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

Chai, Cynthia M.

Gharib, Shahla

Liu, Jonathan

Pokala, Navin

Schwarz, Erich M.

Sternberg, Paul W.

Wang, Han

Caltech Authors - Main

 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. 
Nature Methods: doi:10.1038/nmeth.4109
 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. 
 
 
Nature Methods: doi:10.1038/nmeth.4109
 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. 
 
Nature Methods: doi:10.1038/nmeth.4109
 
 
Supplementary Information  
 
cGAL, a Temperature-Robust GAL4-UAS System for C. elegans 
Han Wang*, Jonathan Liu*, Shahla Gharib, Erich M. Schwarz, Navin Pokala, and Paul W. 
Sternberg 
* These authors contributed to this work equally. 
 
 
 
 
 
 
 
 
  
Nature Methods: doi:10.1038/nmeth.4109
 
 
 
Supplementary Note 1 | Vector Map of the driver construct pHW393 
(pHW393) Prab-3::nls::GAL4-SK(DBD)::VP64::let-858 3’UTR. New driver constructs can be 
generated by cloning new promoters into the MCS, between SphI/NotI/FseI and 
AscI/BssHII/BamHI/SmaI/XmaI/MscI. The backbone of pHW393 (between SphI and KpnI) is 
derived from the plasmid pPD117.01 from the Fire lab C. elegans Vector Kit 1997. pPD117.01 
has a 5’ decoy and the let-858 3'UTR. The 5’ decoy contains a splice acceptor (decoy intron) 
and a short terminated coding region (decoy ORF) upstream of the MCS, which was designed 
to catch any upstream transcription with a 3’ splice site. Adding this 5’ decoy sequence was 
shown to decrease ectopic expression in the posterior gut (A. Fire, personal communication). 
We highly recommend using the pPD117.01 vector for new neuronal driver constructs. 
  
Nature Methods: doi:10.1038/nmeth.4109
 
 
 
 
Supplementary Note 2 | Sequence of the driver construct pHW393 
(pHW393) Prab-3::nls::GAL4-SK(DBD)::VP64::let-858 3’UTR, is available from Addgene 
(www.addgene.org; plasmid no. 85583). The color scheme here matches the vector map of 
pHW393 shown in Supplementary Note 1.  
Nature Methods: doi:10.1038/nmeth.4109
 
 
 
Supplementary Note 3 | Vector map of the effector construct pHW394 
 (pHW394) 15xUAS::Δpes-10::gfp::let-858 3'UTR.New effector constructs can be generated by 
cloning new effector genes into the MCS, between SalI/HincII/KpnI/AgeI and EcoRI/AccIII. The 
backbone of pHW393 (between SphI and KpnI) is derived from the plasmid pPD117.01 from the 
Fire lab C. elegans Vector Kit 1997. pPD117.01 has a 5’ decoy and the let-858 3'UTR. The 5’ 
decoy contains a splice acceptor (decoy intron) and a short terminated coding region (decoy 
ORF) upstream of the MCS, which was designed to catch any upstream transcription with a 3’ 
splice site. Adding this 5’ decoy sequence was shown to decrease ectopic expression in the 
posterior gut (A. Fire, personal communication). 
Nature Methods: doi:10.1038/nmeth.4109
 
 
 
 
 
Supplementary Note 4 | Sequence of the effector construct pHW394 
(pHW394) 15xUAS::Δpes-10::gfp::let-858 3'UTR is available from Addgene (www.addgene.org; 
plasmid no. 85584).The color scheme here matches the vector map of pHW394 shown in 
Supplementary Note 3. 
 
