Overview of Genetic Tools and Techniques to Study Notch Signaling in Mice

  • Thomas GridleyEmail author
  • Andrew K. GrovesEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1187)


Aberrations of Notch signaling in humans cause both congenital and acquired defects and cancers. Genetically engineered mice provide the most efficient and cost-effective models to study Notch signaling in a mammalian system. Here, we review the various types of genetic models, tools, and strategies to study Notch signaling in mice, and provide examples of their use. We also provide advice on breeding strategies for conditional mutant mice, and a protocol for tamoxifen administration to mouse strains expressing inducible Cre recombinase-estrogen receptor fusion proteins.

Key words

Conditional mutations Notch reporter lines Notch receptor-Cre fusions Fate mapping Lineage analysis Domain-swap mice Tamoxifen administration 


  1. 1.
    Guruharsha KG, Kankel MW, Artavanis-Tsakonas S (2012) The Notch signalling system: recent insights into the complexity of a conserved pathway. Nat Rev Genet 13:654–666PubMedCrossRefGoogle Scholar
  2. 2.
    Ilagan MX, Kopan R (2007) SnapShot: Notch signaling pathway. Cell 128:1246PubMedCrossRefGoogle Scholar
  3. 3.
    Kopan R (2012) Notch signaling. Cold Spring Harb Perspect Biol 4:a011213PubMedCrossRefGoogle Scholar
  4. 4.
    Andersson ER, Sandberg R, Lendahl U (2011) Notch signaling: simplicity in design, versatility in function. Development 138:3593–3612PubMedCrossRefGoogle Scholar
  5. 5.
    Bradley A, Anastassiadis K, Ayadi A et al (2012) The mammalian gene function resource: the International Knockout Mouse Consortium. Mamm Genome 23:580–586PubMedCentralPubMedCrossRefGoogle Scholar
  6. 6.
    Bucan M, Eppig JT, Brown S (2012) Mouse genomics programs and resources. Mamm Genome 23:479–489PubMedCrossRefGoogle Scholar
  7. 7.
    White JK, Gerdin AK, Karp NA et al (2013) Genome-wide generation and systematic phenotyping of knockout mice reveals new roles for many genes. Cell 154:452–464PubMedCentralPubMedCrossRefGoogle Scholar
  8. 8.
    Osterwalder M, Galli A, Rosen B et al (2010) Dual RMCE for efficient re-engineering of mouse mutant alleles. Nat Methods 7:893–895PubMedCentralPubMedCrossRefGoogle Scholar
  9. 9.
    Schnütgen F, Ehrmann F, Poser I et al (2011) Resources for proteomics in mouse embryonic stem cells. Nat Methods 8:103–104PubMedCrossRefGoogle Scholar
  10. 10.
    Gridley T (2010) Notch signaling in the vasculature. Curr Top Dev Biol 92:277–309PubMedCentralPubMedCrossRefGoogle Scholar
  11. 11.
    Sternberg N, Hamilton D (1981) Bacteriophage P1 site-specific recombination. I. Recombination between loxP sites. J Mol Biol 150:467–486PubMedCrossRefGoogle Scholar
  12. 12.
    Sauer B, Henderson N (1988) Site-specific DNA recombination in mammalian cells by the Cre recombinase of bacteriophage P1. Proc Natl Acad Sci U S A 85:5166–5170PubMedCentralPubMedCrossRefGoogle Scholar
  13. 13.
    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 265:103–106PubMedCrossRefGoogle Scholar
  14. 14.
    Kwan KM (2002) Conditional alleles in mice: practical considerations for tissue-specific knockouts. Genesis 32:49–62PubMedCrossRefGoogle Scholar
  15. 15.
    Orban PC, Chui D, Marth JD (1992) Tissue- and site-specific DNA recombination in transgenic mice. Proc Natl Acad Sci U S A 89:6861–6865PubMedCentralPubMedCrossRefGoogle Scholar
  16. 16.
    Danielian PS, Muccino D, Rowitch DH et al (1998) Modification of gene activity in mouse embryos in utero by a tamoxifen-inducible form of Cre recombinase. Curr Biol 8:1323–1326PubMedCrossRefGoogle 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 93:10887–10890PubMedCentralPubMedCrossRefGoogle Scholar
  18. 18.
