Of mice and men: Human RNA polymerase III promoter U6 is more efficient than its murine homologue for shRNA expression from a lentiviral vector in both human and murine progenitor cells

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.