, Vol.168 (1), p.104-112
• Main Authors: Gascón, Sergio, Paez-Gomez, Juan A., Díaz-Guerra, Margarita, Scheiffele, Peter, Scholl, Francisco G.
• Format: Article
• Language: English
• Subjects: Animals; Anti-Bacterial Agents - pharmacology; Cells, Cultured; Cerebral Cortex - cytology; Doxycycline - pharmacology; Embryo, Mammalian; Gene Expression Regulation - drug effects; Gene Expression Regulation - physiology; Gene Transfer Techniques; Genetic Vectors - physiology; Inducible-expression; Lentivirus; Lentivirus - physiology; Nerve Tissue Proteins - genetics; Nerve Tissue Proteins - metabolism; Neurons - physiology; Neurons - virology; Primary neurons; Promoter Regions, Genetic - physiology; Rats; RNA, Messenger - metabolism; Synapsin promoter
• ISSN: 0165-0270; 1872-678X
• DOI: 10.1016/j.jneumeth.2007.09.023

Gene transfer methods for efficient co-expression of exogenous proteins in neurons are crucial tools towards the understanding of the molecular basis of the central nervous system. Lentiviruses are retroviral vectors that can transduce a wide variety of cells including differentiated neurons. In this work, we have generated lentiviral vectors containing dual promoters that allow efficient co-expression of exogenous proteins in neurons. We show that insertion of two copies of a human synapsin promoter/WPRE cassette in a single lentiviral vector directs robust co-expression of cDNAs in cultured neurons, while excluding expression in the surrounding glial cells. Furthermore, insertion of the tetracycline-inducible system (Tet-off) controlled by the synapsin promoter results in tightly regulated expression of EGFP when used as a transgene in cultured neurons. Transduction of primary neurons with this inducible system leads to a 100-fold increase in EGFP mRNA levels in the absence of doxycycline. In transduced cultures, EGFP transcription is inhibited within 24 h upon addition of doxycycline. The viral systems we developed here provide neuron-specific and regulated expression mediated by single lentiviral vectors and will prove valuable tools for the study of neuronal function.