The practical use of Cre and loxP technologies in mouse auditory research

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.