Journal of RNAi and Gene Silencing (May 2008), 4(1), 289-294

Short Report

Identification and characterisation of the porcine 7SK RNA polymerase III promoter for short hairpin RNA expression

David Cummins †‡, Timothy J Doran †*, Scott Tyack †, Damian Purcell‡ and Jef Hammond †

ABSTRACT

The RNA polymerase III (pol III) type III promoters U6 and 7SK are routinely used to express short hairpin RNA (shRNA) molecules from a DNA construct. In this study, we identified, characterised and compared the porcine 7SK promoter in porcine (homologous) and non-porcine (heterologous) derived cell lines. The porcine 7SK small nuclear RNA (snRNA) was identified by alignment with known sequences and further characterisation of the upstream regions determined the presence of typical RNA pol III sequence motifs. The porcine 7SK (po7SK) promoter was cloned and a one-step PCR strategy used to construct shRNA expression cassettes. The 7SK promoter activity was quantified by knockdown of the exogenous reporter gene encoding the enhanced green fluorescent protein (EGFP). Results indicated the po7SK promoter was functional in both homologous and heterologous cells lines. The identification and characterisation of the porcine RNA pol III promoter will contribute to the area of RNAi delivery and further develop our understanding of RNA promoter structure and function.

KEYWORDS: RNAi, 7SK, U6, pol III, shRNA

†CSIRO Livestock Industries, Australian Animal Health Laboratory, Geelong 3220, Australia

‡ The University of Melbourne, Department of Microbiology and Immunology, Parkville 3010, Australia

*Correspondence to: Timothy Doran, Email: timothy.doran@csiro.au

© Copyright The Authors

(Received 13 March 2008; Revised 11 April 2008; Accepted 06 May 2008)