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


CONCLUSIONS
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.
Lambeth
Luke S
LS
CSIRO Livestock Industries, Australian Animal Health Laboratory, Geelong, VIC 3220, Australia. Luke.Lambeth@csiro.au
Moore
Robert J
RJ
Muralitharan
Morley
M
Dalrymple
Brian P
BP
McWilliam
Sean
S
Doran
Timothy J
TJ
D000818:Animals; D001483:Base Sequence; D001709:Biotechnology / Q000379:methods*; D002417:Cattle; D002460:Cell Line; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D004247:DNA / Q000737:chemistry; D004258:DNA Polymerase III / Q000235:genetics; D004285:Dogs; D017930:Genes, Reporter; D005821:Genetic Techniques; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000378:metabolism; D066298:In Vitro Techniques; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D012313:RNA / Q000737:chemistry*; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D014162:Transfection; D019076:Transgenes
D034741:RNA, Small Interfering; D049452:Green Fluorescent Proteins; D012313:RNA; D004247:DNA; D004258:DNA Polymerase III