Abstract
BackgroundPrecise temporal and spatial regulation of transgene expression is a critical tool to investigate gene function in developing organisms. The most commonly used technique to achieve tight control of transgene expression, however, requires the use of specific DNA enhancers that are difficult to characterize in non-model organisms. Here, we sought to eliminate the need for this type of sequence-based gene regulation and to open the field of functional genetics to a broader range of organisms.ResultsWe have developed a new laser mediated method to heat shock groups of cells that provides precise spatio-temporal control of gene expression without requiring knowledge of specific enhancer sequences. We tested our laser-system in a transgenic line of Bicyclus anynana butterflies containing the EGFP reporter gene attached to the heat sensitive hsp70 promoter of Drosophila melanogaster. Whole organismal heat shocks demonstrated that this Drosophila promoter can drive gene expression in butterflies, and the subsequent laser heat shocks showed that it was possible to activate cell-specific gene expression in very precise patterns on developing pupal wings.ConclusionThis laser-mediated gene expression system will enable functional genetic investigations, i.e., the ectopic expression of genes and their knock-down in targeted groups of cells in model and non-model organisms with little or no available regulatory data, as long as a compatible heat-shock promoter is used and the target tissue is accessible to a laser beam. This technique will also be useful in evolutionary developmental biology as it will enable the study of the evolution of gene function across a variety of organisms.
Highlights
Precise temporal and spatial regulation of transgene expression is a critical tool to investigate gene function in developing organisms
The use of transgenic animals has helped to produce major advances in the field of functional genetics. These experiments use only model organisms with known regulatory DNA sequences, i.e. enhancers, that drive gene expression at particular times in development and in particular cells. While transposable elements such as piggyBac have enabled transgenic manipulations of increasingly diverse organisms[1], the lack of versatile tools for genetic manipulations in these organisms has hindered their use in functional genetic experiments
Dramatic genetic manipulations utilizing the widely used yeast GAL4/UAS system have increased our understanding of gene function [3]
Summary
Precise temporal and spatial regulation of transgene expression is a critical tool to investigate gene function in developing organisms. The use of transgenic animals has helped to produce major advances in the field of functional genetics. These experiments use only model organisms with known regulatory DNA sequences, i.e. enhancers, that drive gene expression at particular times in development and in particular cells. Researchers must create extensive transgenic lines to bring the GAL4 transcription factor under the control of an appropriate regulatory sequence. This is both time and labor intensive and requires maintenance of a large number of animal stocks. Modifications to the traditional GAL4/UAS system can eliminate the requirement for specific GAL4 lines but do not reproduce the precise spatial control of transgene expression[4]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
