Enhancing the precision of genetic lineage tracing using dual recombinases

Abstract

The Cre–loxP recombination system is the most widely used technology for in vivo tracing of stem or progenitor cell lineages. The precision of this genetic system largely depends on the specificity of Cre recombinase expression in targeted stem or progenitor cells. However, Cre expression in nontargeted cell types can complicate the interpretation of lineage-tracing studies and has caused controversy in many previous studies. Here we describe a new genetic lineage tracing system that incorporates the Dre–rox recombination system to enhance the precision of conventional Cre–loxP-mediated lineage tracing. The Dre–rox system permits rigorous control of Cre–loxP recombination in lineage tracing, effectively circumventing potential uncertainty of the cell-type specificity of Cre expression. Using this new system we investigated two topics of recent debates—the contribution of c-Kit+ cardiac stem cells to cardiomyocytes in the heart and the contribution of Sox9+ hepatic progenitor cells to hepatocytes in the liver. By overcoming the technical hurdle of nonspecific Cre–loxP-mediated recombination, this new technology provides more precise analysis of cell lineage and fate decisions and facilitates the in vivo study of stem and progenitor cell plasticity in disease and regeneration.

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Figure 1: Generation and characterization of an interleaved reporter (IR1) mouse line.
Figure 2: c-Kit+ non-cardiomyocytes do not generate cardiomyocytes.
Figure 3: Hepatocyte-to-ductal cell conversion is uncovered by the DeaLT-IR strategy.
Figure 4: Generation and characterization of a secondary dual reporter system that uses nested recombinase sites.
Figure 5: SOX9+ biliary epithelial cells adopt a ductal cell fate but not a hepatocyte fate after injury.
Figure 6: Generation and characterization of mice with additional interleaved reporters.

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Acknowledgements

We thank B. Wu, G. Chen, Z. Weng and A. Huang for the animal husbandry and W. Bian for technical help. We thank H. Zeng at Allen Institute for sharing mouse lines and K. Anastassiadis for valuable suggestions and insightful advice on this study. We thank Shanghai Model Organisms Center, Inc. (SMOC) and Nanjing Biomedical Research Institute of Nanjing University for the generation of mouse lines. This work was supported by the Strategic Priority Research Program of the Chinese Academy of Sciences (CAS) (grant no. XDB19000000; B.Z.), the National Science Foundation of China (grant no. 31730112 (B.Z.), 91639302 (B.Z.), 31625019 (B.Z.), 31571503 (X.T.), 31501172 (H. Zhang), 31601168 (Q.L.) and 31701292 (L.H.)), the National Key Research and Development Program of China (grant no. 2017YFC1001303 (L.H.) and 2016YFC1300600 (X.T.)), the Youth Innovation Promotion Association of CAS (award no. 2015218; X.T.), the Key Project of Frontier Sciences of CAS (grant no. QYZDB-SSW-SMC003; B.Z.), the International Cooperation Fund of CAS (B.Z.), the National Program for Support of Top-notch Young Professionals (B.Z.), the Shanghai Science and Technology Commission (grant no. 17ZR1449600 (B.Z.) and 17ZR1449800 (X.T.)), the Young Elite Scientists Sponsorship Program by the China Association for Science and Technology (Q.L. and L.H.), the Shanghai Yangfan Project (award no. 15YF1414000 (H. Zhang) and 16YF1413400 (L.H.)) and Rising-Star Program (grant no.15QA1404300; X.T.), the China Postdoctoral Science Foundation (Y.W., Q.L. and J.T.), the President Fund of Shanghai Institutes for Biological Sciences (SIBS) (B.Z.), Astrazeneca (B.Z.), Sanofi-SIBS Fellowship (X.T. and L.H.), Boehringer Ingelheim (B.Z.) and a Royal Society–Newton Advanced Fellowship (B.Z.).

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L.H. and B.Z. designed the study, performed experiments and analyzed the data; Yan Li, Yi Li, W.P., X.H., X.T., Y.W., H. Zhang, Q.L., L.Z., H. Zhao and J.T. bred the mice and performed experiments; D.C., H.J., Zhibo Han, Zhongchao Han, Y.N., S.H., Q.-D.W., R.S., J.F., Y.Y. and H.H. analyzed data, provided technical support and edited the manuscript; F.W. and T.C. provided mouse lines; W.T.P. provided intellectual input and edited the manuscript; B.Z. conceived and supervised the study, analyzed the data and wrote the manuscript.

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Correspondence to Bin Zhou.

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The authors declare no competing financial interests.

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Supplementary Figures 1–11 & Supplementary Table 1 (PDF 11449 kb)

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He, L., Li, Y., Li, Y. et al. Enhancing the precision of genetic lineage tracing using dual recombinases. Nat Med 23, 1488–1498 (2017). https://doi.org/10.1038/nm.4437

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