M165fc fluorescence dissecting microscope

Synchronized L1 animals were obtained by harvesting eggs from hypochlorite-treated gravid adults and overnight starvation in M9 solution (15 (link)). Hatched L1 larvae were transferred on NGM plates seeded with either E. coli OP50 or HT115 bacteria (16 (link)). In order to insure complete RNAi before DSB induction, L1 worms were cultured at 20°C for at least 20 h. Heat-shock driven I-SceI expression was induced by putting the worms at 34°C for 60–180 min, as indicated. After the heat-shock procedure, worms were cultured at 20°C to allow DSB formation, DSB repair and worm development. NHEJ activity was measured by scoring pharyngeal GFP expression using a Leica M165FC fluorescence dissecting-microscope. Experiments were performed in triplicate with 50–200 animals tested for each condition. After GFP quantification, ∼25 adult animals were transferring onto microscope slides with 3% agarose pads and representative pictures were acquired using a Leica DM6000 microscope with 10× objective. SSA activity was measured by scoring animals showing LacZ positive cells in non-pharyngeal somatic tissues (17 (link)). One hour prior fixation/LacZ staining, young adults were heat-shocked at 34°C for 120 min to induce SSA reporter expression.

Dual reporter larvae (XF540) were mutagenized with ethyl methanesulfonate (EMS) using standard procedures (14 (link)). Complex F2 populations, each derived from 50 mutagenized P0s, were bleached and synchronized L1 larvae (F3) were seeded on NGM/OP50 plates. On two consecutive days larvae were heat-shocked at 34°C for 180 min in order to maximize GFP ORF correction. GFPlow F3 animals were selected using a Leica M165FC fluorescence dissecting-microscope and clonal F4 populations were tested again for NHEJ activity. Populations showing reduced GFP expression were fixed and stained with X-gal as described previously (17 (link)).

Dual reporter larvae defective for thoc-5 (XF677) were mutagenized with ethyl methanesulfonate (EMS) using standard procedures (14 (link)). Complex F2 populations, each derived from 10 mutagenized P0s, were bleached and synchronized L1 larvae (F3) were seeded on NGM/OP50 plates. Larvae were heat-shocked for 140 min, which was determined to be the optimal heat exposure to distinguish all NHEJ proficient worms spiked into a NHEJ deficient population. GFPhigh F3 animals were selected using a Leica M165FC fluorescence dissecting-microscope and clonal F4 populations were tested again for NHEJ activity. Populations showing GFP expression were fixed and stained with X-gal as described previously.