FIGURE

Figure 4

ID

ZDB-FIG-230228-354

Publication

Trejo-Reveles et al., 2023 - Identification of Novel Coloboma Candidate Genes through Conserved Gene Expression Analyses across Four Vertebrate Species

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Figure 4

Targeted gene disruption of OFC novel candidates using CRISPR/Cas9 gene targeting in zebrafish embryos. (a) Sanger sequence traces for genomic DNA PCR analysis of CRISPR/Cas9 targeted embryos show successful genetic disruption at the sgRNA site loci for eph3ba, eph3bb, and emx2. PAM sites are indicated. (b) Brightfield microscopy analysis of targeted fish at 3 dpf and 5 dpf compared to uninjected controls and Cas9-only controls. (c) Eye diameter metrics for fish injected with CRISPR/Cas9 at 3 dpf and 5 dpf (**** = p < 0.0001). (d) Confocal analysis of laminin-stained cryosections from gene-edited and control eyes showing persistence of the basement membrane in the optic fissure margin (arrowheads). Colobomas were observed in 27% and 47% of gene-edited embryos analysed for emx2 and ephb3a/b, respectively (n = 15 per target gene).

Expression Data

Antibody:	Ab1-lama1
Fish:	AB AB + CRISPR8-emx2 AB + CRISPR1-ephb3a + CRISPR2-ephb3b
Knockdown Reagents:	CRISPR1-ephb3a CRISPR2-ephb3b CRISPR8-emx2
Anatomical Terms:	eye basement membrane optic furrow basement membrane
Stage:	Protruding-mouth

Expression Detail

Antibody Labeling

Phenotype Data

Fish:	AB + CRISPR8-emx2 AB + CRISPR1-ephb3a + CRISPR2-ephb3b
Knockdown Reagents:	CRISPR1-ephb3a CRISPR2-ephb3b CRISPR8-emx2
Observed In:	closure of optic fissure eye optic fissure optic furrow basement membrane
Stage Range:	Protruding-mouth to Day 5

Phenotype Detail

Acknowledgments

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