Novel fugu U6 promoter driven shRNA expression vector for efficient vector based RNAi in fish cell lines
Section snippets
Materials and methods
Characterization of U6 promoter in fugu. Putative fugu U6 snRNA gene was identified using blast search in fugu genome project web site at http://www.fugu-sg.org/ against 107 bps human U6 snRNA sequence (GenBank Accession No. X59362).
Construction of shRNA expression vector targeting EGFP. All oligonucleotide sequences used in this study were summarized in Table 1. Genomic DNA of fugu was isolated from a small portion of trunk muscle using Labo Pass tissue kit (Cosmo Genentech, Korea), and used as
Characterization of fugu U6 promoter
As a result of blast search of fugu genomic library, three loci with high identity with human U6 snRNA was detected (>92%). In sequence analysis of upstream region of these candidate fugu U6 snRNA, only one locus contained typical U6 promoter elements reported in fugu and other mammalian species. This region predicted to be a U6 promoter was utilized as a promoter for shRNA expression vector construction. The sequence and location of promoter elements of fugu U6 promoter identified in this
Discussion
In fish species, although many studies on the use of RNAi for gene analysis and therapeutic purpose have been reported, almost of them have utilized direct transfection or injection methods of synthesized siRNA [10], [12], [13]. In the present study, we demonstrated that fugu U6 promoter could express shRNA more efficiently than commonly used mouse U6 promoter in various fish cell lines.
In vertebrate, promoter regions of snRNA genes consist of enhancer region referred to as a distal sequence
Acknowledgment
This research was supported by the Korea Research Foundation Grant funded by the Korean Government (MOEHRD, Basic Research Promotion Fund) (KRF-2006-311-F00090).
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