The Cre/Lox System to Assess the Development of the Mouse Brain

  • Claudius F. Kratochwil
  • Filippo M. RijliEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 2047)


Cre-mediated recombination has become a powerful tool to confine gene deletions (conditional knockouts) or overexpression of genes (conditional knockin/overexpression). By spatiotemporal restriction of genetic manipulations, major problems of classical knockouts such as embryonic lethality or pleiotropy can be circumvented. Furthermore, Cre-mediated recombination has broad applications in the analysis of the cellular behavior of subpopulations and cell types as well as for genetic fate mapping. This chapter gives an overview about applications for the Cre/LoxP system and their execution.


Cre recombinase CRISPR-Cas9 Transgenesis Conditional knockout Conditional knockin CreERT2 Flpe recombinase MADM Split-Cre Brainbow 



Work in FMR laboratory in Friedrich Miescher Institute (Basel, Switzerland) is supported by the Swiss National Science Foundation (CRSII5_173868 and 31003A_175776) and the Novartis Research Foundation. C.F.K. is funded by the Baden-Württemberg Foundation and the Deutsche Forschungsgemeinschaft (DFG, KR 4670/2-1).


  1. 1.
    Thomas KR, Capecchi MR (1987) Site-directed mutagenesis by gene targeting in mouse embryo-derived stem cells. Cell 51:503–512CrossRefGoogle Scholar
  2. 2.
    Gu H, Marth JD, Orban PC et al (1994) Deletion of a DNA polymerase beta gene segment in T cells using cell type-specific gene targeting. Science 265:103–106CrossRefGoogle Scholar
  3. 3.
    Lakso M, Sauer B, Mosinger B et al (1992) Targeted oncogene activation by site-specific recombination in transgenic mice. Proc Natl Acad Sci U S A 89:6232–6236CrossRefGoogle Scholar
  4. 4.
    Bechara A, Laumonnerie C, Vilain N et al (2015) Hoxa2 selects Barrelette neuron identity and connectivity in the mouse somatosensory brainstem. Cell Rep 13:783–797. Scholar
  5. 5.
    Renier N, Dominici C, Erzurumlu RS et al (2017) A mutant with bilateral whisker to barrel inputs unveils somatosensory mapping rules in the cerebral cortex. Elife.
  6. 6.
    Kim JC, Dymecki SM (2009) Genetic fate-mapping approaches: new means to explore the embryonic origins of the cochlear nucleus. Methods Mol Biol 493:65–85. Scholar
  7. 7.
    Schwalie PC, Dong H, Zachara M et al (2018) A stromal cell population that inhibits adipogenesis in mammalian fat depots. Nature 559:103–108. Scholar
  8. 8.
    Siegal ML, Hartl DL (1996) Transgene coplacement and high efficiency site-specific recombination with the Cre/loxP system in Drosophila. Genetics 144:715–726PubMedPubMedCentralGoogle Scholar
  9. 9.
    Werdien D, Peiler G, Ryffel GU (2001) FLP and Cre recombinase function in Xenopus embryos. Nucleic Acids Res 29:E53–E53CrossRefGoogle Scholar
  10. 10.
    Pan YA, Freundlich T, Weissman TA et al (2013) Zebrabow: multispectral cell labeling for cell tracing and lineage analysis in zebrafish. Development 140:2835–2846. Scholar
  11. 11.
    Chen H, Luo J, Zheng P et al (2017) Application of Cre-lox gene switch to limit the Cry expression in rice green tissues. Sci Rep 7:14505. Scholar
  12. 12.
    Guo F, Gopaul DN, van Duyne GD (1997) Structure of Cre recombinase complexed with DNA in a site-specific recombination synapse. Nature 389:40–46. Scholar
  13. 13.
    Nakano M, Odaka K, Ishimura M et al (2001) Efficient gene activation in cultured mammalian cells mediated by FLP recombinase-expressing recombinant adenovirus. Nucleic Acids Res 29:E40CrossRefGoogle Scholar
  14. 14.
    Buchholz F, Angrand PO, Stewart AF (1998) Improved properties of FLP recombinase evolved by cycling mutagenesis. Nat Biotechnol 16:657–662. Scholar
  15. 15.