    Feil R, Wagner J, Metzger D et al (1997) Regulation of Cre recombinase activity by mutated estrogen receptor ligand-binding domains. Biochem Biophys Res Commun 237:752–757PubMedCrossRefGoogle Scholar
  19. 19.
    Feil S, Valtcheva N, Feil R (2009) Inducible Cre mice. Methods Mol Biol 530:343–363PubMedCrossRefGoogle Scholar
  20. 20.
    Hayashi S, McMahon AP (2002) Efficient recombination in diverse tissues by a tamoxifen-inducible form of Cre: a tool for temporally regulated gene activation/inactivation in the mouse. Dev Biol 244:305–318PubMedCrossRefGoogle Scholar
  21. 21.
    Kellendonk C, Tronche F, Monaghan AP et al (1996) Regulation of Cre recombinase activity by the synthetic steroid RU 486. Nucleic Acids Res 24:1404–1411PubMedCentralPubMedCrossRefGoogle Scholar
  22. 22.
    Rose MF, Ahmad KA, Thaller C et al (2009) Excitatory neurons of the proprioceptive, interoceptive, and arousal hindbrain networks share a developmental requirement for Math1. Proc Natl Acad Sci U S A 106:22462–22467PubMedCentralPubMedCrossRefGoogle Scholar
  23. 23.
    Dymecki SM, Ray RS, Kim JC (2010) Mapping cell fate and function using recombinase-based intersectional strategies. Methods Enzymol 477:183–213PubMedCrossRefGoogle Scholar
  24. 24.
    Meyers EN, Lewandoski M, Martin GR (1998) An Fgf8 mutant allelic series generated by Cre- and Flp-mediated recombination. Nat Genet 18:136–141PubMedCrossRefGoogle Scholar
  25. 25.
    D’Souza B, Meloty-Kapella L, Weinmaster G (2010) Canonical and non-canonical Notch ligands. Curr Top Dev Biol 92:73–129PubMedCrossRefGoogle Scholar
  26. 26.
    Skarnes WC, Rosen B, West AP et al (2011) A conditional knockout resource for the genome-wide study of mouse gene function. Nature 474:337–342PubMedCentralPubMedCrossRefGoogle Scholar
  27. 27.
    Tu L, Fang TC, Artis D et al (2005) Notch signaling is an important regulator of type 2 immunity. J Exp Med 202:1037–1042PubMedCentralPubMedCrossRefGoogle Scholar
  28. 28.
    Weng AP, Nam Y, Wolfe MS et al (2003) Growth suppression of pre-T acute lymphoblastic leukemia cells by inhibition of notch signaling. Mol Cell Biol 23:655–664PubMedCentralPubMedCrossRefGoogle Scholar
  29. 29.
    Murray SA, Eppig JT, Smedley D et al (2012) Beyond knockouts: cre resources for conditional mutagenesis. Mamm Genome 23:587–599PubMedCentralPubMedCrossRefGoogle Scholar
  30. 30.
    Heffner CS, Herbert Pratt C, Babiuk RP et al (2012) Supporting conditional mouse mutagenesis with a comprehensive cre characterization resource. Nat Commun 3:1218PubMedCentralPubMedCrossRefGoogle Scholar
  31. 31.
    Nagy A, Mar L, Watts G (2009) Creation and use of a cre recombinase transgenic database. Methods Mol Biol 530:365–378PubMedCrossRefGoogle Scholar
  32. 32.
    Chandras C, Zouberakis M, Salimova E et al (2012) CreZOO—the European virtual repository of Cre and other targeted conditional driver strains. Database 2012:bas029PubMedCentralPubMedCrossRefGoogle Scholar
  33. 33.
    Bao J, Ma HY, Schuster A et al (2013) Incomplete cre-mediated excision leads to phenotypic differences between Stra8-iCre; Mov10l1(lox/lox) and Stra8-iCre; Mov10l1(lox/Delta) mice. Genesis 51:481–490PubMedCentralPubMedCrossRefGoogle Scholar
  34. 34.
