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J. Biol. Chem., Vol. 276, Issue 17, 14037-14043, April 27, 2001
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From the Department of Biological Sciences, University of Notre
Dame, Notre Dame, Indiana 46556
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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF331797.
Isolation of a Zebrafish Rod Opsin Promoter to Generate a
Transgenic Zebrafish Line Expressing Enhanced Green Fluorescent Protein
in Rod Photoreceptors*
,
*
This work was supported by a grant from the Foundation for
Fighting Blindness (to D. R. H.).The costs of publication of this article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
Recipient of a postdoctoral fellowship from the Keck Center for
Transgene Research, University of Notre Dame. Present address: Howard
Hughes Medical Inst./Dept. of Biochemistry, P.O. Box 357370, University
of Washington, Seattle, WA 98195.
§
Supported by a National Science Foundation-Research Experience for
Undergraduates award.
¶
To whom correspondence should be addressed. Tel.:
219-631-8054; Fax: 219-631-7413; E-mail: hyde.1@nd.edu.
Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
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