Novel fugu U6 promoter driven shRNA expression vector for efficient vector based RNAi in fish cell lines.

In fish species, although many studies on the use of RNA interference (RNAi) for gene function analysis have been reported, almost of them have utilized in vitro transcribed or synthesized small interfering RNA (siRNA), and there are a few studies which examined vector based RNAi in fish species. In this study, we have identified U6 promoter of fugu (Takifugu rubripes), and utilized it for expression of short hairpin RNA (shRNA) in fish cell lines. Using Northern blot analysis, we confirmed successful transcription of shRNA by fugu U6 promoter in bluegill fry (BF-2) cells. The knock down assay targeting an exogenous EGFP reporter gene demonstrated that fugu U6 promoter expressed shRNA more efficiently than mouse U6 promoter in BF-2, grunt fin (GF), and Chinook salmon embryo (CHSE) cell lines. This study suggests that fugu U6 promoter driven shRNA expression vector can be novel tool for RNAi induction in fish cell lines.