Comparison of tetracycline and temperature sensitive GAL80 transgenes with UAS-grim

In order to assess the ability of GAL80 to repress the expression of a UAS transgene, lethality tests were performed in presence of a UAS-grim reporter (Genesis 34:34, 2002, PeerJ 5:e4167, 2017). The Grim reporter gene encodes a strong pro-apoptotic factor, thus the lethality across development (embryonic, larval, pupal) can be scored to assess the repression ability of GAL80. The UAS-grim lethality test facilitates the determination of the developmental period during which GAL4 activity is not inhibited.

Two different muscle-specific GAL4 drivers, Mef2-Gal4 (BDSC:27390) and DJ694 (BDSC: 8176), were each crossed with a second chromosome insertion of UAS-grim (Cell Death Differ 5:930,1998), UAS-grim recombined with third chromosome insertions of GAL80TET (3.3+1077, PeerJ 5:e4167), and UAS-grim recombined with third chromosome insertions of GAL80ts (BDSC:7017). Negative control (no lethality) was generated by crossing the UAS-grim strain with a strain without any GAL4 transgene (w1118,Cell 36:469, 1984 ). Crosses were maintained, and eggs were collected at 20˚C (Experimental workflow). Parents were obtained from multiple independent cultures to set-up independent crosses (biological replicates, Set indicates parents from the same culture). Crosses were kept 24 to 48h in culture tubes to allow mating before transferring them to egg collectors. Multiple egg collections were done for each parental set (technical replicates, Date indicates dates of collection). Parents were allowed to lay eggs for 12h to 16h. Up to 25 eggs were aligned on a slice of food and up to 4 slices were used for a given collection from a set. Slices of food are then transferred to culture vials and incubated at the indicated temperature. The scoring of the number of first-instar larvae (L1) was done by scoring the number of empty eggs 25-30h (29˚C) or 43-48h (20˚C) after egg alignment. The number of pupae and adults was scored 5-6 days (29˚C) or 8-10 days (20˚C) after the scoring of the previous stage.