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An inducible p21-Cre mouse model to monitor and manipulate p21-highly-expressing senescent cells in vivo

Abstract

The role of senescent cells has been implicated in various tissue dysfunctions associated with aging, obesity and other pathological conditions. Currently, most transgenic mouse models target only p16Ink4a-highly expressing (p16high) cells. In the present technical report, we generated a p21-Cre mouse model, containing a p21 promoter-driving inducible Cre, enabling us to examine p21Cip1-highly expressing (p21high) cells, a previously unexplored cell population exhibiting several characteristics typical of senescent cells. By crossing p21-Cre mice with different floxed mice, we managed to monitor, sort, image, eliminate or modulate p21high cells in vivo. We showed that p21high cells can be induced by various conditions, and percentages of p21high cells varied from 1.5% to 10% across different tissues in 23-month-old mice. Intermittent clearance of p21high cells improved physical function in 23-month-old mice. Our report demonstrates that the p21-Cre mouse model is a valuable and powerful tool for studying p21high cells to further understand the biology of senescent cells.

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Fig. 1: Generation of p21-Cre mouse model.
Fig. 2: Whole-body live imaging of p21high cells in vivo using BLI.
Fig. 3: The p21high cells accumulate in various tissues with aging.
Fig. 4: The p21high cells can be induced by aging, chemotherapy and obesity.
Fig. 5: The p21high cells exhibit features of cellular senescence.
Fig. 6: Inducible elimination of p21high cells using DTA.
Fig. 7: Genetic inhibition of SASP in p21high cells in vivo.
Fig. 8: Clearance of p21high cells improves physical function in old mice.

Data availability

The source data are published with the manuscript and are available from the corresponding author on reasonable request. The link for the Tabula Muris Senis database is https://tabula-muris-senis.ds.czbiohub.org.

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Acknowledgements

We thank colleagues in the UConn Center on Aging for helpful and constructive discussion, Z. Hao for histology services and S. Farkas for administrative assistance. This work was supported in part by the Regenerative Medicine Initiative for Diabetes–Career Development Award from Mayo Clinic (to M.X.), Glenn Foundation for Medical Research and AFAR Grant for Junior Faculty (to M.X.), Robert and Arlene Kogod (to J.L.K.), the Connor Group (to J.L.K.), Robert J. and Theresa W. Ryan (to J.L.K.), the Noaber Foundation (to J.L.K.), Travelers Chair in Geriatrics and Gerontology (to G.A.K.), and National Institutes of Health grants (nos. R37AG013925 (to J.L.K.), P01AG062413 (to J.L.K.), R33AG061456 (to J.L.K., T.T. and G.A.K.), AG063528 (to M.X.), AG066679 (to M.X.) and AG068860 (to M.X.)).

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Authors

Contributions

M.X., J.L.K. and T.T. conceived the p21-Cre mouse model. M.X. designed, generated and validated the p21-Cre mouse model. B.W., L.W., N.S.G., Y.Z., C.G., T.K. and M.X. performed the mouse studies. B.W. and E.R.J. contributed to the FACS analysis. N.S.G., B.W. and Y.Z. contributed to the histological analysis. L.H., S.Y., G.A.K. and J.L.K. contributed to manuscript preparation. M.X. wrote the manuscript with input from all coauthors. M.X. oversaw all experiments, data analysis and manuscript preparation.

Corresponding author

Correspondence to Ming Xu.

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The authors declare no competing financial interests.

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Peer review information Nature Aging thanks Valery Krizhanovsky, and the other, anonymous reviewer(s) for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 p16high and p21high cells are two distinct cell populations in aged tissues.

Uniform manifold approximation and projection (UMAP) plots showing expression levels of p21 (cdkn1a) and p16 (cdkn2a) in visceral fat, liver and heart in 18-30 months old mice. The figures were generated using the Tabula Muris Senis interactive platform (https://tabula-muris-senis.ds.czbiohub.org/).

Extended Data Fig. 2 p21 expression in tdTomato+ and tdTomato- cells in old mice.

(a) Representative images of tdTomato + and tdTomato- SVF cells in old PT mice. (b) p21 staining intensity of tdTomato + and tdTomato- SVF cells detected by flow cytometry. (c) p21 + cells percentage and p21 mean fluorescence intensity (MFI) of tdTomato + and tdTomato- SVF cells. For c, n = 3 for both groups. Results were shown as mean ± s.e.m. * p < 0.05; two-tailed, paired Student’s t-test. p = 0.009 for p21 + cells%; p = 0.028 for p21 MFI.

Source data

Extended Data Fig. 3 Tissue distribution of p21high cells.

Representative images of LUC activity in various tissues from young and old mice. r.l.u., relative luciferase units.

Extended Data Fig. 4 The activity of p21-Cre is low in cells in vitro.

(a) Flow cytometry analysis of tdTomato (T) in MEFs (P/P; T/+and P/+; T/+). Representative image of tdTomato+ cells can be only seen in P/P; T/+MEFs treated with both 4-OHT and DOXO. (b) tdTomato+ cells percentage in MEFs. For b, n = 4 for all groups. Results were shown as mean ± s.e.m. * p = 0.018; two-tailed, unpaired Student’s t-test.

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Unprocessed gels.

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Unprocessed gels.

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Wang, B., Wang, L., Gasek, N.S. et al. An inducible p21-Cre mouse model to monitor and manipulate p21-highly-expressing senescent cells in vivo. Nat Aging 1, 962–973 (2021). https://doi.org/10.1038/s43587-021-00107-6

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