Kennedy, B.N., Vihtelic, T.S., Checkley, L., Vaughan, K.T., and Hyde, D.R. (2001) Isolation of a zebrafish rod opsin promoter to generate a transgenic zebrafish line expressing EGFP in rod photoreceptors. The Journal of biological chemistry. 276(17):14037-14043.
To exploit zebrafish as a transgenic model, tissue-specific promoters must be identified. We isolated a 20-kb zebrafish rod opsin genomic clone, which consists of 18-kb of 5' flanking region, the entire coding region and 0.5-kb of 3' flanking sequence. PCR, Southern blotting and DNA sequencing revealed the rod opsin gene lacks introns. The transcription start site was localized 94 nucleotides upstream of the translation initiation site. Sequence alignment with orthologous promoters revealed conserved cis-elements including glass, NRE, OTX/Bat-1, Ret-1/PCE-1, Ret-4, and TATA box. A 1.2-kb promoter fragment was cloned upstream of the enhanced green fluorescent protein (EGFP) cDNA and microinjected into 1-2 cell-stage zebrafish embryos. EGFP expression was detected in the ventral-nasal eye at 3 days post-fertilization and spread throughout the eye. Progeny of the positive founder fish, which were identified by PCR amplification of fin genomic DNA, exhibited EGFP expression in the retina confirming the germline transmission of the transgene. Frozen eye sections demonstrated the EGFP expression was rod-specific and exhibited a similar developmental expression profile as the rod opsin protein. This stable transgenic line provides a novel tool for identification of genes regulating development and maintenance of rod photoreceptors.