Figure S7. Generation and Characterization of Worms Overexpressing Dicer in the Intestine, Related to Figure 6(A) Endogenous expression of Dicer in the worm intestine and neurons. Microscopy images of L3-L4worms expressing Dicer::GFP fusion protein under the control of Dicer endogenous promoter.(B) Generation of transgenic worms overexpressing Dicer in the intestine from extra-chromosomal arrays comprised of multiple copies of a construct containing the Dicer gene under the transcriptional regulation of an intestine-specific promoter
ges-1 (
pges-1::
dcr-1).(C) Levels of Dicer mRNA and the miRNAs
lin-4,
let-7 and miR-231 are increased in day 3
pges-1::
dcr-1transgenic worms. Multiple independent lines were assessed in this experiment.(D) Intestinal Dicer overexpression prevents the decline of Dicer mRNA expression with aging in worms.Dicer mRNA expression was determined in day 3 (D3) and day 11 (D11) transgenic worms using RT-qPCR.* P < 0.05 vs. D3; # P < 0.05 vs. control. Pools of at least 30 worms were used for the RT-qPCR analyses.(E) Representative figure of a compilation of independent lifespan experiments with the
pges-1::
dcr-1line 6 and control worms at different temperatures (n=19-170). For statistics refer to Table S4.(F) Survival of worms on 50 mM paraquat (n=19-56 per group). For statistics refer to Table S4.(G) and (H) Functional characterization of worms overexpressing Dicer in the intestine. (G) Schematic for generation of strains to test functionality of the Dicer constructs and to generate Dicer rescue strains,i.e. Dicer mutant strains with rescued expression of Dicer in the intestine (
pges-1::
dcr-1) or in the tissues where Dicer is constitutively expressed (
pdcr-1::
dcr-1). (H) Fluorescent microscopy images of L3-L4worms after heat shock (two shocks of 33C for 5h spanned by one day period). Note that worms in which Dicer expression was rescued in the intestine, RNAi is completely restored, as assessed by double stranded RNA-mediated GFP knockdown exclusively in the intestine (arrows).(I) Survival at 30C of worms carrying a Dicer loss-of-function mutation [
dcr-1(
ok247)] and intestinal(
pges-1::
dcr-1) or whole body (
pdcr-1:
dcr-1) Dicer rescue constructs (n=147-204 per group). * P < 0.05vs.
dcr-1 mutant.(J) Levels of
lin-4 in young
pges-1::
lin-4 and
plin-4::
lin-4 transgenic worms. Data are obtained from pools of 30 worms per group. Multiple independent lines were assessed in this experiment.(K) Survival of worms after 21h on 5 mM arsenite (n=5 pools of 10 worms per group). Multiple independent lines were assessed in this experiment. This experiment was repeated twice and in both cases showed identical patterns. * P < 0.05 vs. control.(L) Expression of stress response genes in
pges-1::
dcr-1 and control worms (n=2-5 pools of 30 worms per group). Genes were chosen based on previous reports in the literature that highlight their importance in stress response in worms (Hars et al., 2007; Murphy et al., 2003; Oliveira et al., 2009). Experiments with transgenic lines were performed in parallel with a co-injection marker control line, in which worms were germline transformed with the plasmids pUN24 and pJK590 only. Error bars, SEM.