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Delivery of short hairpin RNAs—triggers of gene silencing—into mouse embryonic stem cells

Nature Methods volume 3, pages 397–400 (2006)Cite this article

  • DMEM knockout medium (Gibco)

  • EGFP_433 RNA shRNA

  • 5′-AGCCACAACGUCUAUAUCAUGGCCGACAAGUUGGCUUGUCGGCC AUGAUAUAGACGUUGUGGCUGU-3′ (Open Biosystems)

  • This sequence was created using a synthetic shRNA design website (maintained by the Sachidanandam lab at CSHL) available online (http://katahdin.cshl.edu/homepage/siRNA/RNAi.cgi?type=shRNA).

  • Gelatin (Sigma; porcine skin, 300 bloom), 0.1% in phosphate-buffered saline (PBS), autoclaved

  • Lipofectamine 2000 (Invitrogen)

  • M15 ES cell medium with leukemia inhibitory factor (LIF): DMEM Knockout (Gibco), 15% fetal bovine serum (FBS; StemCell Technologies or HyClone), 100 μM β-mercaptoethanol, 2 mM L-glutamine, 50 U/ml penicillin, 40 μg/ml streptomycin, 1,000 U/ml LIF (Chemicon)

  • M15 ES cell medium with LIF, without antibiotics

  • Mouse ES cell line (feeder-free adapted; Nanog-GFP cells are shown in Fig. 1)

  • Detailed information for the generation of Nanog-GFP ES cells is available on request; see Figure 1a for a brief description.

  • PBS (pH 7.4)

  • Trypsin solution, 1× (pH 7.6): trypsin (Gibco; 1:250; 2.5 g/l), 120 mM NaCl, 6 mM D-glucose, 1 mM Na2HPO4(7·H2O), 5 mM KCl, 2 mM KH2PO4, 1 mM EDTA, 25 mM Tris base and a few drops of phenol red

DMEM knockout medium (Gibco)

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Figure 1: Delivery of synthetic shRNA into Nanog-GFP mouse ES cells.

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Acknowledgements

This method was adapted from the Cell Signaling Consortium protocol for transfecting 293T cells in suspension (kindly provided by Iain Fraser; California Institute of Technology) and the manufacturer's protocol for Lipofectamine 2000. The BAC recombination protocol and shuttle vector for creating the Nanog-GFP construct were kindly provided by Nathanial Heintz (Rockefeller University). P.J.P. thanks A. Mills and her lab at Cold Spring Harbor Laboratory (CSHL) for help with ES cell culture. P.J.P. is supported by the CSHL President's Council Fund for CSHL fellows.

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Authors and Affiliations

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

    Christoph Schaniel, Feng Li, Xenia L Schafer & Ihor R Lemischka

  2. Open Biosystems, Huntsville, 35806, Alabama, USA

    Troy Moore

  3. Cold Spring Harbor Laboratory, 1 Bungtown Road, Cold Spring Harbor, 11724, New York, USA

    Patrick J Paddison

Authors
  1. Christoph Schaniel
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  2. Feng Li
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  3. Xenia L Schafer
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Correspondence to Patrick J Paddison.

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Schaniel, C., Li, F., Schafer, X. et al. Delivery of short hairpin RNAs—triggers of gene silencing—into mouse embryonic stem cells. Nat Methods 3, 397–400 (2006). https://doi.org/10.1038/nmeth0506-397

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