Gene manipulation, specifically in the hair cells of the inner ear during development and adulthood in mice, is crucial for understanding the physiology of hearing and the pathology of deafness in humans. Recent advances have demonstrated that gene expression can be manipulated in developing mouse hair cells in a spatially and temporally controlled manner. The Cre–loxP system has been widely used for such purposes. Many laboratories, including ours, have developed and characterized transgenic mouse lines that express or induce Cre activity specifically in inner ear hair cells. These Cre lines have been used with high efficiency to inactivate several genes such as Rb in hair cells. Here we discuss the use of these Cre lines in inner ear research with emphasis on practical issues for researchers who are not familiar with these particular techniques but are interested in using these Cre mice and floxed mice to inactivate genes of their interest specifically in inner ear hair cells. We provide detailed protocols for the use of these techniques and reagents. These considerations and protocols can be easily applied to other cell types in the inner ear and other parts of the auditory pathways. Because the NIH Knockout Mouse Project (KOMP) and the European Conditional Mouse Mutant Program (EUCOMM) have initiated plans to create conditional (floxed) knockout strains for every gene in the mouse genome and because numerous Cre-expressing mouse lines have already been created in various systems, including the nervous system, it is our hope that many hearing researchers will benefit from the detailed protocols and practical considerations described in this review.
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Gao, J., Wu, X. and Zuo, J. (2004) Targeting hearing genes in mice. Brain Res. Mol. Brain Res. 132, 192–207.
Holt, J. R., Gillespie, S. K., Provance, D. W., Shah, K., Shokat, K. M., Corey, D. P., Mercer, J. A., and Gillespie, P. G. (2002) A chemical-genetic strategy implicates myosin-1c in adaptation by hair cells. Cell 108, 371–381.
Stauffer, E. A., Scarborough, J. D., Hirono, M., Miller, E. D., Shah, K., Mercer, J. A., Holt, J. R., and Gillespie, P. G. (2005) Fast adaptation in vestibular hair cells requires myosin-1c activity. Neuron 47, 541–553.
Zambrowicz, B. P., Abuin, A., Ramirez-Solis, R., Richter, L. J., Piggott, J., BeltrandelRio, H., et al. (2003) Wnk1 kinase deficiency lowers blood pressure in mice: a gene-trap screen to identify potential targets for therapeutic intervention. Proc. Natl. Acad. Sci. USA 100, 14109–14114.
Coates, C. J., Kaminski, J. M., Summers, J. B., Segal, D. J., Miller, A. D., and Kolb, A. F. (2005) Site-directed genome modification: derivatives of DNA-modifying enzymes as targeting tools. Trends Biotechnol. 23, 407–419.
Tian, Y., James, S., Zuo, J., Fritzsch, B., and Beisel, K. W. (2006) Conditional and inducible gene recombineering in the mouse inner ear. Brain Res 1091, 243–254.
Nagy, A. (2000) Cre recombinase: the universal reagent for genome tailoring. Genesis 26, 99–109.
Zuo, J. (2002) Transgenic and gene targeting studies of hair cell function in mouse inner ear. J. Neurobiol. 53, 286–305.
Kuhn, R., Schwenk, F., Aguet, M., and Rajewsky, K. (1995) Inducible gene targeting in mice. Science 269, 1427–1429.
No, D., Yao, T. P., and Evans, R. M. (1996) Ecdysone-inducible gene expression in mammalian cells and transgenic mice. Proc. Natl. Acad. Sci. USA 93, 3346–3351.
Shimizu, E., Tang, Y. P., Rampon, C., and Tsien, J. Z. (2000) NMDA receptor-dependent synaptic reinforcement as a crucial process for memory consolidation [In Process Citation]. Science 290, 1170–1174.
Utomo, A. R., Nikitin, A. Y., and Lee, W. H. (1999) Temporal, spatial, and cell type-specific control of Cre-mediated DNA recombination in transgenic mice. Nat. Biotechnol. 17, 1091–1096.
Bond, C. T., Herson, P. S., Strassmaier, T., Hammond, R., Stackman, R., Maylie, J., and Adelman, J. P. (2004) Small conductance Ca2+-activated K+channel knock-out mice reveal the identity of calcium-dependent afterhyperpolarization currents. J. Neurosci. 24, 5301–5306.
Hammond, R. S., Bond, C. T., Strassmaier, T., Ngo-Anh, T. J., Adelman, J. P., Maylie, J., and Stackman, R. W. (2006) Small-conductance Ca2+-activated K+ channel type 2 (SK2) modulates hippocampal learning, memory, and synaptic plasticity. J. Neurosci. 26, 1844–1853.
Brocard, J., Warot, X., Wendling, O., Messaddeq, N., Vonesch, J. L., Chambon, P., and Metzger, D. (1997) Spatio-temporally controlled site-specific somatic mutagenesis in the mouse. Proc. Natl. Acad. Sci. USA 94, 14559–14563.
Danielian, P. S., Muccino, D., Rowitch, D. H., Michael, S. K., and McMahon, A. P. (1998) Modification of gene activity in mouse embryos in utero by a tamoxifen-inducible form of Cre recombinase. Curr. Biol. 8, 1323–1326.
Indra, A. K., Warot, X., Brocard, J., Bornert, J. M., Xiao, J. H., Chambon, P., and Metzger, D. (1999) Temporally-controlled site-specific mutagenesis in the basal layer of the epidermis: comparison of the recombinase activity of the tamoxifen-inducible Cre-ER(T) and Cre-ER(T2) recombinases. Nucleic Acids Res. 27, 4324–4327.
Li, M., Indra, A. K., Warot, X., Brocard, J., Messaddeq, N., Kato, S., Metzger, D., and Chambon, P. (2000) Skin abnormalities generated by temporally controlled RXRalpha mutations in mouse epidermis. Nature 407, 633–636.
