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

Lambeth, Luke S, Moore, Robert J, Muralitharan, Morley, Dalrymple, Brian P, McWilliam, Sean and Doran, Timothy J 2005, Characterisation and application of a bovine U6 promoter for expression of short hairpin RNAs, BMC biotechnology, vol. 5, no. 1, pp. 1-13.

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Title Characterisation and application of a bovine U6 promoter for expression of short hairpin RNAs
Author(s) Lambeth, Luke S
Moore, Robert J
Muralitharan, Morley
Dalrymple, Brian P
McWilliam, Sean
Doran, Timothy J
Journal name BMC biotechnology
Volume number 5
Issue number 1
Start page 1
End page 13
Publisher BioMed Central
Place of publication London, England
Publication date 2005
ISSN 1472-6750
Keyword(s) bovinae
biosynthesis
cytology
DNA
genome sequencing
transgenic applications
genetic engineering
Summary 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.
Language eng
Field of Research 060499 Genetics not elsewhere classified
Socio Economic Objective 970106 Expanding Knowledge in the Biological Sciences
HERDC Research category C1 Refereed article in a scholarly journal
ERA Research output type C Journal article
Copyright notice ©2005, BioMed Central
Free to Read? Yes
Persistent URL http://hdl.handle.net/10536/DRO/DU:30003270

Document type: Journal Article
Collections: School of Biological and Chemical Sciences
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Every reasonable effort has been made to ensure that permission has been obtained for items included in DRO. If you believe that your rights have been infringed by this repository, please contact drosupport@deakin.edu.au.