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Efficient introduction of specific TP53 mutations into mouse embryonic fibroblasts and embryonic stem cells

Nature Protocols volume 7, pages 1145–1160 (2012)Cite this article

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Abstract

This protocol describes a rapid, precise method for generating sets of embryonic stem (ES) cells or mouse embryonic fibroblasts (MEFs) harboring point mutations in the p53 tumor suppressor gene (officially known as Trp53). The strategy uses cells from the Trp53 (p53-null) 'platform' mouse, which allows site-specific integration of plasmid DNA into the Trp53 locus. Simple PCR protocols identify correctly targeted clones and immunoblots verify re-expression of the protein. We also present protocol modifications needed for efficient recovery of MEF clones expressing p53 constructs that retain wild-type function, including growth at low (3%) oxygen and transient downregulation of p53 regulators to forestall cell senescence of primary MEFs. A library of cell lines expressing various p53 mutants derived from the same population of primary fibroblasts or platform ES cells can be acquired and screened in less than 1 month.

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Figure 1: Using the TOP system.
Figure 2: Confirming integrase-mediated cassette exchange by PCR.
Figure 3: Generation of related MEF lines expressing missense p53 mutants.
Figure 4: Transient silencing of INK4a/ARF potentiates cell growth and colony formation.
Figure 5: ES cell lines expressing both WT and mutant p53 can be generated from a plf/plf parent line (G1) under feeder-free conditions.

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Acknowledgements

We thank N. Pechlivani, A. Weninger and U. Kloz for technical assistance, and the Yorkshire Cancer Research, the White Rose Foundation and the German Cancer Research Center (Deutsches Krebsforschungszentrum) for funding.

Author information

Author notes

  1. Quan-Xiang Wei and Franciscus van der Hoeven: These authors contributed equally to this work.

Authors and Affiliations

  1. Genetic Alterations in Carcinogenesis (C016), German Cancer Research Center (Deutsches Krebsforschungszentrum), Heidelberg, Germany

    Quan-Xiang Wei & Monica Hollstein

  2. Transgenic Service (W450), German Cancer Research Center (Deutsches Krebsforschungszentrum), Heidelberg, Germany

    Franciscus van der Hoeven

  3. Faculty of Medicine and Health, Leeds Institute of Genetics, Health and Therapeutics (LIGHT) Laboratories, University of Leeds, Leeds, UK

    Monica Hollstein & Adam F Odell

Authors
  1. Quan-Xiang Wei
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  2. Franciscus van der Hoeven
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  3. Monica Hollstein
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  4. Adam F Odell
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Contributions

M.H., A.F.O. and Q.-X.W. designed the experiments; Q.-X.W., A.F.O. and F.v.d.H. conducted the experiments; A.F.O., Q.-X.W. and M.H. analyzed the data; A.F.O. and M.H. supervised the project; A.F.O., M.H. and Q.-X.W. wrote the protocol.

Corresponding authors

Correspondence to Monica Hollstein or Adam F Odell.

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Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Figs. 1

Growth of primary platform MEFs at 3% and 21% O2 conditions (a) Growth rate and (b) proliferation of platform MEFs (plf/plf; CA10 and 182.2) is enhanced by growth at 3% O2. Shown are collated data from 3 independent experiments expressed around the SEM. (PDF 1438 kb)

Supplementary Figs. 2

Transient depletion of Ink4a gene products enhances proliferation of primary platform MEFs. Shown are collated data from 4 independent experiments expressed around the SEM. (PDF 1160 kb)

Supplementary Figs. 3

Functional cell lines are generated following Ink4a siRNA depletion. Analysis of cell lines produced at different O2 tensions following prior Ink4a depletion by immunoblotting. Expression of p21 is restored in cell lines generated with TOP constructs retaining WT p53 function (P72, R72 and S15A) when p16 and p19 are previously depleted. Cells were treated with doxorubicin (4 h, 0.5 µM) prior to lysis for stimulation of serine 15 phosphorylation. (PDF 1902 kb)

Supplementary Figs. 4

Expression of p19 (p19arf; ab80-100) is restored by 7 days post-selection (10 days post-siRNA) in WT p53 (pab1801; sc-98) TOP transfectants grown at 21% O2. Scale bars: 20 µm. (PDF 1676 kb)

Supplementary Fig. 5

Expression of several common markers of pluripotency, Nanog (ab80892), Oct3/4 (sc-5279), Sox2 (ab97959), SSEA1 (ab16285) and Tbx3 (sc-31657) are maintained in ES cells generated to express WT, G245S and A138V p53. Also shown are the parent G1 ES cell line and the heterozygous D2 ES cells. Scale bars: 20 µm. (PDF 3718 kb)

Supplementary Method

Analysis of integrase-mediated cassette exchange by duplex PCR (PDF 18 kb)

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Wei, QX., van der Hoeven, F., Hollstein, M. et al. Efficient introduction of specific TP53 mutations into mouse embryonic fibroblasts and embryonic stem cells. Nat Protoc 7, 1145–1160 (2012). https://doi.org/10.1038/nprot.2012.042

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