-
A
Schematic representation of otoferlin cDNA from otoferlin dual‐AAV‐transduced, wild‐type, and Otof
−/− organs of Corti, displaying binding sites of primers used in PCRs to assess dual‐AAV reassembly.
-
B
Otoferlin PCR amplicons from organ of Corti cDNA. A 1,753‐bp‐long amplicon (*), also present in non‐injected wild‐type controls (WTB6, WTCD1B6F1), indicates successful reassembly of the split otoferlin expression cassette in otoferlin dual‐AAV‐TS‐transduced CD1B6F1‐Otof
−/− organs of Corti (injected ear). In Otof
−/− samples, three shorter products were amplified (a, b and c).
-
C
Sanger sequencing confirmed correct dual‐AAV split‐site assembly (dashed line) as well as the presence of an artificial AccIII restriction site introduced in the dual‐AAV‐TS otoferlin cDNA, which is absent in the wild‐type (WT) and Otof
−/− cDNA (a–c). Amplicons a‐c from Otof
−/− organs of Corti all lack exons 14–15, while bands “b” (1,480 bp) and “c” (1,679 bp) still contain intron 20–21 (b) or intron 23–24 (c), respectively.
-
D
Western blotting on cell lysates of WT and Otof
−/− CD1B6F1 organs of Corti. Two bands of ˜210–230 kDa, corresponding to full‐length otoferlin, were detected in WT but absent in Otof
−/− ears. (**) refers to an unspecific band detected in both samples. GAPDH was used as loading control.
Data information: CDS: coding sequence, Ex: exon, TS: trans‐splicing, Hyb: hybrid, control ear: non‐treated ears, non‐injected ear: contralateral non‐injected
ears.
