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Shunichi Yoshikawa, Xinping C. Zhao; Retrotransposon-based Transgenesis in Zebrafish. Invest. Ophthalmol. Vis. Sci. 2011;52(14):2354.
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© ARVO (1962-2015); The Authors (2016-present)
Transgenic technology has been powerful genetic tools for zebrafish as an emerging model animal for molecular genetics and eye disease studies. Transgenic zebrafish is generally produced by injecting DNA to fertilized eggs, which typically results in highly mosaic integration of the transgene. Therefore, somatic transgenic analysis is difficult in the injected animals. We aim to develop new non-viral, RNA-based transgenic vectors that integrate more uniformly into the zebrafish chromosome than the DNA-based method so that somatic analysis can be performed in the injected animals. This new technology can be used to study development and diseases in the zebrafish eye. The purpose of this study is to examine if an RNA vector derived from a zebrafish LINE retrotransposon functions to generate transgenic zebrafish.
We cloned and sequenced the zebrafish retrotransposon CR1-2_DR, and found all 16 clones had 1 or 2 deletions and multiple point mutations. The reverse transcriptase (RT) gene was reconstructed in vitro. The marker cassette including the ubiquitous bactin1 promoter and GFP was inserted between the RT and the 3' UTR in the inverse direction (RTiG3U). The RTiG3U RNA vector, modified vectors lacking RT (iG3U) or 3' UTR (RTiG) and the RT mRNA were in vitro transcribed by T7 RNA polymerase. The RNA vectors were injected in 1-cell-stage embryos and the GFP signal in the embryo was observed under fluorescence microscope at 3 days post-fertilization or later.
Four groups of injected embryos (RTiG3U, iG3U, RTiG and iG3U plus RT mRNA) were tested and 88% (N=24), 0% (N=54), 0% (N=152) and 38% (N=50) showed GFP expression, respectively. These results indicate that the reconstructed zebrafish RT works in vivo and the 3' UTR is required for integration and reporter expression. One of 2 injected fish showed germline transmission on preliminary screening.
We reconstructed an autonomous retrotransposon that can be used for both somatic and germline transgenic fish. The 3' UTR is essential for integration and the endogenous RT level, if any, is not at a detectable level in transient transgenic analysis. Transgenic efficiency and compatibility with inducible and ocular cell type-specific promoters will be examined in the future.
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