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S. Meng, D. McMahon; Bacteria Artificial Chromosome Modification for Generating Transgenic Zebrafish Lines for Retinal Circadian Study. Invest. Ophthalmol. Vis. Sci. 2008;49(13):187. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Circadian clock mechanisms in the zebrafish retina are of interest both from a comparative aspect and as a model system with unique experimental advantages. The vertebrate retina is profoundly influenced by circadian rhythmicity in a variety of processes, yet knowledge about the mechanisms of the zebrafish retinal circadian clock is limited. To further the study of the zebrafish retinal clock, we are generating transgenic zebrafish in which promoter sequences from the circadian clock gene (Per3) drive dynamic green fluorescent protein (d2GFP) expression.
We started with a BLAST search for bacteria artificial chromosome (BAC) clones which contain at least 10 kb sequence upstream from the start codon as well as the entire coding region of the Per3 gene. A 158 kb BAC clone, CH211-138E4, was chosen and used for further modification by an E.coli-based BAC engineering method (Lee et al., 2001 and Shin et al., 2003). Briefly, the reporter gene (d2gfp) and an SV40 polyadenylation tail (pA) were ligated to the kanamycin resistance (kanr) gene flanked by FRT, the recognition site for flpe-mediated filp recombination. This d2gfp-kanr gene cassette was inserted downstream of the start codon of Per3. Following the targeting recombination, the kanr gene was flipped-out of the recombinant BAC (rBAC). The final modified rBAC DNA was linearized and injected into 1-cell stage zebrafish embryos. The injected embryos were screened for GFP expression.
Restriction digestion analysis and DNA sequencing near the targeting region showed correct recombination of the Per3 BAC with d2gfp inserted after the start codon. Following microinjection of the rBAC DNA (~ 1 nl at 40 ng/ul), transient expression of GFP was observed in the retina and in brain regions at 1-day post fertilization. Among the ~ 4000 injected embryos, about 10% expressed GFP. These embryos are being raised to adulthood and will be screened for germ-line transformed founders.
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