The generation and characterization of a transgenic zebrafish line with lens-specific Cre expression.

PURPOSE

Danio rerio zebrafish constitute a popular model for studying lens development and congenital cataracts. However, the specific deletion of a gene with a Cre/LoxP system in the zebrafish lens is unavailable because of the lack of a lens-Cre-transgenic zebrafish. This study aimed to generate a transgenic zebrafish line in which Cre recombinase was specifically expressed in the lens.

METHODS

The pTol2 :Cre-polyA-:EGFP (enhanced green fluorescent protein) plasmid was constructed and co-injected with Tol2-transposase into one-to-two-cell-stage wild-type (WT) zebrafish embryos. Whole-mount in situ hybridization (ISH), tissue section, hematoxylin and eosin staining, a Western blot, a split-lamp observation, and a grid transmission assay were used to analyze the Cre expression, lens structure, and lens transparency of the transgenic zebrafish.

RESULTS

In this study, we generated a transgenic zebrafish line, zTg(:Cre-:EGFP), in which Cre recombinase and EGFP were driven by the lens-specific promoter. zTg(:Cre-:EGFP) began to express Cre and EGFP specifically in the lens at the 22 hpf stage, and this ectopic Cre could efficiently and specifically delete the red fluorescent protein (RFP) signal from the lens when zTg(:Cre-:EGFP) embryos were injected with the -flanked RFP plasmid. The overexpression of Cre and EGFP did not impair zebrafish development or lens transparency. Accordingly, this zTg(:Cre-:EGFP) zebrafish line is a useful tool for gene editing, specifically with zebrafish lenses.

CONCLUSIONS

We established a zTg(:Cre-:EGFP) zebrafish line that can specifically express an active Cre recombinase in lens tissues. This transgenic zebrafish line can be used as a tool to specifically manipulate a gene in zebrafish lenses.