title: Simplified method for cell-specific gene expression analysis in Caenorhabditis elegans.
abstract: In the neural circuit functional identities of individual neurons are mainly specified by their differential gene expression patterns. Unveiling functional roles of each neuron requires cell-specific interrogation of neural circuitry in the context of gene expressions. The mRNA tagging strategy in Caenorhabditis elegans is a powerful technique, in which cell-specific transcripts can be isolated by co-immunoprecipitating the complexes of mRNAs and epitope-tagged poly(A) binding protein (3× FLAG-PAB-1), expressed in target neurons. However, the conventional protocol requires laborious and time-consuming procedures; chromosomal integration of gene encoding 3× FLAG-PAB-1 and bleaching of obtained integrant animals for the isolation of huge amounts of synchronized animals. In this paper, we have presented a simplified methodology for cell-specific mRNA tagging analysis in C. elegans. We show that mRNA tagging was achieved using transgenic animals expressing 3× FLAG-PAB-1 as an extrachromosomal array under the control of the flp-18 promoter, without the chromosomal integration procedure. Furthermore, we successfully isolated cell-specific mRNAs from adult transgenic animals synchronously grown from eggs laid by gravid adults during a time window of 3h. This simplification facilitates the implementation of cell-specific gene expression analysis of C. elegans, which contributes to the understanding of neural circuitry at a cell-specific resolution.
authors.affiliation: Institute for Integrated Cell-Material Sciences (WPI-iCeMS), Kyoto University, Yoshida-Honmachi, Sakyo-ku, Kyoto 606-8501, Japan; PRESTO, Japanese Science and Technology Agency, 4-1-8 Honcho, Kawaguchi, Japan. Electronic address: tsugi@icems.kyoto-u.ac.jp.
authors.forename: Takuma
authors.initials: T
authors.lastname: Sugi
authors.affiliation: Institute for Integrated Cell-Material Sciences (WPI-iCeMS), Kyoto University, Yoshida-Honmachi, Sakyo-ku, Kyoto 606-8501, Japan.
authors.forename: Yasuko
authors.initials: Y
authors.lastname: Ohtani
mesh_terms: D000818:Animals; D017173:Caenorhabditis elegans / Q000235:genetics / Q000378:metabolism*; D029742:Caenorhabditis elegans Proteins / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D020869:Gene Expression Profiling / Q000379:methods*; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism*; D009474:Neurons / Q000166:cytology* / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics*; D013194:Staining and Labeling
chemical_list: D029742:Caenorhabditis elegans Proteins; D009419:Nerve Tissue Proteins; D012333:RNA, Messenger
keywords: C. elegans; Cell-specific mRNA tagging; Gene expressions