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Silencing cuticular pigmentation genes enables RNA FISH in intact chemosensory appendages

MPG-Autoren
http://pubman.mpdl.mpg.de/cone/persons/resource/persons187908

Pentzold,  Stefan
Department of Bioorganic Chemistry, Prof. Dr. W. Boland, MPI for Chemical Ecology, Max Planck Society;

http://pubman.mpdl.mpg.de/cone/persons/resource/persons76357

Grabe,  Veit
BMBF Research Group Dr. S. Sachse, Olfactory Coding, MPI for Chemical Ecology, Max Planck Society;

http://pubman.mpdl.mpg.de/cone/persons/resource/persons220741

Ogonkov,  Andrei
Department of Bioorganic Chemistry, Prof. Dr. W. Boland, MPI for Chemical Ecology, Max Planck Society;

http://pubman.mpdl.mpg.de/cone/persons/resource/persons185801

Schmidt,  Lydia
Department of Bioorganic Chemistry, Prof. Dr. W. Boland, MPI for Chemical Ecology, Max Planck Society;

http://pubman.mpdl.mpg.de/cone/persons/resource/persons3812

Boland,  Wilhelm
Department of Bioorganic Chemistry, Prof. Dr. W. Boland, MPI for Chemical Ecology, Max Planck Society;

http://pubman.mpdl.mpg.de/cone/persons/resource/persons3825

Burse,  Antje
Department of Bioorganic Chemistry, Prof. Dr. W. Boland, MPI for Chemical Ecology, Max Planck Society;

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BOL698.pdf
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Zitation

Pentzold, S., Grabe, V., Ogonkov, A., Schmidt, L., Boland, W., & Burse, A. (in press). Silencing cuticular pigmentation genes enables RNA FISH in intact chemosensory appendages. bioRxiv. doi:10.1101/318535.


Zitierlink: http://hdl.handle.net/21.11116/0000-0001-464B-D
Zusammenfassung
Optical imaging of gene expression by RNA-fluorescent in situ hybridisation (FISH) in whole-mount sensory appendages of insects is often impeded by their highly pigmented cuticle. Since most chemical bleaching agents are incompatible with imaging fluorescentlabelled nucleotides, we developed a RNA interference-based method for clearing cuticular pigmentation that allows imaging of fluorescent mRNA in whole-mount appendages of insects. Silencing key genes of the tyrosine-derived pigmentation pathway by injecting dsRNA of laccase2 or tyrosine hydroxylase in two leaf beetles species (Chrysomela populi, Phaedon cochleariae) resulted in clearance of the highly pigmented cuticle and in significant decreased light absorbance. Intact chemosensory appendages (palps, antennae and legs) from RNAi-cleared individuals were used to image expression and spatial distribution of antisense mRNA of two chemosensory genes (gustatory receptor, odorant-binding protein) via RNA FISH and confocal laser scanning microscopy. Imaging of these genes did neither work for RNAi-controls (dsGfp) due to retained pigmentation, nor for FISH-controls using sense mRNA. Furthermore, we show that several chemical bleaching agents are not feasible with FISH, either due to significant degradation of polynucleotides, lack of clearing efficacy or long incubation times. Overall, silencing pigmentation genes is a significant improvement over bleaching agents allowing fluorescence imaging in whole-mount appendages and organs.