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.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.


  1. 1.

    et al. Postnatal Isl1+ cardioblasts enter fully differentiated cardiomyocyte lineages. Nature 433, 647–653 (2005).

  2. 2.

    et al. De novo cardiomyocytes from within the activated adult heart after injury. Nature 474, 640–644 (2011).

  3. 3.

    et al. Endothelial cells are progenitors of cardiac pericytes and vascular smooth muscle cells. Nat. Commun. 7, 12422 (2016).

  4. 4.

    et al. Defining a mesenchymal progenitor niche at single-cell resolution. Science 346, 1258810 (2014).

  5. 5.

    et al. Intestinal crypt homeostasis results from neutral competition between symmetrically dividing Lgr5 stem cells. Cell 143, 134–144 (2010).

  6. 6.

    et al. Cardiac lymphatics are heterogeneous in origin and respond to injury. Nature 522, 62–67 (2015).

  7. 7.

    & Site-specific DNA recombination in mammalian cells by the Cre recombinase of bacteriophage P1. Proc. Natl. Acad. Sci. USA 85, 5166–5170 (1988).

  8. 8.

    Cre recombinase: the universal reagent for genome tailoring. Genesis 26, 99–109 (2000).

  9. 9.

    , , & Lost in transgenesis: a user's guide for genetically manipulating the mouse in cardiac research. Circ. Res. 111, 761–777 (2012).

  10. 10.

    , & Cellular origin and developmental program of coronary angiogenesis. Circ. Res. 116, 515–530 (2015).

  11. 11.

    & DNA recombination with a heterospecific Cre homolog identified from comparison of the pac-c1 regions of P1-related phages. Nucleic Acids Res. 32, 6086–6095 (2004).

  12. 12.

    et al. Dre recombinase, like Cre, is a highly efficient site-specific recombinase in E. coli, mammalian cells and mice. Dis. Model. Mech. 2, 508–515 (2009).

  13. 13.

    , & A Cre recombinase transgene with mosaic, widespread tamoxifen-inducible action. Genesis 32, 8–18 (2002).

  14. 14.

    et al. Adult cardiac stem cells are multipotent and support myocardial regeneration. Cell 114, 763–776 (2003).

  15. 15.

    et al. Adult c-Kit+ cardiac stem cells are necessary and sufficient for functional cardiac regeneration and repair. Cell 154, 827–842 (2013).

  16. 16.

    et al. c-Kit+ cells minimally contribute cardiomyocytes to the heart. Nature 509, 337–341 (2014).

  17. 17.

    et al. Stimulatory effects of MSCs on c-Kit+ cardiac stem cells are mediated by SDF1–CXCR4 and SCF–c-Kit signaling pathways. Circ. Res. 119, 921–930 (2016).

  18. 18.

    & Are resident c-Kit+ cardiac stem cells really all that are needed to mend a broken heart? Circ. Res. 113, 1037–1039 (2013).

  19. 19.

    Letter by Molkentin regarding article, “The absence of evidence is not evidence of absence: the pitfalls of Cre knock-ins in the c-Kit locus”. Circ. Res. 115, e21–e23 (2014).

  20. 20.

    et al. Genetic lineage tracing identifies in situ Kit-expressing cardiomyocytes. Cell Res. 26, 119–130 (2016).

  21. 21.

    et al. Robust cellular reprograming occurs spontaneously during liver regeneration. Genes Dev. 27, 719–724 (2013).

  22. 22.

    et al. Mfsd2a+ hepatocytes repopulate the liver during injury and regeneration. Nat. Commun. 7, 13369 (2016).

  23. 23.

    & Vertebrate endoderm development and organ formation. Annu. Rev. Cell Dev. Biol. 25, 221–251 (2009).

  24. 24.

    , & Clonal tracing of Sox9+ liver progenitors in mouse oval cell injury. Hepatology 60, 278–289 (2014).

  25. 25.

    et al. Continuous cell supply from a Sox9-expressing progenitor zone in adult liver, exocrine pancreas and intestine. Nat. Genet. 43, 34–41 (2011).