    Cai T, Seymour ML, Zhang H et al (2013) Conditional deletion of Atoh1 reveals distinct critical periods for survival and function of hair cells in the organ of Corti. J Neurosci 33:10110–10122PubMedCentralPubMedCrossRefGoogle Scholar
  35. 35.
    Reinert RB, Kantz J, Misfeldt AA et al (2012) Tamoxifen-induced Cre-loxP recombination is prolonged in pancreatic islets of adult mice. PLoS One 7:e33529PubMedCentralPubMedCrossRefGoogle Scholar
  36. 36.
    Park EJ, Sun X, Nichol P et al (2008) System for tamoxifen-inducible expression of cre-recombinase from the Foxa2 locus in mice. Dev Dyn 237:447–453PubMedCrossRefGoogle Scholar
  37. 37.
    Leone DP, Genoud S, Atanasoski S et al (2003) Tamoxifen-inducible glia-specific Cre mice for somatic mutagenesis in oligodendrocytes and Schwann cells. Mol Cell Neurosci 22:430–440PubMedCrossRefGoogle Scholar
  38. 38.
    Murtaugh LC, Stanger BZ, Kwan KM et al (2003) Notch signaling controls multiple steps of pancreatic differentiation. Proc Natl Acad Sci U S A 100:14920–14925PubMedCentralPubMedCrossRefGoogle Scholar
  39. 39.
    Varadkar PA, Kraman M, McCright B (2009) Generation of mice that conditionally express the activation domain of Notch2. Genesis 47:573–578PubMedCrossRefGoogle Scholar
  40. 40.
    Oh P, Lobry C, Gao J et al (2013) In vivo mapping of Notch pathway activity in normal and stress hematopoiesis. Cell Stem Cell 13:190–204PubMedCrossRefGoogle Scholar
  41. 41.
    Ong CT, Cheng HT, Chang LW et al (2006) Target selectivity of vertebrate notch proteins. Collaboration between discrete domains and CSL-binding site architecture determines activation probability. J Biol Chem 281:5106–5119PubMedCrossRefGoogle Scholar
  42. 42.
    Mizutani K, Yoon K, Dang L et al (2007) Differential Notch signalling distinguishes neural stem cells from intermediate progenitors. Nature 449:351–355PubMedCrossRefGoogle Scholar
  43. 43.
    Souilhol C, Cormier S, Monet M et al (2006) Nas transgenic mouse line allows visualization of Notch pathway activity in vivo. Genesis 44:277–286PubMedCentralPubMedCrossRefGoogle Scholar
  44. 44.
    DasGupta R, Fuchs E (1999) Multiple roles for activated LEF/TCF transcription complexes during hair follicle development and differentiation. Development 126:4557–4568PubMedGoogle Scholar
  45. 45.
    Maretto S, Cordenonsi M, Dupont S et al (2003) Mapping Wnt/beta-catenin signaling during mouse development and in colorectal tumors. Proc Natl Acad Sci U S A 100:3299–3304PubMedCentralPubMedCrossRefGoogle Scholar
  46. 46.
    Ohtsuka T, Imayoshi I, Shimojo H et al (2006) Visualization of embryonic neural stem cells using Hes promoters in transgenic mice. Mol Cell Neurosci 31:109–122PubMedCrossRefGoogle Scholar
  47. 47.
    Basak O, Taylor V (2007) Identification of self-replicating multipotent progenitors in the embryonic nervous system by high Notch activity and Hes5 expression. Eur J Neurosci 25:1006–1022PubMedCrossRefGoogle Scholar
  48. 48.
    Imayoshi I, Sakamoto M, Yamaguchi M et al (2010) Essential roles of Notch signaling in maintenance of neural stem cells in developing and adult brains. J Neurosci 30:3489–3498PubMedCrossRefGoogle Scholar
  49. 49.
    Fre S, Hannezo E, Sale S et al (2011) Notch lineages and activity in intestinal stem cells determined by a new set of knock-in mice. PLoS One 6:e25785PubMedCentralPubMedCrossRefGoogle Scholar
  50. 50.
    Sale S, Lafkas D, Artavanis-Tsakonas S (2013) Notch2 genetic fate mapping reveals two previously unrecognized mammary epithelial lineages. Nat Cell Biol 15:451–460PubMedCrossRefGoogle Scholar
  51. 51.
