Fig 1.
Schematic representation of the GAL4/GeneSwitchGal4-UAS systems.
(A) The GAL4-UAS system allows spatial control of gene expression. (B) The GeneSwitch (GS) system allows temporal control of gene expression thanks to a modified GAL4 protein that is active only when the synthetic progesterone analogue (mifespristone, RU-486) binds to the fused progesterone steroid receptor. In absence of RU-486, GAL4 activity is maintained at a minimum. Pictures of Drosophila male and female where obtained from: https://commons.wikimedia.org/wiki/File:Biology_Illustration_Animals_Insects_Drosophila_melanogaster.svg.
Fig 2.
No significant leak is detected when expressing protein coding transgenes using GS.
(A) qPCR expression data of NDI1 in induced vs. non-induced flies. Controls without the RNAi transgene are also included (n = 3). (B) Western blot analysis of NDI1 levels in induced vs non-induced flies. (C) Quantification of B (n = 2). (D) Western blot analysis of AOX expression driven by two (++) or one (+) copy of daughterless-GeneSwitch GAL4 (daGS) in flies carrying two (++) or one copy (+) of the AOX transgene in induced vs non-induced groups. (E) Quantification of D (n = 3). (F) Western blot analysis of NDI1 expression driven by two (++) or one (+) copy of daughterless-GeneSwitch GAL4 (daGS) in flies carrying two (++) or one copy (+) of the NDI1 gene in induced vs non-induced groups. (G) Quantification of F (n = 3). (H) Western blot analysis of AOX expression driven by two (++) or one (+) copy of tubulin-GeneSwitch GAL4 (tubGS) in flies carrying two (++) or one copy (+) of the AOX gene in induced vs non-induced groups. (I) Quantification of H (n = 3). (J) Western blot analysis of NDI1 expression driven by two (++) or one (+) copy of tubulin-GeneSwitch GAL4 (tubGS) in flies carrying two (++) or one copy (+) of the NDI1 gene in induced vs non-induced groups. (K) Quantification of J (n = 3). GAPDH or Tubulin is shown as loading control. +/- indicate presence/absence of the transgene or 500 μM RU-486 (inducer).
Fig 3.
Expression of protein coding transgenes correlates with the presence of RU-486 in the fly food.
(A) Western blot analysis of ND-42-HA levels after feeding flies RU-486 for 5 days and after withdrawal of the drug from the food. (B) Quantification of A (n = 3). (C) Western blot analysis of LacZ levels driven by daGS or tubGS GAL4 in induced vs non-induced groups. (D) Quantification of C (n = 3). Tubulin is shown as loading control. daGS, daughterless-GeneSwitch GAL4; tubGS, tubulin-GeneSwitch GAL4; +/- indicate presence/absence of the transgene or 500 μM RU-486 (inducer).
Fig 4.
Use of qPCR to validate knock-down of a gene using GS requires additional controls.
(A) qPCR expression data of ND-39 (CI subunit) in induced vs non-induced groups (n = 3). (B) qPCR expression data of UQCR-C1 (CIII subunit) in induced vs. non-induced groups (n = 3). (C) qPCR expression data of COX5B (CIV subunit) in induced vs. non-induced groups (n = 3). (D) qPCR expression data of Bellwether (CV subunit) in induced vs. non-induced groups (n = 3). daGS, daughterless-GeneSwitch GAL4; tubGS, tubulin-GeneSwitch GAL4; +/- indicate presence/absence of the transgene or 500 μM RU-486 (inducer).
Fig 5.
Expression of RNAi transgenes does not correlate with the presence of RU-486 in the fly food.
(A) Western blot analysis of ND-39, NDI1 and ATP5A levels in control, induced and non-induced groups. (B) Quantification of A (n = 2). (C) Respirometry in tubGS flies with and without an ND-39 RNAi transgene (n = 3–8). (D) Western blot analysis of ND-39 and NDI1 levels in control, induced and non-induced groups. (E) Quantification of D (n = 2–3). (F) Western blot analysis of ND-39 levels in controls and experimental flies fed with RU-486 during development (1 μM), development and adulthood (1 μM and 500 μM respectively) or exclusively during adulthood (500 μM) (n = 2–3). All flies were 5 days old when proteins were extracted. Flies that were exclusively fed during development spent 5 days in food without RU-486. GAPDH is shown as loading control. daGS, daughterless-GeneSwitch GAL4; tubGS, tubulin-GeneSwitch GAL4; +/- indicate presence/absence of the transgene or 500 μM RU-486 (inducer).
Fig 6.
No difference in protein levels between induced and non-induced groups using a strong GS driver.
(A) Western blot analysis of ND-19 levels in control (without the RNAi transgene), induced and non-induced groups. (B) Quantification of A (n = 3). (C) Western blot analysis of ND-39 levels in control (without the RNAi transgene), induced and non-induced groups. (D) Quantification of C (n = 3). (E) Western blot analysis of Sod2 levels in control (without the RNAi transgene), induced and non-induced groups. (D) Quantification of E (n = 3). Tubulin is shown as loading control. daGS, daughterless-GeneSwitch GAL4; tubGS, tubulin-GeneSwitch GAL4; +/- indicate presence/absence of the transgene or 500 μM RU-486 (inducer).