Auwerx, J., Avner, P., Baldock, R., Ballabio, A., Balling, R., Barbacid, M., et al. (2004) The European dimension for the mouse genome mutagenesis program. Nat. Genet. 36, 925–927.
Gong, S., Doughty, M., Harbaugh, C. R., Cummins, A., Hatten, M. E., Heintz, N., and Gerfen, C. R. (2007) Targeting Cre recombinase to specific neuron populations with bacterial artificial chromosome constructs. J. Neurosci. 27, 9817–9823.
Hebert, J. M. and McConnell, S. K. (2000) Targeting of cre to the Foxg1 (BF-1) locus mediates loxP recombination in the telencephalon and other developing head structures. Dev. Biol. 222, 296–306.
Matei, V., Pauley, S., Kaing, S., Rowitch, D., Beisel, K. W., Morris, K., Feng, F., Jones, K., Lee, J., and Fritzsch, B. (2005) Smaller inner ear sensory epithelia in Neurog 1 null mice are related to earlier hair cell cycle exit. Dev. Dyn. 234, 633–650.
Ohyama, T. and Groves, A. K. (2004) Generation of Pax2-Cre mice by modification of a Pax2 bacterial artificial chromosome. Genesis 38, 195–199.
Sage, C., Huang, M., Karimi, K., Gutierrez, G., Vollrath, M. A., Zhang, D. S., Garcia-Anoveros, J., Hinds, P. W., Corwin, J. T., Corey, D. P., and Chen, Z. Y. (2005) Proliferation of functional hair cells in vivo in the absence of the retinoblastoma protein. Science 307, 1114–1118.
Sage, C., Huang, M., Vollrath, M. A., Brown, M. C., Hinds, P. W., Corey, D. P., Vetter, D. E., and Chen, Z. Y. (2006) Essential role of retinoblastoma protein in mammalian hair cell development and hearing. Proc. Natl. Acad. Sci. USA 103, 7345–7350.
Li, M., Tian, Y., Fritzsch, B., Gao, J., Wu, X., and Zuo, J. (2004) Inner hair cell Cre-expressing transgenic mouse. Genesis 39, 173–177.
Tian, Y., Li, M., Fritzsch, B., and Zuo, J. (2004) Creation of a transgenic mouse for hair-cell gene targeting by using a modified bacterial artificial chromosome containing Prestin. Dev. Dyn. 231, 199–203.
Chow, L. M., Tian, Y., Weber, T., Corbett, M., Zuo, J., and Baker, S. J. (2006) Inducible Cre recombinase activity in mouse cerebellar granule cell precursors and inner ear hair cells. Dev. Dyn. 235, 2991–2998.
Editorial (2007) Toxic alert. Nature 449, 378.
Soriano, P. (1999) Generalized lacZ expression with the ROSA26 Cre reporter strain [letter]. Nat. Genet. 21, 70–71.
Novak, A., Guo, C., Yang, W., Nagy, A., and Lobe, C. G. (2000) Z/EG, a double reporter mouse line that expresses enhanced green fluorescent protein upon Cre-mediated excision. Genesis 28, 147–155.
Goodrich, L. V. (2007) A genetic dissection of auditory circuit assembly and function. ARO MidWinter Meeting, 241.
Johnson, K. R. and Zheng, Q. Y. (2002) Ahl2, a second locus affecting age-related hearing loss in mice. Genomics 80, 461–464.
Ohlemiller, K. K. (2004) Age-related hearing loss: the status of Schuknecht’s typology. Curr. Opin. Otolaryngol. Head Neck Surg. 12, 439–443.
Kujawa, S. G. and Liberman, M. C. (2006) Acceleration of age-related hearing loss by early noise exposure: evidence of a misspent youth. J. Neurosci. 26, 2115–2123.
Huynh, K., Edge, R., Gao, J., Zuo, J., Dallos, P., and Cheatham, M. A. (2006) Anatomy and physiology of prestin knockout mice backcrossed to the CBA/CaJ strain. ARO Midwinter Meeting, Abstr. 379.
Kiermayer, C., Conrad, M., Schneider, M., Schmidt, J., and Brielmeier, M. (2007) Optimization of spatiotemporal gene inactivation in mouse heart by oral application of tamoxifen citrate. Genesis 45, 11–16.
Hong, S. B., Furihata, M., Baba, M., Zbar, B., and Schmidt, L. S. (2006) Vascular defects and liver damage by the acute inactivation of the VHL gene during mouse embryogenesis. Lab Invest. 86, 664–675.
Lantinga-van Leeuwen, I. S., Leonhard, W. N., van de Wal, A., Breuning, M. H., Verbeek, S., de Heer, E., and Peters, D. J. (2006) Transgenic mice expressing tamoxifen-inducible Cre for somatic gene modification in renal epithelial cells. Genesis 44, 225–232.
Guo, C., Yang, W., and Lobe, C. G. (2002) A Cre recombinase transgene with mosaic, widespread tamoxifen-inducible action. Genesis 32, 8–18.
Acknowledgements
This work was supported in part by grants from the National Institutes of Health (DC06471, DC05168, DC008800, CA21765) and the American Lebanese Syrian Associated Charities of St. Jude Children’s Research Hospital. J. Zuo is a recipient of a Hartwell Individual Biomedical Research Award.
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Yu, Y., Zuo, J. (2009). The Practical Use of Cre and loxP Technologies in Mouse Auditory Research. In: Sokolowski, B. (eds) Auditory and Vestibular Research. Methods in Molecular Biology™, vol 493. Humana Press. https://doi.org/10.1007/978-1-59745-523-7_6
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