N ethyl n nitrosourea

This screen was performed by mutagenizing a strain (AMJ174) with the transgene T (oxSi487[mex-5p::mCherry::H2B::tbb-2 3’UTR::gpd-2 operon::GFP::H2B::cye-1 3’UTR + unc-119(+)], (29 (link))) silenced for >200 generations after introducing a mutation in lin-2(jam30) (sgRNA (P1), primers (P2, P3, P4) using Cas9-mediated genome editing of AMJ844 (iT; dpy-2(e8), (29 (link))) while correcting the dpy-2(e8) mutation to wild type (creating dpy-2(jam29); sgRNA (P5), primers (P6, P7, P8)). The lin-2 mutation limits brood size (61 (link)) and facilitates screening. Near-starved animals (P0) of all life stages were mutagenized using 1mM N-Ethyl-N-Nitrosourea (ENU, Toronto Research Chemicals) for 4–6 hours. Mutagenized animals were washed four times with wash buffer (0.01% Triton X-100 in M9) and 2–3 adult animals were placed on NG plates seeded with OP50. Over the next 3 weeks, F1, F2, and F3 progeny were screened to isolate mutants that show mCherry fluorescence. These animals were singled out (up to 7 animals from each P0 plate) and tested for the persistence of expression in descendants. Of the 15 viable mutants isolated using this primary screen, five with mutations in mut-16 were analyzed in this study.