Page 69 - Read Online

Page 114 Ghaseminejad et al. J Transl Genet Genom 2022;6:111-25 https://dx.doi.org/10.20517/jtgg.2021.49

probed with anti-rod opsin antibodies mabB630N or mab514-18 (at 1:10 dilution of tissue culture
[21]
supernatant, gifts of W.C. Smith). The mabB630N epitope is contained within residues 3-14 , while the
[22]
epitope for mab514-18 includes residue F13 found in mammalian rod opsins . Secondary antibody was IR-
dye800-conjugated goat anti-mouse used at 1:10000 of 1 mg/mL solution (LI-COR Biosciences). A LI-COR
Odyssey imaging system was used to image and quantify blot signals. Samples were prepared for confocal
microscopy as previously described . Fixed eyes were infiltrated with 20% sucrose for 48 h, embedded in
[23]
OCT (Sakura Finetek), frozen, and stored at -80 °C for sectioning. 12 µM slices were cut using a cryostat
[24]
and labeled overnight with mAb B630N or mAb2B2 antibodies (1:10 dilution of tissue culture
supernatant, mAb2B2 gift of Robert Molday). Similar to mAb514-18, mammalian rod opsin residue F13 is a
[22]
critical component of the mAb2B2 epitope . Secondary antibody was Cy3-conjugated anti-mouse antibody
(1:750 dilution, Jackson Immunoresearch). Sections were counterstained with AlexaFluor 488-conjugated
wheat germ agglutinin (WGA; Life Technologies) and Hoechst 33342 (Sigma-Aldrich) and imaged using a
Zeiss 800 laser scanning confocal microscope with a 40X NA 1.2 water immersion objective. Adobe
Photoshop was used for post-processing and figure assembly. In order to improve image detail, non-linear
adjustments were performed on Hoechst 33342 and WGA labeling channels. Antibody signal was linearly
adjusted for better visualization.

Genomic DNA extraction, PCR, and sequencing
We designed four PCR primers to specifically amplify Rho.L regions of interest without amplifying other
X. laevis Rho genes [Supplementary Material 1]. Genomic DNA was extracted from tadpole tail clips as
previously described . Genomic DNA samples were PCR amplified and analyzed by gel electrophoresis.
[19]
[25]
PCR products were treated with an exoSAP protocol and Sanger sequenced using a commercial service
(Genewiz).

Electroretinogram recordings
Electroretinograms (ERGs) were recorded from dark-adapted animals at age 9 months as previously
[26]
described . Following the recordings, blood samples were collected to identify genotypes (WT vs. WT/
Rho.LΔ11Δ1) via Sanger sequencing. An example of analysis is shown in Supplementary Material 2.

Statistical analyses
GraphPad Prism software (version 9.0.0) was used to perform statistical analyses and generate figures.
Specific tests and numbers of animals used are given for each experiment in the results section.

RESULTS
[12]
In a previously published study , we used CRISPR/Cas9 to generate indels in the X. laevis Rho genes. We
determined that X. laevis have three Rho genes (Rho.L, Rho.2.L, and Rho.S), and that in-frame indels in the
X. laevis Rho genes cause RD, while frame-shifting indels have minimal phenotype. From this initial set of
animals, we identified a line carrying a complex deletion comprised of adjacent 11bp and 1bp deletions that
immediately follow the start codon of the Rho.L gene. The complex deletion could be formally designated
Chr4L:131405006_131405016delAACGGAACAGA,131405030delT using HVGS nomenclature, but is
hereafter referred to as “Rho.LΔ11Δ1” [Figure 1A, Supplementary Materials 1 and 3]. Preliminary data
derived from X. laevis Rho.L knockout tadpoles [Supplementary Material 4] suggests that Rho.L is the
highest expressing of the three X. laevis Rho genes, and that each Rho.L allele contributes no more than 42%
to the total retinal rod opsin.

Initial characterization of the Rho.LΔ11Δ1 X. laevis model for adRP
We characterized X. laevis tadpoles aged 14 dpf generated from a cross between WT and heterozygous WT/
Rho.LΔ11Δ1 animals. Genotypically, the offspring were expected to be half WT and half WT/Rho.LΔ11Δ1. A