RNA interference (RNAi) is the phenomenon of post-transcriptional gene silencing (PTGS) found in C. elegans in 1998 in which dsRNA specifically blocks the expression of its homologous gene. RNAi has been widely applied in many organisms such as plants, lower animals and higher vertebrates as a tool to study gene function, gene regulation and gene therapy. However, as a model organism receiving more and more attention, the study of RNAi application in zebrafish has not developed with convenient technology as that in the lower animals and mouse. So far, reports on the study of RNAi in zebrafish have not reached the consistent conclusions, but even controversial conclusions. Thus, it remains a question that whether RNAi works in zebrafish as a tool to study gene function. In this project we firstly described the cloning, transcription activity analysis and RNAi application of zebrafish H1 and U6 promoters. By ways of experiments in cultured fish cells and zebrafish embryos, shRNA vectors driven by zebrafish H1 and U6 promoters could knock down the transcription levels of foreign and endogenous genes. This study also applied bacteriophage T7 RNA polymerase/T7 promoter system in RNAi study of zebrafish embryos and the results confirmed that this system could work well in zebrafish embryos. Furthermore, shRNA vector driven by T7 promoter inhibited effectively the target genes expression in transgenic zebrafish embryos which expressed stably T7 RNA polymerase. This study provided a good foundation for further application of RNAi in zebrafish and a wide profoundness for study gene function in zebrafish by ways of RNAi.
Keywords: RNA interference, zebrafish, H1 promoter, U6 promoter, T7 RNA polymerase, T7 promoter, shRNA vector