title: A 16 bp upstream sequence from the rat tyrosine hydroxylase promoter supports long-term expression from a neurofilament promoter, in a helper virus-free HSV-1 vector system.
abstract: Helper virus-free Herpes Simplex Virus vector-mediated gene transfer has supported studies on neuronal physiology, and may support specific gene therapies. Long-term, neuron-specific expression is required for many of these applications. A neurofilament heavy gene (NFH) promoter does not support long-term expression. We previously developed a promoter that supports long-term expression by fusing 6.3 kb of upstream sequences from the rat tyrosine hydroxylase (TH) promoter to a NFH promoter, and this promoter has supported physiological studies. The TH promoter fragment contains an enhancer, as it has activity in both orientations and at a distance from the basal promoter. Identifying this enhancer may support further improvements in long-term expression. A previous deletion analysis identified two ~100 bp fragments that each support long-term expression, and are contained within an ~320 bp fragment located ~3 kb from the TH promoter transcription start site. As this analysis used overlapping fragments, the two ~100 bp fragments contained 44 or 23 bp of unique sequence. Here, we used mutagenesis to identify a short sequence that supports long-term expression. We studied a 42 bp sequence, centered on the 23 bp unique sequence. Analysis of the wt sequence, and five mutations containing clustered changes that spanned the sequence, identified two adjacent mutations that do not support long-term expression, which together defined a 16 bp maximum essential sequence. This 16 bp sequence contains a putative E2F-1/DP-1 transcription factor binding site, and this transcription factor is expressed in many brain areas.
authors: Department of Neurology, West Roxbury VA Hospital/Harvard Medical School, W. Roxbury, MA 02132, USA.
authors: Guo-Rong
authors: GR
authors: Zhang
authors: Hua
authors: H
authors: Zhao
authors: Xu
authors: X
authors: Li
authors: Soumya
authors: S
authors: Awasthi
authors: Alfred I
authors: AI
authors: Geller
mesh_terms: D000818:Animals; D002452:Cell Count / Q000379:methods; D002461:Cell Line, Transformed; D003342:Corpus Striatum / Q000378:metabolism; D006224:Cricetinae; D001330:Electronic Data Processing; D005786:Gene Expression Regulation / Q000235:genetics*; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D018259:Herpesvirus 1, Human / Q000378:metabolism; D008297:Male; D008647:Mesocricetus; D008970:Molecular Weight; D009419:Nerve Tissue Proteins / Q000378:metabolism; D016900:Neurofilament Proteins / Q000235:genetics / Q000378:metabolism*; D010751:Phosphopyruvate Hydratase / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics*; D051381:Rats; D020318:Rats, Long-Evans; D014162:Transfection / Q000379:methods; D014446:Tyrosine 3-Monooxygenase / Q000235:genetics* / Q000378:metabolism
chemical_list: D009419:Nerve Tissue Proteins; D016900:Neurofilament Proteins; D014446:Tyrosine 3-Monooxygenase; D010751:Phosphopyruvate Hydratase