Conditional Gene Expression in the Mouse Inner Ear Using Cre-loxP

Abstract

In recent years, there has been significant progress in the use of Cre-loxP technology for conditional gene expression in the inner ear. Here, we introduce the basic concepts of this powerful technology, emphasizing the differences between Cre and CreER. We describe the creation and Cre expression pattern of each Cre and CreER mouse line that has been reported to have expression in auditory and vestibular organs. We compare the Cre expression patterns between Atoh1-CreER TM and Atoh1-CreER T2 and report a new line, Fgfr3-iCreER T2 , which displays inducible Cre activity in cochlear supporting cells. We also explain how results can vary when transgenic vs. knock-in Cre/CreER alleles are used to alter gene expression. We discuss practical issues that arise when using the Cre-loxP system, such as the use of proper controls, Cre efficiency, reporter expression efficiency, and Cre leakiness. Finally, we introduce other methods for conditional gene expression, including Flp recombinase and the tetracycline-inducible system, which can be combined with Cre-loxP mouse models to investigate conditional expression of more than one gene.

Regulation of p27Kip1 by Sox2 Maintains Quiescence of Inner Pillar Cells in the Murine Auditory Sensory Epithelium

“…4G-H’’’). Finally, consistent with the observation that iCre activity in Fgfr3 iCreER+ mice was present in limited number of endogenous OHCs (Cox et al, 2012), a few EGFP+/calbindin+ cells (endogenous HCs with iCre activity) were found in the experimental group. However, the number of EGFP+/calbindin+ cells was similar between the experimental and control groups, suggesting that Sox2-negative OPCs and DCs also did not change fate to become HCs.…”

“…Therefore, we instead characterized the Fgfr3 iCreER+ transgenic mouse (Young et al, 2010). When tamoxifen was given at P0 and P1, inside the organ of Corti, the iCre activity of Fgfr3 iCreER+ is primarily restricted into IPCs, OPCs and DCs (Cox et al, 2012). Of note, iCre+ OHCs, Hensen’s cells or Claudius cells were also observed, but iCre+ IHCs and inner phalangeal cells (IPHs) were never observed.…”

“…Therefore, we deleted Sox2 in SCs at P6 and P7 (defined as juvenile SCs) to determine whether they still need Sox2 to remain quiescent. When tamoxifen was given at P6 and P7, inside the organ of Corti, iCre activity of Fgfr3 iCreER+ were present in almost all IPCs, OPCs and DCs (Cox et al, 2012). Again, only a few iCre+ cells were OHCs, Hensen’s cells and Claudius cells.…”

Sox2 haploinsufficiency primes regeneration and Wnt responsiveness in the mouse cochlea

“…The results of this study underscore the potential confounding effects from haploinsufficiency when using knockin mouse models (79). For example, the Foxg1-Cre mouse line can have brain deficits that probably stem from alterations in proliferation, possibly as a result of haploinsufficiency (80)(81)(82).…”

Endocannabinoid signaling in hypothalamic circuits regulates arousal from general anesthesia in mice

“…For example, the traditional bacteriophage-derived Cre is not optimal for eukaryotes due to its codon usage of prokaryotes, and its 65 CpG dinucleotides may induce epigenetic silencing (38). To overcome these potential problems, we generated vGLUT1-iCreERT2, vGLUT2-iCreERT2, and GAD65-iCreERT2 mutant mice with a knockin of iCreERT2 (38,39) into the Vglut1, Vglut2, or GAD65 locus. Mice received 8 daily injection of tamoxifen 1 week before use (40).…”