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Schlagwörter:
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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.
