Genetic Manipulation Protocols for C. elegans

We followed standard procedures for C. elegans maintenance and other genetic manipulations [64 (link)]. Nematodes were grown at 20 °C unless noted otherwise. The following strains, available at Caenorhabditis Genetics Center (CGC), were used: N2: Wild type Bristol isolate, DA2123: adIs2122 [lgg-1p::GFP::lgg-1 + rol-6(su1006)]), HZ589: bpIs151[sqst-1p::sqst-1::GFP + unc-76(+)], TU3401: sid-1(pk3321) V; uIs69 V, JJ2586: cox4(zu476[cox-4::eGFP::3xFLAG]) I, RW12185: atg-4.1(st12185[atg-4.1::TY1::EGFP::3xFLAG]), PS6187: syEx1155 [myo-3p::tomm-20::mRFP::3xMyc + Cbr-unc-119(+)]. Strains produced in this study by micro-injections [rol-6(su1006)] or microparticle bombardment [Cbr-unc-119(+)]: N2; Ex[psyx-17::SYX-17::mCherry;pRF4], PS6187: syEx1155 [myo-3p::tomm-20::mRFP::3xMyc + Cbr-unc-119(+)] + Ex[pmyo-3::VAMP-7::GFP;pRF4], DA2123: adIs2122 [lgg-1p::GFP::lgg-1 + rol-6(su1006)] + Ex[psyx-17::SYX-17::mCherry;pRF4], N2; Ex[puso-1::USO-1::GFP;pRF4], N2;Ex[psyx-17::SYX-17::mCherry + puso-1::USO-1::GFP;pRF4], N2;Ex[plgg-1::DsRed::LGG-1 + puso-1::USO-1::GFP;pRF4], unc-119 (ed 3) III; Ex[puso-1::USO-1::GFP+cb-unc-119(+)], unc-119 (ed 3) III; Ex[pmyo-3::SNB-6::dsRed+cb-unc-119(+)]. We noted that overexpression of snb-6 and vamp-7 by their endogenous promoters could not be tolerated by the animals, as we obtained F1 transgenics that were not able to produce viable progeny, thus the line could not be sustained. For syx-17 and uso-1, we obtained transgenics with expression under the endogenous promoter, however, their expression levels were low. Moreover, when two transgenes were co-injected, oftentimes they were not expressed in the same cell/tissue. Tissue-specific expression of mitoSNARE components in body wall muscles was better tolerated.