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.
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