  
Nature Methods: doi:10.1038/nmeth.4109
 
 
Supplementary Note 5 | Information for extrachromosomal arrays and integrants 
The co-injection markers used include KP708 (Pttx-3::rfp), KP1369 (Pmyo-2::nls::mCherry), 
KP1106 (Pmyo-2::nls::gfp), unc-119(+) rescue plasmid, Pofm-1::rfp and Punc-122::gfp.  
All initial descriptions of extrachromosomal arrays (syEx####) and integrants (syIs####) are 
highlighted for convenience. All integrants were generated by X-ray irradiation. 
A full list of the available integrated drivers and effectors are listed in Supplementary 
Table 1. 
syEx1452 [15xUAS::Δpes-10::gfp::unc-54 3'UTR, 25ng/µL; Pttx-3::rfp, 40ng/µL; pBlueScript, 35 
ng/µL], injected into N2, used to generate syIs300 and syIs302. 
syEx1431 and syEx1432 [Pmyo-2::GAL4SC::VP16::unc-54 3’UTR, 10ng/µL; unc-119(+), 
50ng/µL; pBlueScript, 40 ng/µL], injected into the strain unc-119(ed3); syIs300. 
syEx1433 and syEx1434 [Pmyo-2::GAL4SC::VP64::unc-54 3’UTR, 10ng/µL; unc-119(+), 
50ng/µL; pBlueScript, 40 ng/µL], injected into the strain unc-119(ed3); syIs300. 
syEx1435 and syEx1436 [Pmyo-2::GAL4SK::VP64::unc-54 3’UTR; 10ng/µL, unc-119(+), 
50ng/µL; pBlueScript, 40 ng/µL], injected into the strain unc-119(ed3); syIs300. 
syEx1437 and syEx1438 [Pmyo-2::gfp::unc-54 3’UTR, 10ng/µL; unc-119(+), 50ng/µL; 
pBlueScript, 40 ng/µL], injected into the strain unc-119(ed3). 
syEx1448 and syEx1449 [Pnlp-40::GAL4SK::VP64::unc-54 3’UTR, 10ng/µL; Pmyo-
2::nls::mCherry, 10ng/µL; pBlueScript, 80ng/µL], injected into syIs302. syEx1449 was used to 
generate syIs318, syIs319 and syIs320 as intestine drivers. 
syEx1450 and syEx1451 [Pmyo-3::GAL4SK::VP64::unc-54 3’UTR, 10ng/µL; Pmyo-
2::nls::mCherry, 10ng/µL; pBlueScript, 80ng/µL], injected into syIs302. syEx1451 was used to 
generate syIs321 as the body wall muscle driver. 
syEx1471 [Punc-47::GAL4SK::VP64::unc-54 3’UTR, 60ng/µL; Pofm-1::rfp, 40ng/µL], syEx1471 
was used to generate syIs322, syIs323, syIs324 and syIs325, as GABAergic neuron drivers 
(These GABAergic drivers were weak, we suggest using drivers built in the pPD117.01 
backbone with the let-858 3’UTR). 
syEx1475, syEx1476, and syEx1477 [5xUAS::Δpes-10::gfp::unc-54 3'UTR, 25ng/µL; unc-
119(+), 50ng/µL; pBlueScript, 25 ng/µL], injected into the strain unc-119(ed3); syIs301. 
syEx1478 and syEx1479 [10xUAS::Δpes-10::gfp::unc-54 3'UTR, 25ng/µL; unc-119(+), 50ng/µL; 
pBlueScript, 25 ng/µL], injected into the strain unc-119(ed3); syIs301. 
syEx1480 and syEx1481 [15xUAS::Δpes-10::gfp::unc-54 3'UTR, 25ng/µL; unc-119(+), 50ng/µL; 
pBlueScript, 25 ng/µL ], injected into the strain unc-119(ed3); syIs301. 
Nature Methods: doi:10.1038/nmeth.4109
 
 
syEx1482 and syEx1483 [20xUAS::Δpes-10::gfp::unc-54 3'UTR, 25ng/µL; unc-119(+), 50ng/µL; 
pBlueScript, 25 ng/µL], injected into the strain unc-119(ed3); syIs301. 
syEx1443 and syEx1444 [15xUAS::Δpes-10::aex-2(+) cDNA::unc-54 3'UTR, 25ng/µL; Pmyo-
2::nls::gfp, 10ng/µL; pBlueScript, 65 ng/µL], injected into the strain aex-2(sa3).  
syEx1433 and syEx1447 [Prab-3::GAL4SK::VP64::let-858 3’UTR, 10 ng/µL; Pofm-1::rfp, 
40ng/µL; pBlueScript, 50 ng/µL], injected into N2. syEx1447 was used to generate syIs334, 
syIs335 and syIs336 as pan-neuronal driver lines. 
syEx1430[Pmyo-2::GAL4SC::VP64::unc-54 3’UTR; 10ng/µL, Pofm-1::rfp 40ng/µL; 1kb DNA 
ladder(NEB), 150 ng/µL], also used to generate the syIs301 as the pharyngeal muscle driver. 
syEx1488 [15xUAS::Δpes-10::gfp::let-858 3’UTR, 25 ng/µL; Pttx-3::rfp, 50 ng/µL; 1 kb ladder 
(NEB), 125 ng/µL], injected into N2, used to generate syIs337 and syIs390 for 
15xUAS::gfp::let-858 3’UTR effector lines.  
syEx1484 [Punc-17:: GAL4SK::VP64::let-858 3’UTR, 25 ng/µL; Punc-17::mCherry, 25 ng/µL; 
unc-119(+), 50 ng/µL], injected into the strain syIs390; unc-119(ed3).  
 
