Abstract
BackgroundThe use of small interfering RNA (siRNA) molecules in animals to achieve double-stranded RNA-mediated interference (RNAi) has recently emerged as a powerful method of sequence-specific gene knockdown. As DNA-based expression of short hairpin RNA (shRNA) for RNAi may offer some advantages over chemical and in vitro synthesised siRNA, a number of vectors for expression of shRNA have been developed. These often feature polymerase III (pol. III) promoters of either mouse or human origin.ResultsTo develop a shRNA expression vector specifically for bovine RNAi applications, we identified and characterised a novel bovine U6 small nuclear RNA (snRNA) promoter from bovine sequence data. This promoter is the putative bovine homologue of the human U6-8 snRNA promoter, and features a number of functional sequence elements that are characteristic of these types of pol. III promoters. A PCR based cloning strategy was used to incorporate this promoter sequence into plasmid vectors along with shRNA sequences for RNAi. The promoter was then used to express shRNAs, which resulted in the efficient knockdown of an exogenous reporter gene and an endogenous bovine gene.ConclusionWe have mined data from the bovine genome sequencing project to identify a functional bovine U6 promoter and used the promoter sequence to construct a shRNA expression vector. The use of this native bovine promoter in shRNA expression is an important component of our future development of RNAi therapeutic and transgenic applications in bovine species.
Highlights
The use of small interfering RNA molecules in animals to achieve doublestranded RNA-mediated interference (RNAi) has recently emerged as a powerful method of sequence-specific gene knockdown
In the current study we describe the characterisation of the bovine U6 small nuclear RNA (snRNA) promoter and its expression of short hairpin RNA (shRNA) molecules in bovine cells
Characterisation of a bovine U6 promoter A bovine BAC clone was identified from GenBank
Summary
The use of small interfering RNA (siRNA) molecules in animals to achieve doublestranded RNA-mediated interference (RNAi) has recently emerged as a powerful method of sequence-specific gene knockdown. BMC Biotechnology 2005, 5:13 http://www.biomedcentral.com/1472-6750/5/13 systems the cellular uptake of long dsRNA induces an antiviral defence mechanism initiated by interferon (IFN), leading to non-specific translational shutdown and apoptosis [10,11,12] This non-specific cellular activity can be circumvented by the direct transfection of either chemically synthesised or in vitro transcribed siRNAs of approximately 21 nt in length into mammalian cells [1,13]. The development of DNAbased vectors for expression of short hairpin RNA (shRNA) molecules that are processed within the cell to produce active siRNA molecules has progressed rapidly [16,17,18] Such DNA expression constructs have achieved highly efficient gene knockdown without induction of the IFN response
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