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Using Cre-recombinase-driven Polylox barcoding for in vivo fate mapping in mice

Nature Protocols volume 14, pages 1820–1840 (2019)Cite this article

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Abstract

Fate mapping is a powerful genetic tool for linking stem or progenitor cells with their progeny, and hence for defining cell lineages in vivo. The resolution of fate mapping depends on the numbers of distinct markers that are introduced in the beginning into stem or progenitor cells; ideally, numbers should be sufficiently large to allow the tracing of output from individual cells. Highly diverse genetic barcodes can serve this purpose. We recently developed an endogenous genetic barcoding system, termed Polylox. In Polylox, random DNA recombination can be induced by transient activity of Cre recombinase in a 2.1-kb-long artificial recombination substrate that has been introduced into a defined locus in mice (Rosa26Polylox reporter mice). Here, we provide a step-by-step protocol for the use of Polylox, including barcode induction and estimation of induction efficiency, barcode retrieval with single-molecule real-time (SMRT) DNA sequencing followed by computational barcode identification, and the calculation of barcode-generation probabilities, which is key for estimations of single-cell labeling for a given number of stem cells. Thus, Polylox barcoding enables high-resolution fate mapping in essentially all tissues in mice for which inducible Cre driver lines are available. Alternative methods include ex vivo cell barcoding, inducible transposon insertion and CRISPR–Cas9-based barcoding; Polylox currently allows combining non-invasive and cell-type-specific labeling with high label diversity. The execution time of this protocol is ~2–3 weeks for experimental data generation and typically <2 d for computational Polylox decoding and downstream analysis.

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Fig. 1: Structure of the Polylox cassette.
Fig. 2: Detection of barcode induction efficiency with Polylox-locus-specific PCR.
Fig. 3: Barcode frequencies in sample repeats.
Fig. 4: Analysis steps of the RPBPBR computational pipeline.
Fig. 5: Selection of thresholds for unique barcodes.

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Code availability

All computational codes and scripts used in this protocol have been deposited in GitHub: https://github.com/hoefer-lab/RPBPBR and https://github.com/hoefer-lab/polylox. The version used for writing this protocol can be found in the Supplementary Software.

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Acknowledgements

We thank C. Quedenau for invaluable help. T.H. is supported by CellNetworks, DKFZ core funding, e:Bio BMBF project FKZ 0316182B (SB-Epo), EU-FP7 ITN QuantI (317040), H2020-MSCA-ITN-2017 grant agreement 764698 and SFB 873-B11; H.-R.R. is supported by ERC Advanced Grant 742883, H2020-MSCA-ITN-2017 grant agreement 764698, SFB 873-B11 and DKFZ core funding.

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Author notes

  1. These authors contributed equally: Weike Pei, Xi Wang, Jens Rössler.

Authors and Affiliations

  1. Division of Cellular Immunology, German Cancer Research Center, Heidelberg, Germany

    Weike Pei, Thorsten B. Feyerabend & Hans-Reimer Rodewald

  2. Division of Theoretical Systems Biology, German Cancer Research Center, Heidelberg, Germany

    Xi Wang, Jens Rössler & Thomas Höfer

  3. Bioquant Center, University of Heidelberg, Heidelberg, Germany

    Xi Wang, Jens Rössler & Thomas Höfer

Authors
  1. Weike Pei
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  4. Thorsten B. Feyerabend
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  5. Thomas Höfer
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Contributions

W.P., X.W., J.R. and T.B.F. contributed to the practical protocol. T.B.F., X.W., T.H. and H.-R.R. wrote the manuscript with input from all authors.

Corresponding authors

Correspondence to Thorsten B. Feyerabend, Thomas Höfer or Hans-Reimer Rodewald.

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The authors declare no competing interests.

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Journal peer review information: Nature Protocols thanks Pavel Osten and other anonymous reviewer(s) for their contribution to the peer review of this work.

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Key reference using this protocol

Pei, W. et al. Nature 548, 456–460 (2017): https://doi.org/10.1038/nature23653

Supplementary information

Supplementary Software

The computational codes and scripts used in writing this protocol.

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Pei, W., Wang, X., Rössler, J. et al. Using Cre-recombinase-driven Polylox barcoding for in vivo fate mapping in mice. Nat Protoc 14, 1820–1840 (2019). https://doi.org/10.1038/s41596-019-0163-5

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