Temporal and spatial control of transgene expression using laser induction of the hsp70 promoter

BACKGROUND: Precise 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. RESULTS: We 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. CONCLUSION: This 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

Distal-less regulates eyespot patterns and melanization in bicyclus butterflies

Butterfly eyespots represent novel complex traits that display substantial diversity in number and size within and across species. Correlative gene expression studies have implicated a large suite of transcription factors, including Distal-less (Dll), Engrailed (En), and Spalt (Sal), in eyespot development in butterflies, but direct evidence testing the function of any of these proteins is still missing. Here we show that the characteristic two-eyespot pattern of wildtype Bicyclus anynana forewings is correlated with dynamic progression of Dll, En, and Sal expression in larval wings from four spots to two spots, whereas no such decline in gene expression ensues in a four-eyespot mutant. We then conduct transgenic experiments testing whether over-expression of any of these genes in a wild-type genetic background is sufficient to induce eyespot differentiation in these pre-patterned wing compartments. We also produce a Dll-RNAi transgenic line to test how Dll down-regulation affects eyespot development. Finally we test how ectopic expression of these genes during the pupal stages of development alters adults color patters. We show that over-expressing Dll in larvae is sufficient to induce the differentiation of additional eyespots and increase the size of eyespots, whereas down-regulating Dll leads to a decrease in eyespot size. Furthermore, ectopic expression of Dll in the early pupal wing led to the appearance of ectopic patches of black scales. We conclude that Dll is a positive regulator of focal differentiation and eyespot signaling and that this gene is also a possible selector gene for scale melanization in butterflies. © 2013 Wiley Periodicals, Inc

Tunable porous photonic bandgap structures for chemical and biological sensing

The development of porous nanostructured materials, such as polymer Bragg gratings, offer an attractive and unique platform for chemical and biological recognition elements. Much of the efforts in polymeric gratings have been focused on holographic polymer dispersed liquid crystal (H-PDLC) gratings with demonstrated applications in switching, lasing, and display devices. Here, we present the application of porous polymer photonic bandgap structures produced using a modified holographic method that includes a solvent as a phase separation fluid. The resulting gratings are simple to fabricate, stable, tunable, and highly versatile. Moreover, these acrylate porous polymer photonic bandgap structures were generated using a simple one-beam setup. In this paper, we describe the application of these nanoporous polymer gratings as a general template for biochemical recognition elements. As a prototype, we developed an oxygen (O ) sensor by encapsulating the fluorophore (tris(4,7-diphenyl-1,10-phenathroline)ruthenium(II) within these nanostructured materials. Thus, the obtained O sensors performed through the full-scale range (0%-100%) with a response time of less than 1 second. Most importantly, the use of the inherent property of these gratings to transmit or reflect a particular wavelength spectrum, based on the grating spacing, enables us to selectively enhance the detection efficiency for the wavelengths of interest