Research Paper Volume 13, Issue 8 pp 11678—11695

Piccolo is essential for the maintenance of mouse retina but not cochlear hair cell function

Generation of Piccolo-/- mouse. (A) Schematic diagram of the CRISPR/Cas9 knockout strategy. (B) Schematic illustration of Cas9-mediated targeting of the mouse Piccolo gene (sgRNA and PAM sequences are underlined) and comparison of Piccolo DNA sequences in wild type and Piccolo-/- (4 bp deletion) mice. (C) Schematic illustration of the frameshift mutation in Piccolo-/- mice. The altered amino acid sequence is marked in red. “*” indicates a premature stop codon. The translation of protein is terminated at the point of the arrow. Also shown are the Piccolo wild type protein sequence and domain organization. (D) Sequencing chromatograms of Piccolo in wild type (WT) mice, heterozygous (HET; Piccolo+/-) mice, and homozygous (Homo; Piccolo-/-) mice. The red box indicates the bases missing in the homozygous (Homo) Piccolo-/- mice. (E) Relative Piccolo expression in Piccolo-/- and WT mice as measured by Q-PCR. (F) Western blot analysis of Piccolo protein in the retina of Piccolo-/- and WT mice. GADPH was used as the loading control.

Figure 1. Generation of Piccolo-/- mouse. (A) Schematic diagram of the CRISPR/Cas9 knockout strategy. (B) Schematic illustration of Cas9-mediated targeting of the mouse Piccolo gene (sgRNA and PAM sequences are underlined) and comparison of Piccolo DNA sequences in wild type and Piccolo-/- (4 bp deletion) mice. (C) Schematic illustration of the frameshift mutation in Piccolo-/- mice. The altered amino acid sequence is marked in red. “*” indicates a premature stop codon. The translation of protein is terminated at the point of the arrow. Also shown are the Piccolo wild type protein sequence and domain organization. (D) Sequencing chromatograms of Piccolo in wild type (WT) mice, heterozygous (HET; Piccolo+/-) mice, and homozygous (Homo; Piccolo-/-) mice. The red box indicates the bases missing in the homozygous (Homo) Piccolo-/- mice. (E) Relative Piccolo expression in Piccolo-/- and WT mice as measured by Q-PCR. (F) Western blot analysis of Piccolo protein in the retina of Piccolo-/- and WT mice. GADPH was used as the loading control.