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Dissecting self-renewal in stem cells with RNA interference

Naturevolume 442pages533538 (2006) | Download Citation

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Abstract

We present an integrated approach to identify genetic mechanisms that control self-renewal in mouse embryonic stem cells. We use short hairpin RNA (shRNA) loss-of-function techniques to downregulate a set of gene products whose expression patterns suggest self-renewal regulatory functions. We focus on transcriptional regulators and identify seven genes for which shRNA-mediated depletion negatively affects self-renewal, including four genes with previously unrecognized roles in self-renewal. Perturbations of these gene products are combined with dynamic, global analyses of gene expression. Our studies suggest specific biological roles for these molecules and reveal the complexity of cell fate regulation in embryonic stem cells.

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Acknowledgements

We thank A. Smith for E14/T cells and the episomal expression system, G. Daley for Aniv15 cells, and D. Baltimore for the FUGW lentiviral expression system. We are grateful to K. A. Moore and members of the Lemischka laboratory for their assistance at various stages of these studies. We also thank A. Smith for constructive criticisms. This work was supported by funding from the NIDDK. Author Contributions N.I. and I.R.L. designed the experiments. N.I., R.L., I.K., J.L., C.D., X.S. and Y.L. performed the experiments. N.I., R.D. and I.R.L. analysed the data. N.I. and I.R.L. wrote the paper.

Author information

Affiliations

  1. Department of Molecular Biology, Princeton University, New Jersey, 08544, Princeton, USA

    • Natalia Ivanova
    • , Radu Dobrin
    • , Rong Lu
    • , Iulia Kotenko
    • , John Levorse
    • , Christina DeCoste
    • , Xenia Schafer
    • , Yi Lun
    •  & Ihor R. Lemischka

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Competing interests

Reprints and permissions information is available at npg.nature.com/reprintsandpermissions. The authors declare no competing financial interests.

Corresponding authors

Correspondence to Natalia Ivanova or Ihor R. Lemischka.

Supplementary information

  1. Supplementary Notes

    This file contains Supplementary Materials, Supplementary Tables 1–4, Supplementary Figures 1–18 and additional references. (PDF 2301 kb)

  2. Supplementary Data 1

    MAS5.0 normalized microarray data (ZIP 16494 kb)

  3. Supplementary Data 2

    MIAME description of microarray data (DOC 55 kb)

  4. Supplementary Data 3

    Description of microarray files (XLS 27 kb)

  5. Supplementary Data 4

    Non-redundant probes used in microarray analysis (XLS 3771 kb)

  6. Supplementary Data 5

    Probes affected at least in one shRNA time-course (XLS 503 kb)

  7. Supplementary Data 6

    901 probes down-regulated in RA differentiation time-course (XLS 732 kb)

  8. Supplementary Data 7

    Information for genes used in the over-expression studies (XLS 43 kb)

  9. Supplementary Data 8

    List of genes affected by Nanog shRNA (XLS 252 kb)

  10. Supplementary Data 9

    List of genes affected by Oct4 shRNA (XLS 189 kb)

  11. Supplementary Data 10

    List of genes affected by Sox2 shRNA (XLS 228 kb)

  12. Supplementary Data 11

    List of genes affected by Esrrb shRNA (XLS 149 kb)

  13. Supplementary Data 12

    List of genes affected by Tbx3 shRNA (XLS 136 kb)

  14. Supplementary Data 13

    List of genes affected by Tcl1 shRNA (XLS 100 kb)

  15. Supplementary Data 14

    List of genes affected by Mm.343880 shRNA (XLS 111 kb)

  16. Supplementary Data 15

    List of genes affected by Dppa4 shRNA (XLS 74 kb)

  17. Supplementary Data 16

    List of genes down-regulated during the Dppa4 shRNA time-course only. (XLS 20 kb)

  18. Supplementary Data 17

    List of genes down-regulated during the Mm.343880 shRNA time-course only. (XLS 17 kb)

  19. Supplementary Data 18

    List of genes up-regulated during the Mm.343880 shRNA time-course only. (XLS 11 kb)

  20. Supplementary Data 19

    List of genes down-regulated during the Nanog and Oct4 shRNA time-courses only. (XLS 13 kb)

  21. Supplementary Data 20

    List of genes down-regulated during the Nanog shRNA time-course only. (XLS 36 kb)

  22. Supplementary Data 21

    List of genes up-regulated during the Nanog shRNA time-course only. (XLS 25 kb)

  23. Supplementary Data 22

    List of genes down-regulated during the Oct4 shRNA time-course only. (XLS 21 kb)

  24. Supplementary Data 23

    List of genes up-regulated during the Oct4 shRNA time-course only. (XLS 20 kb)

  25. Supplementary Data 24

    List of genes up-regulated during the Oct4 and Sox2 shRNA time-courses only. (XLS 14 kb)

  26. Supplementary Data 25

    List of genes down-regulated in Pattern1 (XLS 27 kb)

  27. Supplementary Data 26

    List of genes up-regulated in Pattern1 (XLS 116 kb)

  28. Supplementary Data 27

    List of genes down-regulated in Pattern2 (XLS 48 kb)

  29. Supplementary Data 28

    List of genes up-regulated in Pattern2 (XLS 48 kb)

  30. Supplementary Data 29

    List of genes down-regulated in Pattern3 (XLS 15 kb)

  31. Supplementary Data 30

    List of genes up-regulated in Pattern3 (XLS 49 kb)

  32. Supplementary Data 31

    List of genes down-regulated during the Sox2 shRNA time-course only (XLS 16 kb)

  33. Supplementary Data 32

    List of genes up-regulated during the Sox2 shRNA time-course only (XLS 19 kb)

  34. Supplementary Data 33

    List of genes down-regulated during the Tbx3 shRNA time-course only (XLS 18 kb)

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DOI

https://doi.org/10.1038/nature04915

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