cGAL, a temperature-robust GAL4–UAS system for Caenorhabditis elegans
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.
Additional Information
© 2016 Macmillan Publishers Limited. Received 22 May; Accepted 18 November; Published online 19 December 2016. We are grateful to A. Fire (Stanford University) for sharing unpublished results and to C.T. Hittinger (University of Wisconsin-Madison), D. Sieburth (University of Southern California), E.M. Jorgensen (University of Utah), C.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. Data availability: The source data used for the plots in the main and supplementary figures are included in the supplementary materials. Other data are also available from the corresponding author upon request. The coding sequence of Gal4p from Saccharomyces kudriavzevii is in GenBank (GU299177.1). The plasmids for cGAL driver (pHW393, plasmid no. 85583) and effector (pHW394, plasmid no. 85584) are available from Addgene. Source data for Figures 1, 2, 3 and Supplementary Figures 1 and 2 are available online. Author 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. The authors declare no competing financial interests.Attached Files
Accepted Version - nihms831958.pdf
Supplemental Material - nmeth.4109-S1.pdf
Supplemental Material - nmeth.4109-S2.xlsx
Supplemental Material - nmeth.4109-S3.xlsx
Supplemental Material - nmeth.4109-SF1.jpg
Supplemental Material - nmeth.4109-SF2.jpg
Supplemental Material - nmeth.4109-SF3.jpg
Supplemental Material - nmeth.4109-sv1.mp4
Supplemental Material - nmeth.4109-sv2.mp4
Supplemental Material - nmeth.4109-sv3.mp4
Supplemental Material - nmeth.4109-sv4.mp4
Supplemental Material - nmeth.4109-sv5.mp4
Files
| Name | Size | Download all |
|---|---|---|
|
md5:8048461ccb6514b5f2a6c86633eaf50c
|
25.0 kB | Download |
|
md5:d8e1f36bea0d0e753992e18e03771f30
|
6.7 MB | Download |
|
md5:dd8ab43eb84ba4e344a339c235fbbcf2
|
2.4 MB | Preview Download |
|
md5:8392f3d438585e75380ea72aa7ee445e
|
2.7 MB | Download |
|
md5:433a1165165064e013cb1b028e31cef7
|
7.5 MB | Download |
|
md5:4e6fd8ba66f3e144cf488fbcdf724f75
|
144.9 kB | Preview Download |
|
md5:7ddcaffa6b9b9302d605a978ab3888f4
|
433.4 kB | Preview Download |
|
md5:3a31cc15ec706b0af97e6c12e084334d
|
7.5 MB | Download |
|
md5:cac26db61a7f3a6d023a9c86685cc7d5
|
1.8 MB | Download |
|
md5:790d3760774b5c271dab262b4d894e04
|
1.0 MB | Preview Download |
|
md5:debdbe50626e49ea6edb7a6116ac5614
|
101.2 kB | Download |
|
md5:cc29b6271263a578f2f2cade6aa06f98
|
102.6 kB | Preview Download |
Additional details
- PMCID
- PMC5693259
- Eprint ID
- 72124
- Resolver ID
- CaltechAUTHORS:20161117-125604575
- NIH
- P40 OD010440
- Caltech Beckman Institute
- Arnold and Mabel Beckman Foundation
- Della Martin Foundation
- NIH Predoctoral Fellowship
- T32GM007616
- Howard Hughes Medical Institute (HHMI)
- Created
-
2016-12-20Created from EPrint's datestamp field
- Updated
-
2021-11-11Created from EPrint's last_modified field