Stereocilin (Strctm1Ugds/tm1Udgs, referred to as Strc−/−) and α-tectorin (TectaΔENT/ΔENT, referred to as Tecta−/−) mice were described in previous studies (1214, 26). TectaΔENT/ΔENT mice were provided by G. Richardson (University of Sussex, Falmer, UK). The targeted deletion of exons 2 and 3 in Strc−/− mice results in a frameshifting deletion that would produce an incomplete signal peptide followed by 30 out-of-frame amino acids (13). Targeted integration of a neor cassette into exon 4 of Tecta results in the skipping of this exon, which is predicted to cause a deletion of 96 amino acids in α-tectorin. The protein is not detected by Western blot analysis in TectaΔENT/ΔENT cochleae (26). Strc−/−/Tecta−/− double-knockout mice were produced by breeding Strc−/− mice with Tecta−/− mice and the compound heterozygous offspring with each other. Strc−/−/Tecta−/− mice were mated to Strc+/−/Tecta−/− mice to generate Strc−/−/Tecta−/− and Strc+/−/Tecta−/− littermate controls.

Strc genotyping of experimental animals was performed using two separate polymerase chain reactions (PCRs) with forward primers F1 (5′-GGGCTCTGAGGAGGCTCTTTGGG-3′) [located in exon 2 (reaction 1)] and F2 (5′-TGGGATTTGAACTCAGGTTGCTAGG-3′) [located in intron 1 (reaction 2)], respectively, and reverse primer R2 (5′-CAGAGGCACACCTCTGCTCAGG-3′) (located in exon 4) (13). Because of the targeted deletion of exons 2 and 3, there is no amplification product for the Strc allele in reaction 1, and reaction 2 gives an amplicon size of 1 kb. The size of the products amplified from the wild-type allele is 1250 base pairs (bp; reaction 1) and 2300 bp (reaction 2) (fig. S5, A and B). Tecta genotyping was performed using forward primer 5′-TTACAGGCGTGGTACTGCTG in exon 4 and reverse primer 5′-TGGTGTTGTTTCCTTCAACG spanning the exon 4–intron 4 boundary. Experimental mice (Strc+/− or Strc−/−) are all homozygous for the Tecta allele and thus give a single 2.1-kb amplicon, owing to the insertion of the neor cassette into exon 4. Amplification from a wild-type allele would give a 281-bp fragment (fig. S5, C and D).

Note: The content above has been extracted from a research article, so it may not display correctly.

Please log in to submit your questions online.
Your question will be posted on the Bio-101 website. We will send your questions to the authors of this protocol and Bio-protocol community members who are experienced with this method. you will be informed using the email address associated with your Bio-protocol account.

We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.