Abstract
Purpose:
RK is a photoreceptor-specific gene driven by a short highly active and specific promoter located immediately upstream of the gene. Eyes tested in preclinical studies from a number of mammals show surprisingly precise and robust expression of foreign therapeutic or reporter gene within their photoreceptor when the short promoter is used in the construct. The purpose of this study is to extend the utility and potential therapeutic use of this promising promoter by investigating its controls.
Methods:
Transgenic RK-GFP mice carrying the 200-bp human RK promoter were exposed to varying dark/light regimen and expression of GFP and RK were examined by Q-RT-PCR, immunoblotting, and immunohistology. Endogenous RK expression was also examined in retinoblastoma cells both in the dark and in the presence of thermostated cool LED blue and green light (2000 lux).
Results:
Light exposed mice showed greater than 2 times the levels of RK RNA as did the dark adapted animals once adjusted for any potential circadian rhythm. The GFP levels were also elevated in parallel to the RK expression in the light exposed animals. The Rk RNA levels were also elevated in the WERI cells exposed to thermostated LED lights by 2 folds.
Conclusions:
RK promoter appears to be responsive to light both in vivo and in vitro systems. The mechanism of this response is largely unknown but appears endogenous to photoreceptors as the magnitude and pattern of response is duplicated both in mouse and retinoblastoma cells. The precise mechanism is currently under investigation. Understanding the basis of the light effect on the promoter activity and its cellular mediators could identify additional points of control for therapeutic gene expression driven by RK promoter.
Keywords: 538 gene transfer/gene therapy •
689 retina: distal (photoreceptors, horizontal cells, bipolar cells) •
738 transcription