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X. C. Zhao, S. Yoshikawa, R. W. Yee; Development of an Efficient cre/loxP Monitor System in Zebrafish. Invest. Ophthalmol. Vis. Sci. 2007;48(13):2973.
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The cre/loxP site-specific recombination system is a powerful genetic tool for genome manipulation and gene expression analysis. The objective of this study is determine if this recombination system is useful in the development of eye tissue-specific cre-transgenic lines that can be used for functional analysis of ocular disease genes.
A GR plasmid (Tol2 5’-xef1α-loxP-GFP-STOP-loxP-RFP-pA-Tol2 3’) was constructed by cloning the green fluorescent protein (GFP), the red fluorescent protein (RFP), the transcriptional stop signal sequence (STOP), and two loxP sites into a Tol2 transposon-based plasmid. A second plasmid (NLScre) was generated by cloning the cre recombinase (cre) and the nucleus localization signal (NLS) into pXT7. The GR plasmid was either injected alone or co-injected with the in vitro synthesized capped mRNA from the NLScre plasmid into the embryos of 1-2 cell stage from the wild type and a stable transgenic heat shock cre line (HS-GFPcre). The injected embryos of the HS-GFPcre at 24 hours postfertilization (hpf) were incubated for 1 hour at 38°C. At 48-72 hpf, the injected embryos were examined for GFP and RPF expression and raised to adulthood for generation of stable transgenic fish. The F1 embryos of the HS-GFPcre and GR double transgenic fish were also analyzed.
The Xenopus translation elongation factor 1α promoter (xef1a) expressed GFP ubiquitously in the GR plasmid-injected embryos and stable transgenic lines. Without the NLScre mRNA, the expression of RFP was effectively blocked by the STOP signal. Co-injection of the GR plasmid and the NLScre mRNA resulted in a robustly expression of RFP, indicating that the cre/loxP system worked remarkably well and the GR plasmid was a very useful for testing the efficiency of this site-directed recombination. Transgenic analysis of the GR plasmid in the HS-GFPcre line and the F1 of the HS-GFPcre and GR double transgenic fish showed a substantial reduction in the GFP signals and a strong expression of the RFP in the heat-treated embryos. These results demonstrated that the efficient induction of cre removed the GFP cassette and activated the RFP by site-directed recombination.
The cre/loxP system works well in zebrafish and can be efficiently combined with the inducible heat-shock promoter for temporal and spatial control of gene expression. The GR stable transgenic fish lines are useful for easily monitoring of this site-specific recombination and for developing ocular tissue-specific cre transgenic lines that can be utilized for disease modeling in zebrafish.
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