Cell-Type-Specific and Regulatable Transgenesis in the Adult Brain: Adenovirus-Encoded Combined Transcriptional Targeting and Inducible Transgene Expression
Authors: Joseph R. Smith-Arica, Adrian E. Morelli, Adriana T. Larregina, John Smith, Pedro R. Lowenstein, Maria G. Castro
Journal: Molecular Therapy (2000)

To achieve transient transgenesis within specific areas or cell populations in the adult central nervous system (CNS), we have developed a dual adenoviral vector system encoding for cell-type-specific and regulatable transcription units. To achieve combined cell-type-specific transcriptional targeting and inducible expression, we have engineered the expression of the tetracycline-dependent transcriptional elements (1Yoshida Y. Hamada H. Adenovirus-mediated inducible gene expression through tetracycline-controllable transactivator with nuclear localisation sequence.Biochem. Biophys. Res. Commun. 1997; 230: 426-430Crossref PubMed Scopus (95) Google Scholar) to be under the transcriptional control of either the astrocyte-specific, glial fibrillary acidic protein (GFAP) (2Brenner M. Kisseberth W.C. Su Y. Besnard F. Messing A. GFAP promoter directs astrocyte-specific expression in transgenic mice.J. Neurosci. 1994; 14: 1030-1037Crossref PubMed Google Scholar) or the neuronal specific enolase (NSE) promoter (3Forss-Petter S. et al.Transgenic mice expressing β-galactosidase in mature neurons under neuron-specific enolase promoter.Neuron. 1990; 5: 187-197Abstract Full Text PDF PubMed Scopus (329) Google Scholar) within a dual adenoviral vector system. Cell-type specificity, inducibility, and levels of transgene expression were characterized in vitro in cell lines, and primary neocortical cultures and in the central nervous system (CNS) in vivo, and compared to a powerful pancellular β-actin/CMV promoter. We demonstrate that the GFAP promoter is able to restrict tetracycline-dependent transgene expression to glial cells in cell lines, primary cultures, and in the CNS in vivo. However, although the NSE promoter did not show neuronal restricted transgene expression in vitro, it did so in the CNS in vivo. Our dual viral system also has provided evidence that an excess of transactivator is needed to achieve maximal transgene expression. Administration of doxycycline completely abrogated transgene expression both in vitro and in vivo. Consequently, our strategy demonstrates that combined cell-type specificity and simultaneous regulation of transgene expression can be obtained in the brain using adenoviral vectors. To achieve transient transgenesis within specific areas or cell populations in the adult central nervous system (CNS), we have developed a dual adenoviral vector system encoding for cell-type-specific and regulatable transcription units. To achieve combined cell-type-specific transcriptional targeting and inducible expression, we have engineered the expression of the tetracycline-dependent transcriptional elements (1Yoshida Y. Hamada H. Adenovirus-mediated inducible gene expression through tetracycline-controllable transactivator with nuclear localisation sequence.Biochem. Biophys. Res. Commun. 1997; 230: 426-430Crossref PubMed Scopus (95) Google Scholar) to be under the transcriptional control of either the astrocyte-specific, glial fibrillary acidic protein (GFAP) (2Brenner M. Kisseberth W.C. Su Y. Besnard F. Messing A. GFAP promoter directs astrocyte-specific expression in transgenic mice.J. Neurosci. 1994; 14: 1030-1037Crossref PubMed Google Scholar) or the neuronal specific enolase (NSE) promoter (3Forss-Petter S. et al.Transgenic mice expressing β-galactosidase in mature neurons under neuron-specific enolase promoter.Neuron. 1990; 5: 187-197Abstract Full Text PDF PubMed Scopus (329) Google Scholar) within a dual adenoviral vector system. Cell-type specificity, inducibility, and levels of transgene expression were characterized in vitro in cell lines, and primary neocortical cultures and in the central nervous system (CNS) in vivo, and compared to a powerful pancellular β-actin/CMV promoter. We demonstrate that the GFAP promoter is able to restrict tetracycline-dependent transgene expression to glial cells in cell lines, primary cultures, and in the CNS in vivo. However, although the NSE promoter did not show neuronal restricted transgene expression in vitro, it did so in the CNS in vivo. Our dual viral system also has provided evidence that an excess of transactivator is needed to achieve maximal transgene expression. Administration of doxycycline completely abrogated transgene expression both in vitro and in vivo. Consequently, our strategy demonstrates that combined cell-type specificity and simultaneous regulation of transgene expression can be obtained in the brain using adenoviral vectors.