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  Silencing cuticular pigmentation genes enables RNA FISH in intact insect appendages

Pentzold, S., Grabe, V., Ogonkov, A., Schmidt, L., Boland, W., & Burse, A. (2018). Silencing cuticular pigmentation genes enables RNA FISH in intact insect appendages. Journal of Experimental Biology, 221: jeb185710. doi:10.1242/jeb.185710.

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http://dx.doi.org/10.1242/jeb.185710 (Publisher version)
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 Creators:
Pentzold, Stefan1, Author           
Grabe, Veit2, Author           
Ogonkov, Andrei1, Author           
Schmidt, Lydia1, Author           
Boland, Wilhelm1, Author           
Burse, Antje1, Author           
Affiliations:
1Department of Bioorganic Chemistry, Prof. Dr. W. Boland, MPI for Chemical Ecology, Max Planck Society, ou_24028              
2Microscopy Service, Dr. Veit Grabe, MPI for Chemical Ecology, Max Planck Society, ou_3171478              

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 Abstract: Optical imaging of gene expression by fluorescence in situ hybridisation (FISH) in insects is often impeded by their pigmented cuticle. Since most chemical bleaching agents are incompatible with FISH, we developed a RNA interference-based method for clearing cuticular pigmentation which enables using whole-mount body appendages for RNA FISH. Silencing laccase2 or tyrosine hydroxylase in two leaf beetles species (Chrysomela populi, Phaedon cochleariae) cleared their pigmented cuticle and decreased light absorbance. Subsequently, intact appendages (palps, antennae, 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). Imaging did neither work for RNAi-controls due to retained pigmentation, nor for FISH-controls (sense mRNA). Several bleaching agents were incompatible with FISH, either due to degradation of RNA, lack of clearing efficacy or long incubation times. Overall, silencing pigmentation genes is a significant improvement over bleaching agents enabling FISH in intact appendages.

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 Dates: 2018-05-102018-07-192018-09-26
 Publication Status: Published in print
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 Identifiers: Other: BOL698
DOI: 10.1242/jeb.185710
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Project name : Marie Skłodowska-Curie
Grant ID : 705151
Funding program : Horizon 2020 (H2020)
Funding organization : European Commission (EC)

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Title: Journal of Experimental Biology
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Publ. Info: London : Published for the Company of Biologists Ltd. by the Cambridge University Press
Pages: - Volume / Issue: 221 Sequence Number: jeb185710 Start / End Page: - Identifier: ISSN: 0022-0949
CoNE: https://pure.mpg.de/cone/journals/resource/110992357319088