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J. R. Leheste, G. Matthews; A Novel Vector System Facilitating Cell-Specific Gene Expression in Zebrafish Retina. Invest. Ophthalmol. Vis. Sci. 2007;48(13):2972.
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© ARVO (1962-2015); The Authors (2016-present)
One of the greatest challenges in reverse genetics is the identification of precise promoter elements that drive expression of a desired transgene in a well-defined fashion. The analysis of such elements is tedious and the subsequent generation of a transgenic animal model time-consuming. Injection of bacterial artificial chromosomes (BACs) as vehicle for the transgene addresses these pitfalls but creates new problems: The resulting animal model is transient and triploid for all the genes residing on that particular BAC. Our alternative approach facilitates the generation of genetically modified zebrafish with retina-specific expression of a stably integrated transgene using large natural promoter sequences.
We developed a DNA vector system suitable for homology-based uptake of large DNA-sequences from BACs via gap-repair (recombineering). As a test, ~10kb of the zebrafish mGluR6 promoter (ON bipolar cell specific) were retrieved to control expression of a fluorescence reporter (EYFP). This expression cassette was flanked by I-SceI meganuclease recognition sites to facilitate subsequent integration into the zebrafish genome. Unicellular zebrafish embryos were injected with a mixture of the assembled DNA construct and I-SceI meganulease enzyme. Five days postfertilization (pf) transgenic fish were selected based on retina-specific EYFP expression and further analyzed.
Approximately 60% of the founder generation (F0) of potentially transgenic fish showed some degree of retinal fluorescence. Fish with robust retinal expression of EYFP were used for propagation. Southern-blot analysis revealed the random genomic integration of one copy of the transgene in all except one line. Cryosections of fixed retinas from adult transgenic fish were analyzed by confocal microscopy. Immunostaining with PKC, a marker for ON bipolar cells, revealed an 85-95% overlap of native EYFP fluorescence and PKC expression, which demonstrates efficient and selective transgenic targeting of ON bipolar cells in the neural retina.
The findings demonstrate the effectiveness of our newly developed vector system for a cell-specific gene targeting approach in zebrafish. The system facilitates the use of large natural gene regulators without requiring their detailed analysis and provides a general tool for spatial or temporal expression of transgenes.
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