Article ID | Journal | Published Year | Pages | File Type |
---|---|---|---|---|
10754858 | Biochemical and Biophysical Research Communications | 2014 | 5 Pages |
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 (3Ã FLAG-PAB-1), expressed in target neurons. However, the conventional protocol requires laborious and time-consuming procedures; chromosomal integration of gene encoding 3Ã 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 3Ã 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 3Â h. 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.
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Authors
Takuma Sugi, Yasuko Ohtani,