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Shunichi Yoshikawa, John O'Brien; The promoter analysis and the targeted mutagenesis of the zebrafish cx50.5 gene. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):2524.
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© ARVO (1962-2015); The Authors (2016-present)
Gap junctions support intercellular transport of small molecules. In the ocular lens they are essential for the nutrition supply since the lens does not have blood vessels. Mutations of gap junction protein GJA8 (also known as CX50) are reported in heritable cataract patients. Our long-term goal is to generate an animal model of GJA8 mutant that is useful to study the mechanism of cataract and provides a drug screening system. We report here the zebrafish cx50.5 gene, the ortholog of GJA8. We (1) analyzed gene regulation in vivo by transgenic zebrafish, and (2) generated targeted mutants by CRISPR/Cas9.
To analyze the promoter and enhancer, the 5' flanking region of cx50.5 was cloned to the GFP expression vector. The plasmid was injected into 1 cell-stage zebrafish embryos and the GFP expression was observed during early development in living animals. To obtain targeted mutants of cx50.5, the guide RNA that targeted the 306th-325th nucleotides downstream of the initiation ATG and Cas9 mRNA were co-injected to the embryos. The targeted region contained the BsrBI recognition sequence and the disruption of this site was used for mutant screening.
In transient transgenic embryos with a 5.3-kb upstream fragment, the GFP signal was observed in the lens at 3 days post-fertilization (pdf). An additional signal was detected in neurons at the midbrain-hindbrain boundary at 5 dpf. We tested shorter fragments and found 120-, 80- and 40-bp fragments showed 61%, 21% and 0% positive embryos, respectively. The important region contained TFAP2A, GATA2 and SOX9/10 binding consensus sequences. The transgenic constructs with mutations in these sites suggested AP2 and SOX were important for the lens-specific expression while GATA2 was dispensable. To obtain the heritable mutants lines, F1 animals were screened and we found that 9 out of 26 F0 CRISPR/Cas9-injected fish showed BsrBI-resistant cx50.5 alleles. We confirmed by sequencing that all 9 alleles were mutated by deletions and/or insertions.
The cis-regulatory element of the zebrafish cx50.5 gene was located in 120 bp upstream region and the transcriptional factors AP2 and SOX were necessary for the lens-specific expression during early development. We successfully generated the cx50.5 mutant zebrafish by CRISPR/Cas9. They will be examined if they have similar symptom of the human cataract to serve as a model animal.
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