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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NCBI Reference Sequence

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We are grateful to A. Fire (Stanford University) for sharing unpublished results and to C.T. Hittinger (University of Wisconsin-Madison), D. Sieburth (University of Southern California), E.M. Jorgensen (University of Utah), C.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

    • Erich M Schwarz

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

    • Han Wang
    •  & Jonathan Liu

    These authors contributed equally to this work.

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

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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.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Paul W Sternberg.

Integrated supplementary information

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–3 and Supplementary Notes 1–6.

Excel files

  1. 1.

    Supplementary Table 1

    Integrated cGAL drivers and effectors

  2. 2.

    Supplementary Table 2

    Plasmids and oligos used in this study

Videos

  1. 1.

    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

  2. 2.

    Negative control of a ChR2 effector without driver

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

  3. 3.

    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

  4. 4.

    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

  5. 5.

    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

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DOI

https://doi.org/10.1038/nmeth.4109

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