Article ID | Journal | Published Year | Pages | File Type |
---|---|---|---|---|
6264734 | Brain Research | 2011 | 10 Pages |
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 6.3Â 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 ~Â 100Â bp fragments that each support long-term expression, and are contained within an ~Â 320Â bp fragment located ~Â 3Â kb from the TH promoter transcription start site. As this analysis used overlapping fragments, the two ~Â 100Â bp fragments contained 44 or 23Â bp of unique sequence. Here, we used mutagenesis to identify a short sequence that supports long-term expression. We studied a 42Â bp sequence, centered on the 23Â 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 16Â bp maximum essential sequence. This 16Â bp sequence contains a putative E2F-1/DP-1 transcription factor binding site, and this transcription factor is expressed in many brain areas.
⺠Promoters that support long-term expression are desirable. ⺠A 16 bp sequence that supports long-term expression was identified. ⺠This 16 bp enhancer sequence is from the TH promoter. ⺠This 16 bp sequence contains a putative E2F-1/DP-1 binding site.