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

https://doi.org/10.1016/j.bbrc.2008.04.116Get rights and content

Abstract

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.

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).

References (34)

  • P.J. Paddison et al.

    Short hairpin RNAs (shRNAs) induce sequence-specific silencing in mammalian cells

    Genes Dev.

    (2002)
  • J.Y. Yu et al.

    RNA interference by expression of short-interfering RNAs and hairpin RNAs in mammalian cells

    Proc. Natl. Acad. Sci. USA

    (2002)
  • L.S. Lambeth et al.

    Characterisation and application of a bovine U6 promoter for expression of short hairpin RNAs

    BMC Biotechnol.

    (2005)
  • L.S. Lambeth et al.

    Comparison of bovine RNA polymerase III promoters for short hairpin RNA expression

    Anim. Genet.

    (2006)
  • T. Kudo et al.

    Usage of putative chicken U6 promoters for vector-based RNA interference

    J. Reprod. Dev.

    (2005)
  • T.G. Wise et al.

    Characterization and Comparison of Chicken U6 Promoters for the Expression of Short Hairpin RNAs

    Anim. Biotechnol.

    (2007)
  • L. Schramm et al.

    Recruitment of RNA polymerase III to its target promoters

    Genes Dev.

    (2002)
  • Cited by (0)

    View full text