Objective: RNA interference mediated by transcription of short hairpin RNAs (shRNAs) from lentiviral expression vectors has emerged as an efficient method to effectively and specifically silence gene expression in a vast variety of mammalian cells. shRNA expression is routinely driven by a RNA polymerase III promoter, most often by the U6 promoter. Here we demonstrate that U6 promoter activity-and consequently gene silencing success-differs significantly among species.
Materials and methods: We have modified pLeGO-G, an HIV-based third-generation lentivector, to express a 19nt shRNA sequence against the human transcription factor nuclear factor erythroid 2 or against its murine homologue, as well as an shRNA against murine JAK2, from either the human or the murine U6 promoter. Gene silencing efficiency was analyzed in a human erythroleukemic cell line, in primary human CD34(+) cells, as well as in a murine erythroleukemic cell line and in primary murine bone marrow.
Results: ShRNA expression from the human U6 promoter resulted in a fourfold increase in knockdown efficiency compared to expression from the murine U6 promoter in both human and murine cells.
Conclusions: The U6 promoter constitutes an important determinant for efficient gene silencing by shRNAs.