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Exp Hematol. 2010 Sep;38(9):792-7. doi: 10.1016/j.exphem.2010.05.005. Epub 2010 May 26.

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.

Author information

1
Department of Experimental Anaesthesiology, University Hospital Freiburg, Center for Clinical Research, Freiburg, Germany.

Abstract

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.

PMID:
20685233
DOI:
10.1016/j.exphem.2010.05.005
[Indexed for MEDLINE]

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