Abstract
Commonly used visible markers for transgenesis use fluorescent proteins expressed at the surface of the body, such as in eyes. One commonly used marker is the 3xP3-EGFP cassette containing synthetic binding sites for the eyeless/Pax6 conserved transcription factor. This marker cassette leads to fluorescent eyes in a variety of animals tested so far. Here we show that upon reaching adulthood, transgenic Bicyclus anynana butterflies containing this marker cassette exponentially loose fluorescence in their eyes. After 12 days, transgenic individuals are no longer distinguishable from wild type individuals. The decreased eye fluorescence is likely due to significantly decreased or halted eyeless/Pax6 expression observed in wild type animals upon adult emergence. Implications from these findings include care in screening transgenic animals before these reach adulthood, or shortly thereafter, and in using adult animals of the same age for quantitative screening of likely homozygote and heterozygote individuals.
Highlights
Transgenesis is a powerful technique to study the function or the regulatory information contained in DNA
Genomic insertions of test candidate DNA sequences are accompanied by the insertion of a marker gene that helps in the identification of transgenic individuals
Wild type as well as transgenic B. anynana butterflies containing the 3xP3-Enhanced Green Fluorescent Protein (EGFP) marker cassette were used for comparisons of eye brightness across time
Summary
Transgenesis is a powerful technique to study the function or the regulatory information contained in DNA. Genomic insertions of test candidate DNA sequences are accompanied by the insertion of a marker gene that helps in the identification of transgenic individuals. Some of the most widely used markers of transgenesis are fluorescent proteins, such as Enhanced Green Fluorescent Protein (EGFP), Ds Red and others. These are inert proteins that are clearly visualized upon light excitation in live organisms once expressed, and allow the clear identification of a transgenic organism [1,2,3]. An ideal regulatory sequence should lead to fluorescent marker gene expression early during development, at the surface of the body, in a small region of the body, PLOS ONE | DOI:10.1371/journal.pone.0132882. An ideal regulatory sequence should lead to fluorescent marker gene expression early during development, at the surface of the body, in a small region of the body, PLOS ONE | DOI:10.1371/journal.pone.0132882 July 14, 2015
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