    Nowotschin S, Xenopoulos P, Schrode N et al (2013) A bright single-cell resolution live imaging reporter of Notch signaling in the mouse. BMC Dev Biol 13:15PubMedCentralPubMedCrossRefGoogle Scholar
  52. 52.
    Vooijs M, Ong CT, Hadland B et al (2007) Mapping the consequence of Notch1 proteolysis in vivo with NIP-CRE. Development 134:535–544PubMedCentralPubMedCrossRefGoogle Scholar
  53. 53.
    Soriano P (1999) Generalized lacZ expression with the ROSA26 Cre reporter strain. Nat Genet 21:70–71PubMedCrossRefGoogle Scholar
  54. 54.
    Smith E, Claudinot S, Lehal R et al (2012) Generation and characterization of a Notch1 signaling-specific reporter mouse line. Genesis 50:700–710PubMedCrossRefGoogle Scholar
  55. 55.
    Govindarajan V, Harrison WR, Xiao N et al (2005) Intracorneal positioning of the lens in Pax6-GAL4/VP16 transgenic mice. Mol Vis 11:876–886PubMedGoogle Scholar
  56. 56.
    Liu Z, Turkoz A, Jackson EN et al (2011) Notch1 loss of heterozygosity causes vascular tumors and lethal hemorrhage in mice. J Clin Invest 121:800–808PubMedCentralPubMedCrossRefGoogle Scholar
  57. 57.
    Liu Z, Obenauf AC, Speicher MR et al (2009) Rapid identification of homologous recombinants and determination of gene copy number with reference/query pyrosequencing (RQPS). Genome Res 19:2081–2089PubMedCentralPubMedCrossRefGoogle Scholar
  58. 58.
    Liu Z, Chen S, Boyle S et al (2013) The extracellular domain of Notch2 increases its cell-surface abundance and ligand responsiveness during kidney development. Dev Cell 25:585–598PubMedCentralPubMedCrossRefGoogle Scholar
  59. 59.
    Morimoto M, Liu Z, Cheng HT et al (2010) Canonical Notch signaling in the developing lung is required for determination of arterial smooth muscle cells and selection of Clara versus ciliated cell fate. J Cell Sci 123:213–224PubMedCentralPubMedCrossRefGoogle Scholar
  60. 60.
    Liu Z, Liu Z, Walters BJ et al (2013) In vivo visualization of Notch1 proteolysis reveals the heterogeneity of Notch1 signaling activity in the mouse cochlea. PLoS One 8:e64903PubMedCentralPubMedCrossRefGoogle Scholar
  61. 61.
    Luche H, Weber O, Nageswara Rao T et al (2007) Faithful activation of an extra-bright red fluorescent protein in “knock-in” Cre-reporter mice ideally suited for lineage tracing studies. Eur J Immunol 37:43–53PubMedCrossRefGoogle Scholar
  62. 62.
    Saito M, Iwawaki T, Taya C et al (2001) Diphtheria toxin receptor-mediated conditional and targeted cell ablation in transgenic mice. Nat Biotechnol 19:746–750PubMedCrossRefGoogle Scholar
  63. 63.
    Kraman M, McCright B (2005) Functional conservation of Notch1 and Notch2 intracellular domains. FASEB J 19:1311–1313PubMedGoogle Scholar
  64. 64.
    Donahue LR, Hrabe de Angelis M, Hagn M et al (2012) Centralized mouse repositories. Mamm Genome 23:559–571PubMedCentralPubMedCrossRefGoogle Scholar
  65. 65.
    Gaj T, Gersbach CA, Barbas CF 3rd (2013) ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering. Trends Biotechnol 31:397–405PubMedCentralPubMedCrossRefGoogle Scholar
  66. 66.
    Wang H, Yang H, Shivalila CS et al (2013) One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering. Cell 153:910–918PubMedCentralPubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  1. 1.Center for Molecular MedicineMaine Medical Center Research InstituteScarboroughUSA
  2. 2.Department of Neuroscience, Program in Developmental Biology, Department of Molecular and Human GeneticsBaylor College of MedicineHoustonUSA

Personalised recommendations