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, Jef Hammond
J RNAi Gene Silencing | Published : 2008
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 exoge..View full abstract