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Knock-In Approaches

  • Anton J. M. RoebroekEmail author
  • Philip L. S. M. Gordts
  • Sara Reekmans
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 693)

Abstract

Molecular genetic strategies to study gene function in mice or to generate a mouse model for a human disease are continuously under development. The application and importance of knock-in approaches are increasing. This chapter elaborates on novel developments for the generation of knock-in mice. Special emphasis is given to recombinase-mediated cassette exchange, a new emerging knock-in strategy that enables easy generation of a series of different knock-in mutations within one gene.

Key words

Knock-in Mutation Site-specific recombinase LoxP Cre FRT Flp att ΦC31 RMCE FLEX switch 

References

  1. 1.
    Allen, J. P., Hathway, G. J., Clarke, N. J., Jowett, M. I., Topps, S., Kendrick, K. M., Humphrey, P. P., Wilkinson, L. S., and Emson, P. C. (2003) Somatostatin receptor 2 knockout/lacZ knockin mice show impaired motor coordination and reveal sites of somatostatin action within the striatum. Eur J Neurosci 17, 1881–1895PubMedCrossRefGoogle Scholar
  2. 2.
    Makita, R., Uchijima, Y., Nishiyama, K., Amano, T., Chen, Q., Takeuchi, T., Mitani, A., Nagase, T., Yatomi, Y., Aburatani, H., Nakagawa, O., Small, E. V., Cobo-Stark, P., Igarashi, P., Murakami, M., Tominaga, J., Sato, T., Asano, T., Kurihara, Y., and Kurihara, H. (2008) Multiple renal cysts, urinary concentration defects, and pulmonary emphysematous changes in mice lacking TAZ. Am J Physiol Renal Physiol 294, F542–F553PubMedCrossRefGoogle Scholar
  3. 3.
    Reichardt, H. M., Kaestner, K. H., Tuckermann, J., Kretz, O., Wessely, O., Bock, R., Gass, P., Schmid, W., Herrlich, P., Angel, P., and Schutz, G. (1998) DNA binding of the glucocorticoid receptor is not essential for survival. Cell 93, 531–541PubMedCrossRefGoogle Scholar
  4. 4.
    Friend, L. D., Shah, D. D., Deppong, C., Lin, J., Bricker, T. L., Juehne, T. I., Rose, C. M., and Green, J. M. (2006) A dose-dependent requirement for the proline motif of CD28 in cellular and humoral immunity revealed by a targeted knockin mutant. J Exp Med 203, 2121–2133PubMedCrossRefGoogle Scholar
  5. 5.
    Satyanarayana, A., Berthet, C., Lopez-Molina, J., Coppola, V., Tessarollo, L., and Kaldis, P. (2008) Genetic substitution of Cdk1 by Cdk2 leads to embryonic lethality and loss of meiotic function of Cdk2. Development 135, 3389–3400PubMedCrossRefGoogle Scholar
  6. 6.
    Mihara, K., Smit, M. J., Krajnc-Franken, M., Gossen, J., Rooseboom, M., and Dokter, W. (2005) Human CXCR2 (hCXCR2) takes over functionalities of its murine homolog in hCXCR2 knockin mice. Eur J Immunol 35, 2573–2582PubMedCrossRefGoogle Scholar
  7. 7.
    Menalled, L. B., Sison, J. D., Dragatsis, I., Zeitlin, S., and Chesselet, M. F. (2003) Time course of early motor and neuropathological anomalies in a knock-in mouse model of Huntington’s disease with 140 CAG repeats. J Comp Neurol 465, 11–26PubMedCrossRefGoogle Scholar
  8. 8.
    Simmons, D. A., Rex, C. S., Palmer, L., Pandyarajan, V., Fedulov, V., Gall, C. M., and Lynch, G. (2009) Up-regulating BDNF with an ampakine rescues synaptic plasticity and memory in Huntington’s disease knockin mice. Proc Natl Acad Sci U S A 106, 4906–4911PubMedCrossRefGoogle Scholar
  9. 9.
    Mercher, T., Raffel, G. D., Moore, S. A., Cornejo, M. G., Baudry-Bluteau, D., Cagnard, N., Jesneck, J. L., Pikman, Y., Cullen, D., Williams, I. R., Akashi, K., Shigematsu, H., Bourquin, J. P., Giovannini, M., Vainchenker, W., Levine, R. L., Lee, B. H., Bernard, O. A., and Gilliland, D. G. (2009) The OTT-MAL fusion oncogene activates RBPJ-mediated transcription and induces acute megakaryoblastic leukemia in a knockin mouse model. J Clin Invest 119, 852–864PubMedGoogle Scholar
  10. 10.
    Beffert, U., Weeber, E. J., Durudas, A., Qiu, S., Masiulis, I., Sweatt, J. D., Li, W. P., Adelmann, G., Frotscher, M., Hammer, R. E., and Herz, J. (2005) Modulation of synaptic plasticity and memory by Reelin involves differential splicing of the lipoprotein receptor Apoer2. Neuron 47, 567–579PubMedCrossRefGoogle Scholar
  11. 11.
    Zhou, D., Ren, J. X., Ryan, T. M., Higgins, N. P., and Townes, T. M. (2004) Rapid tagging of endogenous mouse genes by recombineering and ES cell complementation of tetraploid blastocysts. Nucleic Acids Res 32, e128PubMedCrossRefGoogle Scholar
  12. 12.
    Backman, C. M., Malik, N., Zhang, Y., Shan, L., Grinberg, A., Hoffer, B. J., Westphal, H., and Tomac, A. C. (2006) Characterization of a mouse strain expressing Cre recombinase from the 3′ untranslated region of the dopamine transporter locus. Genesis 44, 383–390PubMedCrossRefGoogle Scholar
