The 5' flanking sequence of the human retGC1 gene acquires aphotoreceptor cell restricted activity pattern over the course ofretinal development

[摘要] Purpose: Specific mutations of the retinal guanylyl cyclase-1(retGC1) gene have been linked to Leber congenital amaurosis type 1(LCA1) and cone-rod dystrophies in humans, diseases that are amenable totreatments using molecular based therapies. As a step towards developinga therapeutic transgene for LCA1, we analyzed the cell specific anddevelopmental activity profiles of fragments of the human retGC1 5'flanking region in vivo.Methods: We generated self inactivating lentiviral vector constructscarrying three different fragments of the human retGC1 promoter fused toa nuclear localized β-galactosidase reporter gene (nlacZ). Thetransgenes were packaged into lentiviral vectors, which were then usedto transduce retinal progenitor cells of the developing chick. Wemonitored the expression of nlacZ in the retina over the course ofdevelopment and in the retina, brain and pineal gland just prior tohatching.Results: A 1.8 kb fragment of the retGC1 5' flanking region upstreamof Exon 2 was capable of targeting nlacZ expression to photoreceptorcells in vivo and its activity was augmented by the presence of intron1. We also demonstrated that the cell specific activity of this fragmentarises, at least in part, by silencing expression in non-photoreceptorcells during the final stages of retinal development.Conclusions: We have identified a human retGC1 promoter fragmentthat exhibits photoreceptor cell specific activity in vivo. Our resultssuggest that an element located in the proximal promoter may play a rolein silencing expression of this gene in non-photoreceptor cells, therebyby shaping the restricted expression pattern of GC1 in the retina.

[学科分类] 生物化学/生物物理