Outer hair cell-specific prestin-CreERT2 knockin mouse lines

Abstract

Outer hair cells (OHCs) in the cochlea are crucial for the remarkable hearing sensitivity and frequency tuning. To understand OHC physiology and pathology, it is imperative to use mouse genetic tools to manipulate gene expression specifically in OHCs. Here, we generated two prestin knockin mouse lines: (1) the prestin-CreERT2 line, with an internal ribosome entry site-CreERT2-FRT-Neo-FRT cassette inserted into the prestin locus after the stop codon, and (2) the prestin-CreERT2-NN line, with the FRT-Neo-FRT removed subsequently. We characterized the inducible Cre activity of both lines by crossing them with the reporter lines CAG-eGFP and Ai6. Cre activity was induced with tamoxifen at various postnatal ages and only detected in OHCs, resembling the endogenous prestin expression pattern. Moreover, prestin-CreERT2+/-(heterozygotes) and +/+(homozygotes) as well as prestin-CreERT2-NN+/-mice displayed normal hearing. These two prestin-CreERT2 mouse lines are therefore useful tools to analyze gene function in OHCs in vivo.

FIG. 1. Generation of prestin-CreER^T2 knock-in mice

(a) Schematic diagram of generation of prestin-CreER^T2 +/− and prestin-CreER^T2–NN (No Neo cassette) knock-in mice. Solid blue rectangles represent exon18–20 of the prestin gene. The external 3′-Southern probe is shown as the black bar. Arrows indicate the approximate positions of the PCR genotyping primers. Abbreviations: IRES, internal ribosome entry site; CreER^T2, Cre recombinase fused to a mutant estrogen ligand-binding domain; Neo, PGK-neo cassette; FRT, FLP recombinase target. (b) Southern blot genotyping analysis of prestin-CreER^T2 +/− and prestin-CreER^T2 +/+ mice. Genomic tail DNA was digested with SpeI and a 3′-external probe indicated in (a) was used to identify the 23-kb wild-type and the 10-kb targeted DNA fragments. (c) PCR genotyping shows 300-bp product (wild type) and 228-bp product (prestin-CreER^T2 +/− mice).

FIG. 2

Inducible Cre recombination in prestin-CreER^T2 +/− mice. Images of a whole cochlea (P7) from a prestin-CreER^T2 +/−; Ai6 mouse without (a–c) and with (d–f) tamoxifen injection at P2–4.

FIG. 3

Cre recombination in all OHCs at various postnatal ages. (a-c″) prestin-CreER^T2 +/− shows a gradual decrease pattern in reporter expression. prestin-CreER^T2 +/−; CAG-eGFP mice were injected with tamoxifen (3mg/40g body weight) at P0–2. (d-d‴) prestin-CreER^T2 +/−; Ai6 mice were injected with tamoxifen at P5–7. Images of GFP (d), anti-prestin (d′), anti-myo VIIa (d″), and merger (d‴). IHCs: Inner hair cells, OHCs: outer hair cells. Scale bar: 20 μm.

FIG. 4

Auditory brainstem response (ABR) thresholds of prestin-CreER^T2 +/− and prestin-CreER^T2 +/+ and wild-type mice at 4–6 weeks. n, number of mice for ABR testing for each genotype. Bars: standard error of mean.

FIG. 5

Cre activity and hearing ability of prestin-CreER^T2-NN +/− mice. Images of P28 prestin-CreER^T2-NN +/−; Ai6 mouse inducted tamoxifen (9mg/40g) at P21–22, DAPI (a, b, c, at the nuclear level) and GFP (d, e, f at the cell body level), in each cochlear turn. Scale bar: 20 μm. (g) ABR thresholds of prestin-CreER^T2-NN +/−, prestin-CreER^T2-NN +/+ and wild-type mice at 4–6 weeks. N, number of mice for ABR testing for each genotype. Bars: standard error of mean. *: P < 0.05.