Nebuilder system

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.

A human renal cell carcinoma cell line, UM-RC-2 cell (ECACC 08090511), was obtained and cultured as described in the Experimental Model And Subject Details section. Full-length cDNA fragments of the human APLN gene were inserted in pcDNA6-myc/His A plasmids (Thermo Fisher Scientific) using the NEbuilder system (E5520, New England Biolabs, MA, USA) to generate human APLN-overexpressed UM-RC-2 cells. Sanger sequencing was then used to confirm this inserted sequence. The plasmid containing human APLN cDNA and the corresponding empty vector was transfected as well into UM-RC-2 cells using FuGene HD (E2311, Promega, WI, USA) according to the manufacture’s protocol. 48 h after the transfection, Blasticidin (R21001, Thermo Fisher Scientific) was added to the culture medium for the drug selection of transfected cells.