  26. 26.

    et al. Hybrid periportal hepatocytes regenerate the injured liver without giving rise to cancer. Cell 162, 766–779 (2015).

  27. 27.

    et al. Resident c-Kit+ cells in the heart are not cardiac stem cells. Nat. Commun. 6, 8701 (2015).

  28. 28.

    et al. c-Kit+ cardiac progenitors of neural crest origin. Proc. Natl. Acad. Sci. USA 112, 13051–13056 (2015).

  29. 29.

    et al. Embryonic ductal plate cells give rise to cholangiocytes, periportal hepatocytes and adult liver progenitor cells. Gastroenterology 141, 1432–1438 (2011).

  30. 30.

    et al. Adult hepatocytes are generated by self-duplication rather than stem cell differentiation. Cell Stem Cell 15, 340–349 (2014).

  31. 31.

    , , , & Cryptic boundaries in roof plate and choroid plexus identified by intersectional gene activation. Nat. Genet. 35, 70–75 (2003).

  32. 32.

    et al. Islet1 derivatives in the heart are of both neural crest and second heart field origin. Circ. Res. 110, 922–926 (2012).

  33. 33.

    et al. Redefining the serotonergic system by genetic lineage. Nat. Neurosci. 11, 417–419 (2008).

  34. 34.

    et al. Expanding the power of recombinase-based labeling to uncover cellular diversity. Development 142, 4385–4393 (2015).

  35. 35.

    , & A knock-in allele of En1 expressing Dre recombinase. Genesis 54, 447–454 (2016).

  36. 36.

    et al. Cell fusion is the principal source of bone-marrow-derived hepatocytes. Nature 422, 897–901 (2003).

  37. 37.

    et al. Fusion of bone-marrow-derived cells with Purkinje neurons, cardiomyocytes and hepatocytes. Nature 425, 968–973 (2003).

  38. 38.

    , & Transplanted bone marrow regenerates liver by cell fusion. Nature 422, 901–904 (2003).

  39. 39.

    et al. Deletion of the developmentally essential gene Atr in adult mice leads to age-related phenotypes and stem cell loss. Cell Stem Cell 1, 113–126 (2007).

  40. 40.

    et al. Temporally regulated and tissue-specific gene manipulations in the adult and embryonic heart using a tamoxifen-inducible Cre protein. Circ. Res. 89, 20–25 (2001).

  41. 41.

    et al. c-Kit+ cells adopt vascular endothelial but not epithelial cell fates during lung maintenance and repair. Nat. Med. 21, 866–868 (2015).

  42. 42.

    et al. A robust and high-throughput Cre reporting and characterization system for the whole mouse brain. Nat. Neurosci. 13, 133–140 (2010).

  43. 43.

    et al. Endocardium contributes to cardiac fat. Circ. Res. 118, 254–265 (2016).

  44. 44.

    et al. Endocardium minimally contributes to coronary endothelium in the embryonic ventricular free walls. Circ. Res. 118, 1880–1893 (2016).

  45. 45.

    et al. Genetic lineage tracing identifies endocardial origin of liver vasculature. Nat. Genet. 48, 537–543 (2016).

  46. 46.

    et al. Genetic targeting of sprouting angiogenesis using Apln–CreER. Nat. Commun. 6, 6020 (2015).

  47. 47.

    et al. Sub-epicardial endothelial cells invade the embryonic ventricle wall to form coronary arteries. Cell Res. 23, 1075–1090 (2013).

  48. 48.

    et al. Genetic lineage tracing discloses arteriogenesis as the main mechanism for collateral growth in the mouse heart. Cardiovasc. Res. 109, 419–430 (2016).

  49. 49.

    et al. Adult mouse epicardium modulates myocardial injury by secreting paracrine factors. J. Clin. Invest. 121, 1894–1904 (2011).

  50. 50.

    et al. Bile duct ligation in mice: induction of inflammatory liver injury and fibrosis by obstructive cholestasis. JoVE 96, e52438 (2015).

