Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Enhancing the precision of genetic lineage tracing using dual recombinases


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.

This is a preview of subscription content

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

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.


  1. 1

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

    CAS  PubMed  PubMed Central  Google Scholar 

  2. 2

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

    CAS  PubMed  PubMed Central  Google Scholar 

  3. 3

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

    CAS  PubMed  PubMed Central  Google Scholar 

  4. 4

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

    PubMed  PubMed Central  Google Scholar 

  5. 5

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

    CAS  PubMed  Google Scholar 

  6. 6

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

    CAS  PubMed  PubMed Central  Google Scholar 

  7. 7

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

    CAS  PubMed  Google Scholar 

  8. 8

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

    CAS  PubMed  Google Scholar 

  9. 9

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

    CAS  PubMed  PubMed Central  Google Scholar 

  10. 10

    Tian, X., Pu, W.T. & Zhou, B. Cellular origin and developmental program of coronary angiogenesis. Circ. Res. 116, 515–530 (2015).

    CAS  PubMed  Google Scholar 

  11. 11

    Sauer, B. & McDermott, J. 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).

    CAS  PubMed  PubMed Central  Google Scholar 

  12. 12

    Anastassiadis, K. 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).

    CAS  PubMed  Google Scholar 

  13. 13

    Guo, C., Yang, W. & Lobe, C.G. A Cre recombinase transgene with mosaic, widespread tamoxifen-inducible action. Genesis 32, 8–18 (2002).

    CAS  PubMed  Google Scholar 

  14. 14

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

    CAS  PubMed  Google Scholar 

  15. 15

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

    CAS  PubMed  Google Scholar 

  16. 16

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

    CAS  PubMed  PubMed Central  Google Scholar 

  17. 17

    Hatzistergos, K.E. 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).

    CAS  PubMed  PubMed Central  Google Scholar 

  18. 18

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

    CAS  PubMed  Google Scholar 

  19. 19

    Molkentin, J.D. 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).

    CAS  PubMed  PubMed Central  Google Scholar 

  20. 20

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

    CAS  PubMed  Google Scholar 

  21. 21

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

    CAS  PubMed  PubMed Central  Google Scholar 

  22. 22

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

    CAS  PubMed  PubMed Central  Google Scholar 

  23. 23

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

    CAS  PubMed  PubMed Central  Google Scholar 

  24. 24

    Tarlow, B.D., Finegold, M.J. & Grompe, M. Clonal tracing of Sox9+ liver progenitors in mouse oval cell injury. Hepatology 60, 278–289 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  25. 25

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

    CAS  PubMed  Google Scholar 

  26. 26

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

    CAS  PubMed  PubMed Central  Google Scholar 

  27. 27

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

    CAS  PubMed  PubMed Central  Google Scholar 

  28. 28

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

    CAS  PubMed  Google Scholar 

  29. 29

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

    CAS  PubMed  PubMed Central  Google Scholar 

  30. 30

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

    CAS  PubMed  PubMed Central  Google Scholar 

  31. 31

    Awatramani, R., Soriano, P., Rodriguez, C., Mai, J.J. & Dymecki, S.M. Cryptic boundaries in roof plate and choroid plexus identified by intersectional gene activation. Nat. Genet. 35, 70–75 (2003).

    CAS  PubMed  Google Scholar 

  32. 32

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

    CAS  PubMed  PubMed Central  Google Scholar 

  33. 33

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

    CAS  PubMed  PubMed Central  Google Scholar 

  34. 34

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

    CAS  PubMed  PubMed Central  Google Scholar 

  35. 35

    Plummer, N.W., de Marchena, J. & Jensen, P. A knock-in allele of En1 expressing Dre recombinase. Genesis 54, 447–454 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  36. 36

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

    CAS  PubMed  Google Scholar 

  37. 37

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

    CAS  PubMed  Google Scholar 

  38. 38

    Vassilopoulos, G., Wang, P.R. & Russell, D.W. Transplanted bone marrow regenerates liver by cell fusion. Nature 422, 901–904 (2003).

    CAS  PubMed  Google Scholar 

  39. 39

    Ruzankina, Y. 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).

    CAS  PubMed  PubMed Central  Google Scholar 

  40. 40

    Sohal, D.S. 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).

    CAS  PubMed  Google Scholar 

  41. 41

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

    CAS  PubMed  Google Scholar 

  42. 42

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

    CAS  PubMed  Google Scholar 

  43. 43

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

    CAS  PubMed  Google Scholar 

  44. 44

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

    CAS  PubMed  Google Scholar 

  45. 45

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

    PubMed  Google Scholar 

  46. 46

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

    CAS  PubMed  PubMed Central  Google Scholar 

  47. 47

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

    CAS  PubMed  PubMed Central  Google Scholar 

  48. 48

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

    CAS  PubMed  PubMed Central  Google Scholar 

  49. 49

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

    CAS  PubMed  PubMed Central  Google Scholar 

  50. 50

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

    Google Scholar 

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




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.

Corresponding author

Correspondence to Bin Zhou.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

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

Life Sciences Reporting Summary (PDF 171 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

He, L., Li, Y., Li, Y. et al. Enhancing the precision of genetic lineage tracing using dual recombinases. Nat Med 23, 1488–1498 (2017).

Download citation

Further reading


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing