Lentivirus-delivered stable gene silencing by RNAi in primary cells (original) (raw)

  1. SHEILA A. STEWART1,7,
  2. DEREK M. DYKXHOORN2,7,
  3. DEBORAH PALLISER2,7,
  4. HANA MIZUNO1,
  5. EVAN Y. YU5,
  6. DONG SUNG AN6,
  7. DAVID M. SABATINI1,4,
  8. IRVIN S.Y. CHEN6,
  9. WILLIAM C. HAHN5,
  10. PHILLIP A. SHARP2,3,4,
  11. ROBERT A. WEINBERG1,4, and
  12. CARL D. NOVINA2
  13. 1Whitehead Institute for Biomedical Research, Cambridge, Massachusetts 02142, USA
  14. 2Center for Cancer Research,
  15. 3McGovern Institute for Brain Research, and
  16. 4Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA
  17. 5Department of Medical Oncology, Dana-Farber Cancer Institute, and Departments of Medicine, Brigham and Women’s Hospital and Harvard Medical School, Boston, Massachusetts 02115, USA
  18. 6University of California, Los Angeles, AIDS Institute, David Geffen School of Medicine, Department of Microbiology, Immunology, Molecular Genetics & Medicine, Los Angeles, California 90025, USA

Abstract

Genome-wide genetic approaches have proven useful for examining pathways of biological significance in model organisms such as Saccharomyces cerevisiae, Drosophila melanogastor, and Caenorhabditis elegans, but similar techniques have proven difficult to apply to mammalian systems. Although manipulation of the murine genome has led to identification of genes and their function, this approach is laborious, expensive, and often leads to lethal phenotypes. RNA interference (RNAi) is an evolutionarily conserved process of gene silencing that has become a powerful tool for investigating gene function by reverse genetics. Here we describe the delivery of cassettes expressing hairpin RNA targeting green fluorescent protein (GFP) using Moloney leukemia virus-based and lentivirus-based retroviral vectors. Both transformed cell lines and primary dendritic cells, normally refractory to transfection-based gene transfer, demonstrated stable silencing of targeted genes, including the tumor suppressor gene TP53 in normal human fibroblasts. This report demonstrates that both Moloney leukemia virus and lentivirus vector-mediated expression of RNAi can achieve effective, stable gene silencing in diverse biological systems and will assist in elucidating gene functions in numerous cell types including primary cells.

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