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cGAL, a temperature-robust GAL4–UAS system for Caenorhabditis elegans

Nature Methods volume 14, pages 145–148 (2017)Cite this article

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Abstract

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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Figure 1: Optimization of activation domain and UAS copy number.
Figure 2: Designing a temperature-robust GAL4 driver via evolutionary analysis.
Figure 3: Functional studies with the cGAL system.

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Acknowledgements

We are grateful to A. Fire (Stanford University) for sharing unpublished results and to C.T. Hittinger (University of Wisconsin-Madison), D. Sieburth (University of Southern California), E.M. Jorgensen (University of Utah), C.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.

Author information

Author notes

  1. Erich M Schwarz

    Present address: Present address: Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York, USA.,

  2. Han Wang and Jonathan Liu: These authors contributed equally to this work.

Authors and Affiliations

  1. Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, California, USA

    Han Wang, Jonathan Liu, Shahla Gharib, Cynthia M Chai, Erich M Schwarz & Paul W Sternberg

  2. Howard Hughes Medical Institute, California Institute of Technology, Pasadena, California, USA

    Han Wang, Jonathan Liu, Shahla Gharib, Cynthia M Chai, Erich M Schwarz & Paul W Sternberg

  3. Department of Life Sciences, New York Institute of Technology, Old Westbury, New York, USA

    Navin Pokala

Authors
  1. Han Wang
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Contributions

H.W., J.L. and P.W.S. conceived the project. H.W. and J.L. performed the experiments, analyzed the data and wrote the paper. S.G. helped with molecular cloning and strain handling. E.M.S. devised the idea of trying Gal4p from yeast species with lower growth temperatures. C.M.C. and N.P. contributed reagents.

Corresponding author

Correspondence to Paul W Sternberg.

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Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Neither driver nor effector alone displays expression of GFP

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

Supplementary Figure 2 Performance of different DBDs from Gal4 proteins at room temperature

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

Supplementary Figure 3 Functional verification of integrated effectors

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 information

Supplementary Text and Figures

Supplementary Figures 1–3 and Supplementary Notes 1–6. (PDF 2321 kb)

Supplementary Table 1

Integrated cGAL drivers and effectors (XLSX 24 kb)

Supplementary Table 2

Plasmids and oligos used in this study (XLSX 98 kb)

Functional verification of a ChR2 effector with a GABAergic driver

Blue light induces paralysis in transgenic animals carrying a GABAergic driver and a channelrhodopsin (ChR2) effector (MP4 6543 kb)

Negative control of a ChR2 effector without driver

No response to blue light in transgenic animals only carrying a channelrhodopsin (ChR2) effector (MP4 7305 kb)

Functional verification of a GCaMP6s effector with a body wall muscle driver

Calcium imaging in body wall muscles of animals carrying a body wall muscle driver and a GCaMP6s::SL2::mKate2 effector (MP4 2681 kb)

Functional verification of a HisCl1 effector with a body wall muscle driver

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)

Functional verification of a TeTx effector with a GABAergic driver

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)

Source data

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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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