Pcfj1662

C. elegans expression plasmids utilized the Pmec-7 promoter from the mec-7 gene for touch neuron expression[68 (link)]. Vector details are available upon request. Cloning was performed using the Gateway in vitro recombination system (Invitrogen, Carlsbad, CA) using Grant lab-modified versions of MiniMos enabled vectors pCFJ1662 (Hygromycin resistant; Addgene #51482) and pCFJ910 (G418 resistant; Addgene #44481) (gifts of Erik Jorgensen, University of Utah): pCFJ1662 Pmec-7 GTWY mNeonGreen let858 (34F6); pCFJ1662 Pmec-7 mNeonGreen GTWY let858 (34D4); pCFJ1662 Pmec-7 GTWY oxGFP let858 (36G3); pCFJ910 Pmec-7 mScarleti GTWY let858 (33B6); and pCFJ910 pmec-7 GTWY mScarleti (35D2). pDONR221 entry vectors containing coding regions for rab-7, lmp-1, lgg-1, clic-1, snx-1, rme-8, were recombined into neuronal destination vectors by Gateway LR clonase II reaction to generate C-/N- terminal fusions. Single-copy integrations were obtained by MiniMOS technology [69 (link)].

Plasmids for C. elegans coleomocyte expression were produced by standard methods including in vitro recombination via the Gateway system (ThermoFisher) and/or Gibson Assembly using the Nebuilder system (NEB). Plasmid backbones were pCFJ1662 and pCFJ910 (Addgene) and used promoters from the snx-1 or cup-4 genes. Our minimos protocol for single copy transgene integration is based on a protocol found on wormbuilder.org as described in [39 (link)]. The gonad arms of first day gravid adults were microinjected with plasmid mixtures including 10ng/ul drug resistant expression plasmid (G418 or Hygromycin) [40 (link),41 (link)] 10ng/μl pGH8, 2.5ng/μl pCFJ90, 65ng/μl pCFJ601, and 10ng/ul pMA122. 2–3 injected animals per plate were incubated at 25°C, with selection drug added between 24–72 hrs post injection. Plates were screened for single copy integrated transformants after a minimum of 10 days of growth, focused on populations that survive drug selection and lack extrachromosomal arrays visualized with the mCherry co-injection markers. Candidate single-copy integrants were passaged on drug containing plates and analyzed for expression. Lines displaying 100% transmission of the expressed transgene without drug selection were frozen and used for experiments.

Plasmids for C. elegans hyp7-specific expression used the promoter from semo-1/Y37A1B.5 (P_hyp-7) [120 (link)]. Vector details are available upon request. Cloning was performed using the Gateway system (Invitrogen) and modified versions of hygromycin-resistant and miniMos-enabled vector pCFJ1662 (gift from Erik Jorgensen, University of Utah; Addgene #51482). pDONR221 entry vectors containing coding regions for rab-5, rab-7, mig-14, tgn-38, aman-2, daf-4, and sma-6 were transferred into hyp7 destination vectors with the Gateway LR clonase II reaction to generate C-/N-terminal fusions. Single-copy integrations were obtained using miniMos technology [121 (link)].