Isolation of a zebrafish rod opsin promoter to generate a transgenic zebrafish line expressing enhanced green fluorescent protein in rod photoreceptors.
To exploit zebrafish as a transgenic model, tissue-specific promoters must be identified. We isolated a 20-kilobase (kbp) zebrafish rod opsin genomic clone, which consists of 18 kbp of 5'-flanking region, the entire coding region, and 0.5 kbp of 3'-flanking sequence. Polymerase chain reaction, 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-kbp promoter fragment was cloned upstream of the enhanced green fluorescent protein (EGFP) cDNA and microinjected into 1- to 2-cell stage zebrafish embryos. EGFP expression was detected in the ventral-nasal eye at 3 days postfertilization and spread throughout the eye. Progeny of the positive founder fish, which were identified by polymerase chain reaction 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.
Kennedy
B N
BN
Department of Biological Sciences, University of Notre Dame, Indiana 46556, USA.
Vihtelic
T S
TS
Checkley
L
L
Vaughan
K T
KT
Hyde
D R
DR
D000595:Amino Acid Sequence; D000818:Animals; D030801:Animals, Genetically Modified; D001483:Base Sequence; D015152:Blotting, Northern; D015139:Blotting, Southern; D003001:Cloning, Molecular; D017124:Conserved Sequence; D018076:DNA, Complementary / Q000378:metabolism; D015723:Gene Library; D049452:Green Fluorescent Proteins; D017403:In Situ Hybridization; D007438:Introns; D008164:Luminescent Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D010786:Photoreceptor Cells / Q000737:chemistry / Q000378:metabolism* / Q000502:physiology; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D012160:Retina / Q000737:chemistry / Q000378:metabolism / Q000502:physiology; D017948:Retinal Rod Photoreceptor Cells / Q000737:chemistry* / Q000502:physiology; D017299:Rod Opsins / Q000096:biosynthesis* / Q000235:genetics*; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D014158:Transcription, Genetic; D015027:Zebrafish
D018076:DNA, Complementary; D008164:Luminescent Proteins; D017299:Rod Opsins; D049452:Green Fluorescent Proteins