Download references


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.).

Author information


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

    • Lingjuan He
    • , Yan Li
    • , Yi Li
    • , Wenjuan Pu
    • , Xiuzhen Huang
    • , Xueying Tian
    • , Yue Wang
    • , Hui Zhang
    • , Qiaozhen Liu
    • , Libo Zhang
    • , Huan Zhao
    • , Juan Tang
    • , Hongbin Ji
    •  & Bin Zhou
  2. Key Laboratory of Nutrition and Metabolism, Institute for Nutritional Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai, China.

    • Lingjuan He
    • , Yan Li
    • , Yi Li
    • , Wenjuan Pu
    • , Xiuzhen Huang
    • , Xueying Tian
    • , Yue Wang
    • , Hui Zhang
    • , Qiaozhen Liu
    • , Libo Zhang
    • , Huan Zhao
    • , Juan Tang
    •  & Bin Zhou
  3. School of Life Science and Technology, ShanghaiTech University, Shanghai, China.

    • Hongbin Ji
    •  & Bin Zhou
  4. Key Laboratory of Regenerative Medicine of Ministry of Education, Institute of Aging and Regenerative Medicine, Jinan University, Guangzhou, China.

    • Dongqing Cai
    •  & Bin Zhou
  5. State Key Laboratory of Experimental Hematology, Institute of Hematology and Hospital of Blood Disease, Chinese Academy of Medical Science and Peking Union Medical College, Tianjin, China.

    • Zhibo Han
    •  & Zhongchao Han
  6. State Key Laboratory of Cardiovascular Disease, Fuwai Hospital, National Center for Cardiovascular Disease, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China.

    • Yu Nie
    •  & Shengshou Hu
  7. Bioscience Heart Failure, Cardiovascular and Metabolic Diseases, IMED Biotech Unit, AstraZeneca, Gothenburg, Sweden.

    • Qing-Dong Wang
  8. Shanghai Model Organisms Center, Inc., Shanghai, China.

    • Ruilin Sun
    •  & Jian Fei
  9. National Institute of Biological Sciences, Beijing, China.

    • Fengchao Wang
    •  & Ting Chen
  10. Zhongshan Hospital, Fudan University, Shanghai, China.

    • Yan Yan
  11. International Peace Maternity and Child Health Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China.

    • Hefeng Huang
  12. Department of Cardiology, Boston Children's Hospital, Boston, Massachusetts, USA and Harvard Stem Cell Institute, Harvard University, Cambridge, Massachusetts, USA.

    • William T Pu
  13. Collaborative Innovation Center of Tianjin for Medical Epigenetics, Tianjin Medical University, Tianjin, China.

    • Bin Zhou


  1. Search for Lingjuan He in:

  2. Search for Yan Li in:

  3. Search for Yi Li in:

  4. Search for Wenjuan Pu in:

  5. Search for Xiuzhen Huang in:

  6. Search for Xueying Tian in:

  7. Search for Yue Wang in:

  8. Search for Hui Zhang in:

  9. Search for Qiaozhen Liu in:

  10. Search for Libo Zhang in:

  11. Search for Huan Zhao in:

  12. Search for Juan Tang in:

  13. Search for Hongbin Ji in:

  14. Search for Dongqing Cai in:

  15. Search for Zhibo Han in:

  16. Search for Zhongchao Han in:

  17. Search for Yu Nie in:

  18. Search for Shengshou Hu in:

  19. Search for Qing-Dong Wang in:

  20. Search for Ruilin Sun in:

  21. Search for Jian Fei in:

  22. Search for Fengchao Wang in:

  23. Search for Ting Chen in:

  24. Search for Yan Yan in:

  25. Search for Hefeng Huang in:

  26. Search for William T Pu in:

  27. Search for Bin Zhou in:


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.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Bin Zhou.

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–11 & Supplementary Table 1

  2. 2.

    Life Sciences Reporting Summary

About this article

Publication history






Further reading