Abstract
The efficiency of small interfering RNA (siRNA)-mediated gene silencing is highly variable. The process depends on a number of factors including the efficiency of siRNA uptake by cells/tissues as well as the half-lives of the RNA transcript and the target protein. While investigators are searching for suitable siRNA delivery methods for therapeutic purposes, reporter proteins are typical used to evaluate the result of gene silencing. In the present study, we demonstrate that the photoconvertible fluorescent protein, Kaede, can serve as an effective reporter to study gene silencing. Unlike the conventional approach of waiting for the natural turnover of reporters, we actively induced the green-to-red conversion of Kaede to erase the residual reporter activity prior to siRNA administration. The low residual background led to an increase in sensitivity of detecting the de novo synthesis of Kaede, which served as an effective indicator of gene silencing. Using this approach, we were able to initiate a significant 2.1-fold difference in fluoresce signals between the Kaede-specific and scramble siRNAs transfected cells twelve hours post administration. In comparison, only 0.4-fold difference was observed while using GFP as the reporter. Our work demonstrates a new application of photoconvertible fluorescent protein Kaede as the gene-silencing reporter, which can be a useful tool for monitoring the mechanism of RNA interference and assessment of appropriate methods for siRNA delivery.