Ablation of Whirlin Long Isoform Disrupts the USH2 Protein Complex and Causes Vision and Hearing Loss (original) (raw)
Figure 1
Whirlin knockout mice were generated.
(A) A schematic diagram illustrating the long and short isoforms of whirlin. The dashed lines indicate the deletion regions of the whirlin gene in whirlin knockout (whirlin−/−) and whirler (whirlinwi_/wi_) mice. The asterisks indicate the mutations of the whirlin gene in humans. The bottom solid lines indicate the antigen regions of various whirlin antibodies. PDZ, postsynaptic density 95/discs large/zonula occludens 1; PR, proline-rich region. (B) Targeting strategy for disrupting the whirlin gene. PCR primers for identification of the mutant and wild-type alleles are shown as arrowheads. E1, exon 1; neo, neomycin, the positive selective marker; DTA, diphtheria toxin expression cassette, the negative selective marker. (C) Identification of the mutant allele by genomic PCR using primers G1, G5r and 3A. (D) RT-PCR analysis shows loss of the first exon of whirlin transcripts in the homozygous mutant retina. Whirlin mRNA transcripts were reverse transcribed and amplified using primers located on exon 1 and 6 (top panel), exon 2 and 6 (middle panel), and exon 11 and 12 (bottom panel). Exon 12 is the last exon of the whirlin gene. NC, negative controls with water instead of DNA samples. (E) The whirlin long isoform was completely knocked out in the retina of homozygous mutants as shown by immunoblotting. γ–tubulin served as a loading control. +/+, wild-types; −/−, whirlin knockout homozygotes; +/−, whirlin knockout heterozygotes.