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Genetic recording of in vivo cell proliferation by ProTracer

Nature Protocols volume 18, pages 2349–2373 (2023)Cite this article

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Abstract

The ability to experimentally measure cell proliferation is the basis for understanding the sources of cells that drive organ development, tissue regeneration and repair. Recently, we generated a genetic approach to detect cell proliferation: we used genetic lineage–tracing technologies to achieve seamless recording of in vivo cell proliferation in a tissue-specific manner. We provide a detailed protocol (generation of mouse lines, characterization of mouse lines, mouse line crossing and cell-proliferation tracing) for using this genetic system to study cell proliferation. This cell-proliferation tracing system, which we term ‘ProTracer’ (Proliferation Tracer), permits lifelong noninvasive monitoring of cell proliferation of specific cell lineages in live animals. Compared with other short-term strategies that require execution of animals, ProTracer does not require sampling or animal sacrifice for tissue processing. To highlight these features, we used ProTracer to study the proliferation of hepatocytes during liver homeostasis and after tissue injury in mice. We show that the protocol is applicable to study any in vivo cell proliferation, which takes ~9 months to finish from mouse generation to data analysis. This protocol can easily be carried out by researchers skilled in mouse-related experiments.

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Fig. 1: Lineage tracing of Ki67+ cells by traditional and dual-recombinase–mediated genetic tracing methods.
Fig. 2: The workflow of fate mapping cell proliferation by ProTracer.
Fig. 3: The ProTracer mouse breeding strategy.
Fig. 4: Characterization of the specificity and leakiness of Ki67-CrexER.
Fig. 5: Characterization of R26-DreER and Alb-DreER mice.
Fig. 6: Hepatocyte proliferation captured by R26-DreER primed ProTracer.
Fig. 7: Proliferative cells captured by ubiquitous ProTracer in multiple tissues.
Fig. 8: Hepatocyte proliferation traced by ProTracer and EdU incorporation.
Fig. 9: Hepatocyte proliferation traced by Alb-DreER primed ProTracer.
Fig. 10: Examination of hepatocyte proliferation after PHx by hepatocyte-specific ProTracer.
Fig. 11: Monitoring hepatocyte proliferation by noninvasive bioluminescence imaging.

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Data availability

The main data of this protocol are from the studies published in the supporting primary research paper by He et al.27.

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Acknowledgements

This study was supported by the National Science Foundation of China (grant nos. 82088101 and 32050087 to B.Z.), the National Key Research & Development Program of China (grant nos. 2019YFA0110403 and 2019YFA0802000 to B.Z. and grant nos. 2021YFA0805100 and 2018YFA0108100 to L.H.), Shanghai Pilot Program for Basic Research – Chinese Academy of Science, Shanghai Branch (grant no. JCYJ-SHFY-2021-0 to B.Z.), CAS Project for Young Scientists in Basic Research (grant no. YSBR-012 to B.Z.), the Pearl River Talent Recruitment Program of Guangdong Province (grant no. 2017ZT07S347 to B.Z.), the XPLORER PRIZE (to B.Z.), Benyuan Young Investigator Program (to L.H.), New Cornerstone Science Foundation (to B.Z.), AstraZeneca and the Shanghai Municipal Science and Technology Major Project. We also thank Shanghai Model Organisms Center, Inc., for generating mice; members of the animal facility and cell platform in CEMCS; the National Center for Protein Science Shanghai for assistance in microscopy; and the Genome Tagging Project (GTP) Center, CEMCS, CAS for technical support.

Author information

Author notes

  1. These authors contributed equally: Xiuxiu Liu, Wendong Weng.

Authors and Affiliations

  1. State Key Laboratory of Cell Biology, Shanghai Institute of Biochemistry and Cell Biology, Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai, China

    Xiuxiu Liu, Wendong Weng & Bin Zhou

  2. School of Life Sciences, Westlake University, Hangzhou, China

    Lingjuan He

  3. School of Life Science and Technology, ShanghaiTech University, Shanghai, China

    Bin Zhou

  4. Key Laboratory of Systems Health Science of Zhejiang Province, School of Life Science, Hangzhou Institute for Advanced Study, University of Chinese Academy of Sciences, Hangzhou, China

    Bin Zhou

  5. New Cornerstone Science Laboratory, Shenzhen, China

    Bin Zhou

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Contributions

B.Z. supervised the project. X.L., W.W., L.H. and B.Z. designed and performed the experiments. X.L. and B.Z. wrote the manuscript. W.W. and L.H. edited the manuscript and provided valuable comments.

Corresponding authors

Correspondence to Lingjuan He or Bin Zhou.

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Nature Protocols thanks Natalie Porat-Shliom, Sean M. Wu and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Key reference using this protocol

He, L. et al. Science 371, eabc4346 (2021): https://doi.org/10.1126/science.abc4346

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Liu, X., Weng, W., He, L. et al. Genetic recording of in vivo cell proliferation by ProTracer. Nat Protoc 18, 2349–2373 (2023). https://doi.org/10.1038/s41596-023-00833-8

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