    Rodríguez CI, Buchholz F, Galloway J et al (2000) High-efficiency deleter mice show that FLPe is an alternative to Cre-loxP. Nat Genet 25:139–140. Scholar
  16. 16.
    Feil R, Wagner J, Metzger D, Chambon P (1997) Regulation of Cre recombinase activity by mutated estrogen receptor ligand-binding domains. Biochem Biophys Res Commun 237:752–757. Scholar
  17. 17.
    Feil R, Brocard J, Mascrez B et al (1996) Ligand-activated site-specific recombination in mice. Proc Natl Acad Sci U S A 93:10887–10890CrossRefGoogle Scholar
  18. 18.
    Kawano F, Okazaki R, Yazawa M, Sato M (2016) A photoactivatable Cre-loxP recombination system for optogenetic genome engineering. Nat Chem Biol 12:1059–1064. Scholar
  19. 19.
    Hirrlinger J, Scheller A, Hirrlinger PG et al (2009) Split-cre complementation indicates coincident activity of different genes in vivo. PLoS One 4:e4286. Scholar
  20. 20.
    Farago AF, Awatramani RB, Dymecki SM (2006) Assembly of the brainstem cochlear nuclear complex is revealed by intersectional and subtractive genetic fate maps. Neuron 50:205–218. Scholar
  21. 21.
    Zong H, Espinosa JS, Su HH et al (2005) Mosaic analysis with double markers in mice. Cell 121:479–492. Scholar
  22. 22.
    Tasic B, Miyamichi K, Hippenmeyer S et al (2012) Extensions of MADM (mosaic analysis with double markers) in mice. PLoS One 7:e33332. Scholar
  23. 23.
    Lee T, Luo L (1999) Mosaic analysis with a repressible cell marker for studies of gene function in neuronal morphogenesis. Neuron 22:451–461CrossRefGoogle Scholar
  24. 24.
    Hippenmeyer S, Youn YH, Moon HM et al (2010) Genetic mosaic dissection of Lis1 and Ndel1 in neuronal migration. Neuron 68:695–709. Scholar
  25. 25.
    Livet J, Weissman TA, Kang H et al (2007) Transgenic strategies for combinatorial expression of fluorescent proteins in the nervous system. Nature 450:56–62. Scholar
  26. 26.
    Soriano P (1999) Generalized lacZ expression with the ROSA26 Cre reporter strain. Nat Genet 21:70–71. Scholar
  27. 27.
    Madisen L, Zwingman TA, Sunkin SM et al (2010) A robust and high-throughput Cre reporting and characterization system for the whole mouse brain. Nat Neurosci 13:133–140. Scholar
  28. 28.
    Luo L (2007) Fly MARCM and mouse MADM: genetic methods of labeling and manipulating single neurons. Brain Res Rev 55:220–227. Scholar
  29. 29.
    Potter CJ, Tasic B, Russler EV et al (2010) The Q system: a repressible binary system for transgene expression, lineage tracing, and mosaic analysis. Cell 141:536–548. Scholar
  30. 30.
    Zhang Y, Buchholz F, Muyrers JP, Stewart AF (1998) A new logic for DNA engineering using recombination in Escherichia coli. Nat Genet 20:123–128. Scholar
  31. 31.
    Sauer B (1998) Inducible gene targeting in mice using the Cre/lox system. Methods 14:381–392. Scholar
  32. 32.
    Schnütgen F, Doerflinger N, Calléja C et al (2003) A directional strategy for monitoring Cre-mediated recombination at the cellular level in the mouse. Nat Biotechnol 21:562–565. Scholar
  33. 33.
    Zheng B, Sage M, Sheppeard EA et al (2000) Engineering mouse chromosomes with Cre-loxP: range, efficiency, and somatic applications. Mol Cell Biol 20:648–655CrossRefGoogle Scholar
  34. 34.
    Visel A, Minovitsky S, Dubchak I, Pennacchio LA (2007) VISTA Enhancer Browser—a database of tissue-specific human enhancers. Nucleic Acids Res 35:D88–D92. Scholar
  35. 35.
    Dousse A, Junier T, Zdobnov EM (2016) CEGA—a catalog of conserved elements from genomic alignments. Nucleic Acids Res 44:D96–D100. Scholar
  36. 36.
    Kratochwil CF, Meyer A (2015) Closing the genotype-phenotype gap: emerging technologies for evolutionary genetics in ecological model vertebrate systems. BioEssays 37:213–226. Scholar
  37. 37.
    Visel A, Blow MJ, Li Z et al (2009) ChIP-seq accurately predicts tissue-specific activity of enhancers. Nature 457:854–858. Scholar
