Cell-type-specific and regulatable transgenesis in the adult brain: adenovirus-encoded combined transcriptional targeting and inducible transgene expression.
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 (1) to be under the transcriptional control of either the astrocyte-specific, glial fibrillary acidic protein (GFAP) (2) or the neuronal specific enolase (NSE) promoter (3) 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 beta-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.
Smith-Arica
J R
JR
Molecular Medicine and Gene Therapy Unit, AstraZeneca, Room 1.302, Stopford Building, Manchester, Cheshire, M13 9PT, England.
Morelli
A E
AE
Larregina
A T
AT
Smith
J
J
Lowenstein
P R
PR
Castro
M G
MG
D000256:Adenoviridae / Q000235:genetics*; D000818:Animals; D030801:Animals, Genetically Modified; D005786:Gene Expression Regulation / Q000187:drug effects / Q000235:genetics*; D018390:Gene Targeting; D005904:Glial Fibrillary Acidic Protein / Q000235:genetics; D008297:Male; D051379:Mice; D051381:Rats; D017207:Rats, Sprague-Dawley; D013752:Tetracycline / Q000494:pharmacology; D014158:Transcription, Genetic / Q000235:genetics*; D019076:Transgenes; D014407:Tumor Cells, Cultured
D005904:Glial Fibrillary Acidic Protein; D013752:Tetracycline