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A.C. Morris, J.M. Fadool; Progress on the Development of Transposon-Based Transgenic Technology for the Identification of Novel Retinal Genes . Invest. Ophthalmol. Vis. Sci. 2003;44(13):2272.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: The identification of new genes involved in the development of the visual system will be greatly facilitated by "enhancer trap" genetic screens employing the mariner transposable element from Drosophila. The mariner element is 1200 bp long, has inverted repeats (IRs) at its 5’ and 3’ termini, and codes for a single gene, the transposase. Mariner moves from one spot to another in the genome via a simple cut-and-paste mechanism; the transposase enzyme recognizes and binds to the terminal inverted repeats, creates staggered, double-strand cuts to excise the element, then catalyzes reintegration of the element at a new location. The transposon typically integrates into the genome at a TA dinucleotide. When the transposon excises, it leaves behind a 2-4 bp "footprint" from one of the transposon ends. Previously, insertion of mariner into the genome of the vertebrate model organism Danio rerio (the zebrafish) was demonstrated. However, it was not known if mariner could be mobilized from zebrafish genomic DNA once it had integrated. Methods: Zebrafish embryos homozygous for an insertion of the mariner transposon were injected at the 1-2 cell stage with transposase mRNA. DNA was extracted from surviving embryos at three days post fertilization and subjected to PCR to determine if transposon excision had occurred. DNA from reactions showing evidence of transposon excision was cloned and sequenced. In a parallel experiment, injected embryos were raised to adulthood to determine if heritable, germline excision had taken place. Results: Exposure of zebrafish embryos to transposase mRNA caused somatic excision of mariner from the zebrafish genome with a frequency of approximately 22%. DNA sequence at the site of excision contained a duplicated TA dinucleotide and a 3-4 bp transposon footprint, confirming that the mariner transposase induced bona fide transposon excision in zebrafish. Conclusions: Our findings demonstrate that the mariner transposon is capable of integration and excision from vertebrate genomic DNA in much the same way as it does in invertebrates, suggesting that it will be a useful tool for conducting genetic screens for retinal genes in this organism.
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