Gfi1Cre mice have early onset progressive hearing loss and induce recombination in numerous inner ear non-hair cells

Abstract

Studies of developmental and functional biology largely rely on conditional expression of genes in a cell type-specific manner. Therefore, the importance of specificity and lack of inherent phenotypes for Cre-driver animals cannot be overemphasized. The Gfi1Cre mouse is commonly used for conditional hair cell-specific gene deletion/reporter gene activation in the inner ear. Here, using immunofluorescence and flow cytometry, we show that the Gfi1Cre mice produce a pattern of recombination that is not strictly limited to hair cells within the inner ear. We observe a broad expression of Cre recombinase in the Gfi1Cre mouse neonatal inner ear, primarily in inner ear resident macrophages, which outnumber the hair cells. We further show that heterozygous Gfi1Cre mice exhibit an early onset progressive hearing loss as compared with their wild-type littermates. Importantly, vestibular function remains intact in heterozygotes up to 10 months, the latest time point tested. Finally, we detect minor, but statistically significant, changes in expression of hair cell-enriched transcripts in the Gfi1Cre heterozygous mice cochleae compared with their wild-type littermate controls. Given the broad use of the Gfi1Cre mice, both for gene deletion and reporter gene activation, these data are significant and necessary for proper planning and interpretation of experiments.

Gfi1‐GCE inducible Cre line for hair cell‐specific gene manipulation in mouse inner ear

“…Compared to the controls, no hearing deficiency was detected in Gfi1 GCE/+ heterozygotes or Gfi1 GCE/GCE homozygotes across five frequencies tested (8, 12, 16, 24, and 32 kHz) ( p > .05) (Figure c). In contrast, Gfi1 Cre/+ heterozygotes showed significantly elevated hearing thresholds at 12, 24, and 32 kHz ( p ≤ .05), consistent with the previous report of progressive hearing loss in Gfi1 Cre/+ heterozygotes (Matern et al, ). Thus, unlike Gfi1 Cre knock‐in, in‐frame fusion of self‐cleavage GCE in Gfi1 GCE mice does not affect GFI1 function and preserves the normal development and function of hair cells.…”

“…Gfi1 haploinsufficiency in the hair cells causes Gfi1 Cre/+ heterozygotes to develop progressive hearing loss (Matern et al, ). Unlike Gfi1 Cre mice in which Gfi1 is inactivated by Cre knock‐in (Yang, Gan, et al, ), using the “self‐cleaving” P2A peptide in Gfi1 GCE inducible Cre mice could avoid disrupting Gfi1 as Figure demonstrates the effective self‐cleavage to generate GFI1 and GCE separately from a single transcript.…”

“…In addition to inner ear hair cells, Gfi1 ‐Cre activities revealed by conditional tdTomato reporter gene expression have been observed in non‐hair cells at P1, and these tdTomato+ non‐hair cells are identified as CD326‐CD45+ immune cells and are observed at the basal, middle, and apical regions of the cochlea (Matern et al, ). We thus compared Gfi1 ‐Cre and Gfi1 ‐GCE activities in the cochleae via the conditional tdTomato reporter gene expression.…”

“…Previously, we have generated Gfi1 Cre knock‐in mouse line by replacing Exons 1–5 of Gfi1 with Cre coding sequence, and have shown that Gfi1‐ Cre is highly efficient for hair cell‐specific gene manipulations during inner ear development (Yang, Gan, et al, ). Recently, it has been reported that Gfi1 Cre/+ heterozygotes display progressive hearing loss in mature mice (Matern et al, ), suggesting a Gfi1 haploinsufficiency in postnatal hair cells and making Gfi1 Cre unsuitable for gene manipulation in postnatal hair cells. In order to generate a Cre recombinase mouse line that circumvents the haploinsufficiency issue and permits more precise spatiotemporal gene manipulation in hair cells, we have chosen to knock in an inducible CreERT2 at Gfi1 locus without disrupting Gfi1 activity through the use of the self‐cleaving P2A peptide.…”

Exosomes mediate sensory hair cell protection in the inner ear

“…To label hair cells with mGFP and supporting cells with mtdTomato, hair cell-specific Gfi1 Cre/+ mice (generated by Lin Gan, University of Rochester, Rochester, New York, USA, and provided by Ronna Hertzano, University of Maryland, College Park, Maryland, USA) were used. Gfi1 Cre/+ mice express Cre recombinase in the Gfi1 locus, which is required for hair cell differentiation and survival (78). Offspring were backcrossed with ROSA-26 mTmG/mTmG to produce Gfi1 Cre/+ ROSA26 mTmG/mTmG mice.…”