The GAL4–UAS system is a powerful tool for manipulating gene expression, but its application in Caenorhabditis elegans has not been described. Here we systematically optimize the system's three main components to develop a temperature-optimized GAL4–UAS system (cGAL) that robustly controls gene expression in C. elegans from 15 to 25 °C. We demonstrate this system's utility in transcriptional reporter analysis, site-of-action experiments and exogenous transgene expression; and we provide a basic driver and effector toolkit.
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We are grateful to A. Fire (Stanford University) for sharing unpublished results and to C.T. Hittinger (University of Wisconsin-Madison), D. Sieburth (University of Southern California), E.M. Jorgensen (University of Utah), C. Bargmann (Rockefeller University) and A. Fire (Stanford University) for reagents. We thank H. Korswagen (Hubrecht Institute) for thoughtful discussion. N.P. thanks C. Bargmann for her support. We thank M. Bao, Y.M. Kim, D. Leighton, J. DeModena and G. Medina for technical assistance; and WormBase for technical support. We also thank M. Kato, H. Schwartz, D. Angeles-Albores and other members of the Sternberg lab for editorial comments on the manuscript. Some strains were provided by the CGC, which is funded by the NIH Office of Research Infrastructure Programs (grant P40 OD010440). Some imaging was performed at the Caltech Biological Imaging Facility with the support of the Caltech Beckman Institute and the Arnold and Mabel Beckman Foundation. H.W. is supported by the Della Martin Fellowship. J.L. was supported by NIH grant T32GM007616. This work is supported by the Howard Hughes Medical Institute, with which P.W.S. is an investigator.
The authors declare no competing financial interests.
Integrated supplementary information
Comparison of GFP fluorescence in a driver- only strain (a) or an effector-only strain (b) to their driver + effector combination controls. One-tailed t-test with Welch's correction. Source data
Quantification of GFP fluorescence in the pharynx of transgenic worms with either Pmyo‑2::GAL4SC::VP64 or Pmyo‑2::GAL4SK::VP64 drivers injected into a strain carrying an integrated 15xUAS::gfp transgene (syIs300) at room temperature (22‑23°C). The drivers were both injected at 10 ng/μL. Strains with a direct Pmyo‑2::gfp fusion array at 10 ng/μL was measured for comparison. Two independent lines were imaged for each genotype. n = 20 ‑ 30 for each line. Bars are mean ± SEM. * p<0.05. ns, not significant. One‑way ANOVA with Tukey’s post-test. a.u., artificial units. Source data
Expression of integrated drivers (left column) or integrated effectors alone (middle column) shows no basal expression. Only the combination (right column) show expression of cytoplasmic or nuclear-localized reporters, or death of appropriate cells. DIC, Differential interference contrast; Green, green filter; Red, red filter. Scale bar is 20 μm.
Supplementary Figures 1–3 and Supplementary Notes 1–6. (PDF 2321 kb)
Integrated cGAL drivers and effectors (XLSX 24 kb)
Plasmids and oligos used in this study (XLSX 98 kb)
Blue light induces paralysis in transgenic animals carrying a GABAergic driver and a channelrhodopsin (ChR2) effector (MP4 6543 kb)
No response to blue light in transgenic animals only carrying a channelrhodopsin (ChR2) effector (MP4 7305 kb)
Calcium imaging in body wall muscles of animals carrying a body wall muscle driver and a GCaMP6s::SL2::mKate2 effector (MP4 2681 kb)
Expressing a histamine-gated chloride channel HisCl1 in body wall muscle induces flaccid paralysis on histamine plates. Worms with either the driver or the effector alone fail to respond to histamine (MP4 7339 kb)
Expressing a tetanus toxin light chain (TeTx) in GABAergic neurons blocks neurotransmission and leads to the characteristic “shrink” phenotype. Transgenic worms with either the driver or the effector don't display the “shrink” phenotype (MP4 1785 kb)
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Wang, H., Liu, J., Gharib, S. et al. cGAL, a temperature-robust GAL4–UAS system for Caenorhabditis elegans. Nat Methods 14, 145–148 (2017). https://doi.org/10.1038/nmeth.4109
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