Generation and characterization of transgenic zebrafish lines using different ubiquitous promoters
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Two commonly used promoters to ubiquitously express transgenes in zebrafish are the Xenopus laevis elongation factor 1 α promoter (XlEef1a1) and the zebrafish histone variant H2A.F/Z (h2afv) promoter. Recently, transgenes utilizing these promoters were shown to be silenced in certain adult tissues, particularly the central nervous system. To overcome this limitation, we cloned the promoters of four zebrafish genes that likely are transcribed ubiquitously throughout development and into the adult. These four genes are the TATA box binding protein gene, the taube nuss-like gene, the eukaryotic elongation factor 1-gamma gene, and the beta-actin-1 gene. We PCR amplified approximately 2.5 kb upstream of the putative translational start site of each gene and cloned each into a Tol2 expression vector that contains the EGFP reporter transgene. We used these four Tol2 vectors to independently generate stable transgenic fish lines for analysis of transgene expression during development and in the adult. We demonstrated that all four promoters drive a very broad pattern of EGFP expression throughout development and the adult. Using the retina as a well-characterized component of the CNS, all four promoters appeared to drive EGFP expression in all neuronal and non-neuronal cells of the adult retina. In contrast, the h2afv promoter failed to express EGFP in the adult retina. When we examined EGFP expression in the various cells of the blood cell lineage, we observed that all four promoters exhibited a more heterogenous expression pattern than either the XlEef1a1 or h2afv promoters. While these four ubiquitous promoters did not express EGFP in all the adult blood cells, they did express EGFP throughout the CNS and in broader expression patterns in the adult than either the XlEef1a1 or h2afv promoters. For these reasons, these four promoters will be valuable tools for expressing transgenes in adult zebrafish.
KeywordsZebrafish Central nervous system Ubiquitous Promoter Tol2 Adult expression pattern
Enhanced green fluorescent protein
Reverse transcriptase polymerase chain reaction
Polymerase chain reaction
The authors thank the Freimann Life Science Center Staff for the care and maintenance of the zebrafish facility and Suzyanne Guzicki for the injection of the expression constructs into zebrafish embryos. We also thank Koichi Kawakami for the generous gift of the pT2KXIG construct containing the Tol2 transposable element and Tol2 transposase gene (pCSTZ2.8) and the Campos-Ortega lab for the gift of the H2A.F/Z promoter. This work was supported by the National Institute of Health (R21-EY017134 to D.R.H).
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