  13. 13.
    Branda, C. S. and Dymecki, S. M. (2004) Talking about a revolution: the impact of site-specific recombinases on genetic analyses in mice. Dev Cell 6, 7–28PubMedCrossRefGoogle Scholar
  14. 14.
    Wirth, D., Gama-Norton, L., Riemer, P., Sandhu, U., Schucht, R., and Hauser, H. (2007) Road to precision: recombinase-based targeting technologies for genome engineering. Curr Opin Biotechnol 18, 411–419PubMedCrossRefGoogle Scholar
  15. 15.
    Oumard, A., Qiao, J., Jostock, T., Li, J., and Bode, J. (2006) Recommended method for chromosome exploitation: RMCE-based cassette-exchange systems in animal cell biotechnology. Cytotechnology 50, 93–108PubMedCrossRefGoogle Scholar
  16. 16.
    Seibler, J. and Bode, J. (1997) Double-reciprocal crossover mediated by FLP-recombinase: a concept and an assay. Biochemistry 36, 1740–1747PubMedCrossRefGoogle Scholar
  17. 17.
    Seibler, J., Schubeler, D., Fiering, S., Groudine, M., and Bode, J. (1998) DNA cassette exchange in ES cells mediated by Flp recombinase: an efficient strategy for repeated modification of tagged loci by marker-free constructs. Biochemistry 37, 6229–6234PubMedCrossRefGoogle Scholar
  18. 18.
    Schlake, T. and Bode, J. (1994) Use of mutated FLP recognition target (FRT) sites for the exchange of expression cassettes at defined chromosomal loci. Biochemistry 33, 12746–12751PubMedCrossRefGoogle Scholar
  19. 19.
    Cesari, F., Rennekampff, V., Vintersten, K., Vuong, L. G., Seibler, J., Bode, J., Wiebel, F. F., and Nordheim, A. (2004) Elk-1 knock-out mice engineered by Flp recombinase-mediated cassette exchange. Genesis 38, 87–92PubMedCrossRefGoogle Scholar
  20. 20.
    Roebroek, A. J., Reekmans, S., Lauwers, A., Feyaerts, N., Smeijers, L., and Hartmann, D. (2006) Mutant Lrp1 knock-in mice generated by recombinase-mediated cassette exchange reveal differential importance of the NPXY motifs in the intracellular domain of LRP1 for normal fetal development. Mol Cell Biol 26, 605–616PubMedCrossRefGoogle Scholar
  21. 21.
    Reekmans, S. M., Pflanzner, T., Gordts, P. L., Isbert, S., Zimmermann, P., Annaert, W., Weggen, S., Roebroek, A. J., and Pietrzik, C. U. (2009) Inactivation of the proximal NPXY motif impairs early steps in LRP1 biosynthesis. Cell Mol Life Sci 67, 135–145PubMedCrossRefGoogle Scholar
  22. 22.
    Gordts, P. L., Reekmans, S., Lauwers, A., Van Dongen, A., Verbeek, L., and Roebroek, A. J. (2009) Inactivation of the LRP1 intracellular NPxYxxL motif in LDLR-deficient mice enhances postprandial dyslipidemia and atherosclerosis. Arterioscler Thromb Vasc Biol 29, 1258–1264PubMedCrossRefGoogle Scholar
  23. 23.
    Sato, T., Kawamura, Y., Asai, R., Amano, T., Uchijima, Y., Dettlaff-Swiercz, D. A., Offermanns, S., Kurihara, Y., and Kurihara, H. (2008) Recombinase-mediated cassette exchange reveals the selective use of Gq/G11-dependent and -independent endothelin 1/endothelin type A receptor signaling in pharyngeal arch development. Development 135, 755–765PubMedCrossRefGoogle Scholar
  24. 24.
    Toledo, F., Liu, C. W., Lee, C. J., and Wahl, G. M. (2006) RMCE-ASAP: a gene targeting method for ES and somatic cells to accelerate phenotype analyses. Nucleic Acids Res 34, e92PubMedCrossRefGoogle Scholar
  25. 25.
    Shmerling, D., Danzer, C. P., Mao, X., Boisclair, J., Haffner, M., Lemaistre, M., Schuler, V., Kaeslin, E., Korn, R., Burki, K., Ledermann, B., Kinzel, B., and Muller, M. (2005) Strong and ubiquitous expression of transgenes targeted into the beta-actin locus by Cre/lox cassette replacement. Genesis 42, 229–235PubMedCrossRefGoogle Scholar
  26. 26.
    Hitz, C., Wurst, W., and Kuhn, R. (2007) Conditional brain-specific knockdown of MAPK using Cre/loxP regulated RNA interference. Nucleic Acids Res 35, e90PubMedCrossRefGoogle Scholar
  27. 27.
    Prosser, H. M., Rzadzinska, A. K., Steel, K. P., and Bradley, A. (2008) Mosaic complementation demonstrates a regulatory role for myosin VIIa in actin dynamics of stereocilia. Mol Cell Biol 28, 1702–1712PubMedCrossRefGoogle Scholar
  28. 28.
    Schnutgen, F., Doerflinger, N., Calleja, C., Wendling, O., Chambon, P., and Ghyselinck, N. B. (2003) A directional strategy for monitoring Cre-mediated recombination at the cellular level in the mouse. Nat Biotechnol 21, 562–565PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Anton J. M. Roebroek
    • 1
    Email author
  • Philip L. S. M. Gordts
    • 1
  • Sara Reekmans
    • 1
  1. 1.Laboratory for Experimental Mouse GeneticsCenter for Human Genetics, K.U. LeuvenLeuvenBelgium

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