|Harrell Ii, Robert - UNIV OF FL, GAINESVILLE|
Submitted to: Biotechniques
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: June 1, 2001
Publication Date: December 1, 2001
Citation: Handler, A.M., Harrell Ii, R.A. 2001. A Polyubiquitin-regulated DsRed marker for transgenic insects. Biotechniques. 31:820-828. Interpretive Summary: The ability to achieve gene transfer in economically important insects will provide a major tool for pest management. Improvement and expanded utilization of this technology to study and control insect pests depends on the discovery and analysis of efficient marking systems for the selection of transformed insects, for use as reporters of gene expression, and as genetic markers for detection of released insects. The primary marker used in insects is the green fluorescent protein (GFP) gene from a jellyfish, that is detected under ultraviolet light. Additional and improved markers are now necessary. Scientists from the Center for Medical, Agricultural and Veterinary Entomology, USDA, ARS, Gainesville, FL report the first testing in insects of the DsRed fluorescent protein from the coral, Discosoma striata. To test DsRed we used it in the piggyBac gene transfer system to genetically transform Drosophila melnaogaster, which were then tested for transformant selection. Additional transformations tested DsRed as a reporter in a gene expression system. For both transformant selection and reporter function, DsRed was compared to the GFP marker, and was shown to be highly effective and in some ways superior to GFP which is encouraging for its widespread use in insects.
Technical Abstract: Genetic transformation of most insect systems requires dominant-acting markers that do not depend on reverting a mutant phenotype in a host strain, and for this purpose GFP has proven to be useful in several insect orders. However, detection of multiple transgenes and reporters for gene expression requires the development of new visible markers that can be unambiguously detected when co-expressed with GFP. The DsRed fluorescent protein has spectral characteristics that are most distinct from GFP and GFP variants and we have explored the use of DsRed as a selectable marker for piggyBac transformation in Drosophila melanogaster, and its use as a reporter when co-expressed with GFP. Transformants marked with polyubiquitinregulated DsRedl were detected at a relatively high frequency throughout development, and they exhibited brighter fluorescence than transformants marked with EGFP. Use of a Texas Red filter set eliminated detection of EGFP fluorescence and autofluorescence, and DsRed expressed from a reporter construct could be unambiguously detected when co-expressed with EGFP. DsRed should prove to be a highly efficient marker system for the selection of transformant insects and as a reporter in gene expression studies.