  38. 38.
    Yee SP, Rigby PW (1993) The regulation of myogenin gene expression during the embryonic development of the mouse. Genes Dev 7:1277–1289CrossRefGoogle Scholar
  39. 39.
    Di Meglio T, Kratochwil CF, Vilain N et al (2013) Ezh2 orchestrates topographic migration and connectivity of mouse precerebellar neurons. Science 339:204–207. Scholar
  40. 40.
    Daigle TL, Madisen L, Hage TA et al (2018) A suite of transgenic driver and reporter mouse lines with enhanced brain-cell-type targeting and functionality. Cell 174:465–480.e22. Scholar
  41. 41.
    Hirrlinger J, Requardt RP, Winkler U et al (2009) Split-CreERT2: temporal control of DNA recombination mediated by split-Cre protein fragment complementation. PLoS One 4:e8354. Scholar
  42. 42.
    Hope IA, Struhl K (1987) GCN4, a eukaryotic transcriptional activator protein, binds as a dimer to target DNA. EMBO J 6:2781–2784CrossRefGoogle Scholar
  43. 43.
    Wang P, Chen T, Sakurai K et al (2012) Intersectional Cre driver lines generated using split-intein mediated split-Cre reconstitution. Sci Rep 2:497. Scholar
  44. 44.
    Espinosa JS, Luo L (2008) Timing neurogenesis and differentiation: insights from quantitative clonal analyses of cerebellar granule cells. J Neurosci 28:2301–2312. Scholar
  45. 45.
    Hippenmeyer S (2013) Dissection of gene function at clonal level using mosaic analysis with double markers. Front Biol 8:557–568. Scholar
  46. 46.
    Hippenmeyer S, Johnson RL, Luo L (2013) Mosaic analysis with double markers reveals cell-type-specific paternal growth dominance. Cell Rep 3:960–967. Scholar
  47. 47.
    Ivanova A, Signore M, Caro N et al (2005) In vivo genetic ablation by Cre-mediated expression of diphtheria toxin fragment A. Genesis 43:129–135. Scholar
  48. 48.
    Mu X, Fu X, Sun H et al (2005) Ganglion cells are required for normal progenitor-cell proliferation but not cell-fate determination or patterning in the developing mouse retina. Curr Biol 15:525–530. Scholar
  49. 49.
    Kim CK, Adhikari A, Deisseroth K (2017) Integration of optogenetics with complementary methodologies in systems neuroscience. Nat Rev Neurosci 18:222–235. Scholar
  50. 50.
    Bitzenhofer SH, Ahlbeck J, Hanganu-Opatz IL (2017) Methodological approach for optogenetic manipulation of neonatal neuronal networks. Front Cell Neurosci 11:239. Scholar
  51. 51.
    Lin MZ, Schnitzer MJ (2016) Genetically encoded indicators of neuronal activity. Nat Neurosci 19:1142–1153. Scholar
  52. 52.
    Shimozono S, Iimura T, Kitaguchi T et al (2013) Visualization of an endogenous retinoic acid gradient across embryonic development. Nature 496:363–366. Scholar
  53. 53.
    Sengupta R, Mendenhall A, Sarkar N et al (2017) Viral Cre-LoxP tools aid genome engineering in mammalian cells. J Biol Eng 11:45. Scholar
  54. 54.
    Mali P, Yang L, Esvelt KM et al (2013) RNA-guided human genome engineering via Cas9. Science 339:823–826. Scholar
  55. 55.
    Yang H, Wang H, Shivalila CS et al (2013) One-step generation of mice carrying reporter and conditional alleles by CRISPR/Cas-mediated genome engineering. Cell 154:1370–1379. Scholar
  56. 56.
    Ma X, Chen C, Veevers J et al (2017) CRISPR/Cas9-mediated gene manipulation to create single-amino-acid-substituted and floxed mice with a cloning-free method. Sci Rep 7:42244. Scholar
  57. 57.
    Chen J, Du Y, He X et al (2017) A convenient Cas9-based conditional knockout strategy for simultaneously targeting multiple genes in mouse. Sci Rep 7:517. Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  1. 1.Zoology and Evolutionary Biology, Department of BiologyUniversity of KonstanzKonstanzGermany
  2. 2.Friedrich Miescher Institute for Biomedical ResearchBaselSwitzerland

Personalised recommendations