Rescuing ATAT-2 Expression in C. elegans

For atat-2 rescues with the native promoter, the atat-2 locus (including promoter, open reading frame, and 3’ UTR) was PCR amplified from N2 genomic DNA. Primer sequences used for cloning are available upon request. For atat-2 rescue with mechanosensory neuron promoters, atat-2 cDNA was amplified from C. elegans RNA and TOPO cloned into pCR8 Gateway entry vector (Invitrogen) to generate pBG-GY896. pBG-GY896 was recombined into a destination vector containing the mec-7 promoter, pBG-GY119, to generate pBG-GY897 (P_mec-7ATAT-2). Site-directed mutagenesis was performed on pBG-GY896 to change two glycine residues into tryptophan (G125W and G127W) resulting in pBG-GY898. Mutation of these conserved glycine residues in the ATAT-2 paralog MEC-17 (G121W and G123W) was previously shown to render MEC-17 catalytically inactive (Topalidou et al., 2012 (link)). After mutagenesis, pBG-GY898 was recombined with the Pmec-7 destination vector to yield pBG-GY899 (P_mec-7ATAT-2 dead). For expression of SYD-2::mScarlet, syd-2 genomic DNA was cloned from C. elegans and TOPO cloned into pCR8 Gateway entry vector to generate pBG-GY699. pBG-GY699 was recombined into a destination vector containing the mec-7 promoter and a C-terminal mScarlet tag, pBG-GY880, to generate pBG-GY936 (P_mec-7SYD-2::mScarlet).