syEx1485 [Punc-47::GAL4SK::VP64::let-858 3’UTR, 25 ng/µL; Punc-47:: mCherry, 25 ng/µL; 
unc-119(+), 50 ng/µL], injected into the strain syIs390; unc-119(ed3).  
 
syEx1486 [Peat-4::GAL4SK::VP64::let-858 3’UTR, 25 ng/µL; Peat-4:: mCherry, 25 ng/µL; unc-
119(+), 50 ng/µL], injected into the strain syIs390; unc-119(ed3). 
 
syEx1460 [15xUAS::Δpes-10::hChR2(H134R)::eyfp::let-858 3'UTR, 25ng/µL; Pttx-3::rfp, 
40ng/µL; pBlueScript, 35 ng/µL],  injected into N2, used to generate syIs340, syIs341 and 
syIs342 for 15xUAS::hChR2(H134R)::eyfp::let-858 3’UTR effector lines. 
syEx1487 [Punc-47::GAL4SK::VP64::let-858 3’UTR, 25 ng/µL; Pofm-1::rfp, 40 ng/µL; 1 kb ladder 
(NEB), 35 ng/µL], injected into the strain syIs341. 
  
Nature Methods: doi:10.1038/nmeth.4109
 
 
Supplementary Note 6 | Strains used in the study 
For detailed information about arrays and integrants, see Supplementary Note 5 and 
Supplementary Table 1.  
Wild type N2 
PS6041 unc-119(ed3) III 
Figure 1b, 1c and Supplementary Figure 1a:  
PS6843 syIs300 V 
PS6932 unc-119(ed3); syIs300 
PS6900 syEx1431; unc-119; syIs300  
PS6901 syEx1432; unc-119(ed3); syIs300  
PS6902 syEx1433; unc-119(ed3); syIs300  
PS6903 syEx1434; unc-119(ed3); syIs300  
Figure 1d, 1e and Supplementary Figure 1b 
PS6844 syIs301 V 
PS6964 unc-119(ed3); syIs301 
PS7007 syEx1475; unc-119(ed3); syIs301 
PS7008 syEx1476; unc-119(ed3); syIs301 
PS7009 syEx1477; unc-119(ed3); syIs301 
PS7010 syEx1478; unc-119(ed3); syIs301 
PS7012 syEx1480; unc-119(ed3); syIs301 
PS7013 syEx1481; unc-119(ed3); syIs301 
PS7014 syEx1482; unc-119(ed3); syIs301 
PS7015 syEx1483; unc-119(ed3); syIs301 
Figure 2 and Supplementary Figure 2: 
PS6902 syEx1433; unc-119(ed3); syIs300 
PS6903 syEx1434; unc-119(ed3); syIs300 
PS6904 syEx1435; unc-119(ed3); syIs300 
PS6905 syEx1436; unc-119(ed3); syIs300 
PS6906 syEx1437; unc-119(ed3)  
PS6907 syEx1438; unc-119(ed3)   
Figure 3a-3f: 
PS6933 syIs318 syIs302 III 
PS7067 syIs321; syIs300 
PS6987 syIs337; syIs334 
PS7149 syIs390 
PS7184 syIs390; unc-119(ed3) 
PS7018 syEx1484; syIs390; unc-119(ed3)  
Nature Methods: doi:10.1038/nmeth.4109
 
 
PS7019 syEx1485; syIs390; unc-119(ed3)  
PS7020 syEx1486; syIs390; unc-119(ed3)  
Figure 3h: 
JT3 aex-2(sa3) X 
PS6975 syEx1443; aex-2(sa3) 
PS6976 syEx1444; aex-2(sa3) 
PS6936 syIs321 
PS6935 syIs320 
PS6938 syIs323 
The exact genotypes used for the quantification of the defecation assay in Figure 3h are (from 
left to right): 
N2 
aex-2(sa3) 
syEx1444; aex-2(sa3) 
syIs323/+; aex-3(sa3) 
syEx1444; syIs323/+; aex-2(sa3) 
syEx1444; syIs321/+; aex-2(sa3) 
syEx1444; syIs320/+; aex-2(sa3) 
 
Figure 3i and Supplementary Video 1-2: 
PS7021 syEx1487; syIs341 
PS7044 syIs341 
Supplementary Figure 3 and Supplementary Video 3-5: 
PS6872 syIs302 III  
PS6844 syIs301 V  
PS6965 syIs301; syIs302 
PS7186 syIs407 
PS7154 syIs391 IV 
PS7136 syIs378 V 
PS7190 syIs409 X 
PS7167 syIs396 syIs337 III 
PS7192 syIs413 IV 
PS6936 syIs321 
PS7205 syIs424 III 
PS7199 syIs371 
PS7201 syIs421 
Nature Methods: doi:10.1038/nmeth.4109


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

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

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