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

Abstract:

BACKGROUND:The 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. RESULTS:To 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. CONCLUSION:We 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.

journal_name

BMC Biotechnol

journal_title

BMC biotechnology

authors

Lambeth LS,Moore RJ,Muralitharan M,Dalrymple BP,McWilliam S,Doran TJ

doi

10.1186/1472-6750-5-13

keywords:

subject

Has Abstract

pub_date

2005-05-11 00:00:00

pages

13

issn

1472-6750

pii

1472-6750-5-13

journal_volume

5

pub_type

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