Abstract
Abstract: :
Purpose: We used a self-inactivating lentiviral vector system to examine the activity of the human retinal guanylyl cyclase-1 (GC1) promoter in the developing chicken retina. Methods: VSV-G pseudotyped viruses were prepared using a lentiviral vector system that contains a nuclear-localized ß-galactosidase reporter gene driven by one of three selected fragments of the human GC1 promoter (+1 = ATG): -700/+6 (GCE1), -1745/+6 (GCE7), or –1745/-386 (GCE8). GCE1 and GCE7 both included intron 1 that separates the first two exons of the GC1 gene. GCE8 was identical to GCE7, except it lacked the intron and the first six nucleotides of exon 2. Virus was injected into the neural tubes of stage 10-12 chicken embryos. Promoter activity was assessed at various developmental stages using routine X-gal staining and histological analyses. In some cases, brains and pineal glands were also examined. Results: In normal chicken retina, GC1 transcripts are first detected on embryonic day (E) 13.5, reaching adult levels by E18.5 when the retina is fully differentiated. In the adult, expression of GC1 is restricted to photoreceptor cells and pinealocytes. GCE7 showed strong expression in all retinal cell layers on E10 and E13.5, but this activity was restricted to the outer nuclear layer (ONL) at E18.5. Similar results were obtained for GCE8; however, staining in the inner nuclear layer (INL) was more restricted at E10 and E13.5, and overall the intensity of staining was lower. In contrast, GCE1 activity was only observed in E18.5 retina and was limited to weakly stained cells within the ONL and the INL. At E18.5, none of the promoters exhibited activity in the brain, and only the GCE7 promoter fragment showed activity in cells within the pineal gland. Conclusions: Our results show that a 1.4 kb region of the human GC1 promoter located upstream of exon 1 contains cis-acting promoter elements necessary to direct gene expression to the photoreceptor cells at the final stage of retinal development. In addition, the activity of the GC1 promoter in vivo appears to be augmented by the presence of intron 1. The activities of the GC1 promoter fragments tested in vivo confirm our results obtained previously by in vitro analyses of these promoters in human retinoblastoma cell lines.
Keywords: gene/expression • photoreceptors • gene transfer/gene therapy