TABLE 2

DMPP resistance and developmental speed in animals with induced tissue-specific daf-2 expression

Genotype% dauera (no. of animals, experiments)DMPPRb (no. of animals, experiments)L2 duration in hr (no. of lines scored)c
+0 ± 0 (66, 3)0 ± 0 (130, 5)11
daf-2(m41)100 ± 0 (236, 10)98 ± 2 (87, 6)23
Control (Pdpy-30-FRT-mCherry-terminator-FRT-gfp-SL2-daf-2)
daf-2(m41); krEx837-840 (four lines)85 ± 5 (169, 6)97 ± 1 (214, 5)22 (2)
Induced panneuronal daf-2 (Pdpy-30-FRT-mCherry-terminator-FRT-gfp-SL2-daf-2; Prab-3-flp)
daf-2(m41); krEx705,707 (two lines)65 ± 8d (277, 11)99 ± 1 (147, 3)22 (2)
Induced intestinal daf-2 (Pdpy-30-FRT-mCherry-terminator-FRT-gfp-SL2-daf-2; Pvha-6-flp)
daf-2(m41); krEx691,693 (two lines)64 ± 7d (278, 12)99 ± 1 (140, 5)25 (1)
Induced hypodermal daf-2 (Pdpy-30-FRT-mCherry-terminator-FRT-gfp-SL2-daf-2; Pdpy-7-flp)
daf-2(m41); krEx704,746 (two lines)43 ± 6d (268, 10)99 ± 1 (177, 3)24 (2)
Induced panneuronal, intestinal, and hypodermal daf-2 (Pdpy-30-FRT-mCherry-terminator-FRT-gfp-SL2-daf-2; Prab-3-flp; Pvha-6-flp; Pdpy-7-flp)
daf-2(m41); krEx841-844 (four lines)38 ± 5d (241, 3)99 ± 0 (313, 4)22 (2)
  • FLP-inducible daf-2 constructs were designed essentially as in Davis et al. (2008), using the Multisite Gateway system from Invitrogen (Carlsbad, CA). The transgenes in the “off” configuration express the mCherry reporter under the control of ubiquitous dpy-30 promoter, and the let-858 transcriptional terminator prevents transcription of the downstream elements. Downstream of the transcriptional terminator is an artificial operon leading to the expression of gfp and daf-2 mRNA in the same cells, where the GFP coding sequence is fused to the SL2 splice leader acceptor site (Gendrel et al. 2009), followed by the daf-2 cDNA. FLP expression under one of the tissue-specific promoters brings the gfp and daf-2 sequences under the control of the tissue-specific promoter. In all the strains above, the Pdpy-30-FRT-mCherry-terminator-FRT-gfp-SL2-daf-2 construct was injected at 10 ng/μl and the FLP-expressing constructs were injected at 5 ng/μl into daf-2(m41) animals. These low concentrations were used because of high toxicity observed with transgenes expressing DAF-2 both conditionally and nonconditionally (data not shown).

  • a Eggs were laid at 15° overnight. Adults were removed and plates were shifted to 25°. Percentage of non-dauer L4 or adult animals was scored 72 hr postshift by visual inspection.

  • b Percentage of survival on 0.75 mm DMPP at 20° (average ± SEM).

  • c L2 stage duration at 20° was determined as in Ruaud and Bessereau (2007).

  • d P ≤ 0.05 when compared to the Pdpy-30-FRT-mCherry-terminator-FRT-gfp-SL2-daf-2 control lines (Student's t-test).