Description
Several bipartite systems have been described for use with
C. elegans
(Wei
et al.
, 2012; Wang
et al.
, 2017; Wang
et al.
, 2018; Mao
et al.
, 2019; Nonet, 2020)
.
However, little manipulation of the drivers or reporters has been performed to optimize them for
C. elegans
. Mao. et. al. 2019 demonstrated that the QF activation domain is a more potent activator than either VP64 or the hybrid VP64-p65-Rta tripartite activatorVPR. Here, I describe modifications of linker region between the activation domains of both GAL4
SK
and TetR drivers that increase the activity of these reporters. Although these studies are not comprehensive, I opted to describe them herein because the modification of the GAL4
SK
driver increases the activity of this driver substantially such that it is now on par with the LexA, TetR and QF2 drivers.
I used an efficient RMCE protocol (Nonet, 2020) to create the transgenic animals. Modified versions of a
mec-4
promoter GAL4
SK
-QF
AD
, GAL4
SK
-VP64 and TetR-QF
AD
constructs were created in an RMCE integration vector using a Golden Gate cloning approach, then integrated on Chr IV using a standard injection protocol. After outcrossing to the appropriate reporter, the expression level of GFP at steady state in PLM and ALM soma of L4 animals was quantified (Figure 1).
Previously, I described drivers consisting of
C. elegans
codon optimized synthetic GAL4
SK
, TetR and LexA DNA binding domains fused to the QF activation domain based on the observations of Mao.
et al.
2019. Although the GAL4
SK
construct was modestly active, the LexA and TetR construct were incapable of activating the reporter (supplemental methods of Nonet, 2020). Replacement of a 49 amino acid portion of central domain of QF, which separates the TetR and LexA DNA binding domains and the QF activation domain in the original constructs, with a 12 amino acid flexible linker converted both into much stronger drivers than the GAL4
SK
-QF
AD
driver (Nonet, 2020). Here I show that insertion of a similar linker also greatly increases the potency of the GAL4
SK
-QF
AD
driver. I also extended the linker of the TetR-L-QF
AD
and this further improved activity of this driver. In Nonet, 2020, I also tested the functionality of a GAL4
SK
-VP64 construct (Wang
et al.
2017) in single copy and found it was incapable of expressing the GFP reporter. To test if the failure of GAL4
SK
-VP64 was also the result of insufficient domain separation, I inserted a 40 amino acid flexible linker in between GAL4 and VP64. Although the linker containing driver activates transcription, it does so extremely poorly in comparison to the GAL4
SK
-QF
AD
drivers. I speculate this is likely due to the loss of the MED25 subunit of the mediator complex in the nematode lineage (Grants
et al.
, 2015). VP64 (a 4X VP16) is known to activate transcription in part through interaction with MED25 (Mittler
et al.
, 2003) and the loss of this interaction could account for the observed weak activation properties of VP64 and related activators in worms (Mao
et al.
2019 and herein).
In addition to the modification of the linker domain of the transgenes I characterize herein, some of the transgenes also differ in other ways that could theoretically impact my conclusions. First, some driver transgenes contain a
tbb-2
3′ UTR and others use an
act-4
3′ UTR. I consider it very unlikely these differences impact GFP expression for two reasons. First, my lab has previously demonstrated that
mec-4
promoter driven GFP-C1 transgenes employing the
tbb-2
3′ UTR and the
act-4
3′ UTR express at very similar levels in ALM and PLM (Dour and Nonet, 2021). More importantly, I previously demonstrated that the activity of the both a GAL4-QF
AD
driver and a LexA-L-QF
AD
driver are insensitive to dosage of the driver (Figure 5 of Nonet, 2020). Specifically, GFP levels observed in GAL4
SK
/+; UAS::GFP ~= GAL
SK
; UAS::GFP. Thus, the level of expression of the driver is unlikely to be determining the GFP signal level. Rather, I speculate that GAL4
SK
is saturating the UAS binding sites in all transgenes and that the inherent activation properties of the driver determines the expression level.
Second, all drivers were integrated at
jsTi1493
except for the inactive linkerless TetR-QF
AD
driver. I present data on integration of NMp3730
(mec-4p tetR-QF
AD
tbb-2 3′ UTR)
at
jsTi1490
(Nonet, 2020). I also previously created insertions of the same plasmid at
jsTi1493
IV and
jsTi1492
II (
jsSi1531
[mec-4p tetR-QF
AD
tbb-2 3′] IV
and
jsSi1534 [mec-4p tetR-QF
AD
tbb-2 3′] II
; Table S1 and Table S5 in Nonet, 2020). The GFP expression was undetectable in
jsSi1531/+; jsSi1519 [7X tetO ∆pes-10p GFP]/+
and
jsSi1534/+;jsSi1519/+
double heterozygote animals. However, only the
jsTi1490
transgene was homozygosed with the reporter. Thus, I quantified that insertion. However, this difference is highly unlikely to impact my findings as the other two insertions are qualitatively completely inactive.
The improvements to GAL4
SK
-QF
AD
by insertion of a flexible linker is an important addition to the
C. elegans
bipartite expression toolkit since the GAL4 system is so extensively developed in
Drosophila
. Using multi-copy lines and a VP64 activator, Wang
et al.
(2018) have already shown that the split GAL4
SK
system is functional in worms. Incorporation of a similar flexible linker and a QF activation domain into those tools should permit development of a robust single copy split-GAL4
SK
system. Other GAL4 system tools previously developed for
Drosophila
such as GAL80ts and GAL4-PR tools (Caygill and Brand, 2016) which provide temporal control in addition to spatial control could also easily be incorporated into the worm toolbox.
In addition, further manipulation of the size and properties of the linker domain separating the DNA binding domain and activation domains could yield drivers with even stronger activation proper which would likely also be applicable to the LexA, QF and Tet ON/OFF driver/reporter systems.
Methods
Methods
C. elegans
was maintained on NGM agar plates spotted with OP50 at 22.5°C or at 25°C during the RMCE protocol.
RMCE transgenesis
Inserts were cloned into pLF3FShC (Addgene # 153083; Nonet, 2020) and injected at ~50 ng/µl into
jsTi1493
young adults. Integrants were identified and isolated as described in detail in Nonet (2020). The GAL4
SK
drivers were crossed to
jsSi1518 [11X UAS ∆pes-10 GFP-C1]
and the TetR strains were crossed to
jsSi1543 [7X tetO ∆mec-7p GFP-C1]
.
Microscopy
For quantification of GFP signals, homozygous L4 hermaphrodite animals were mounted on 2% agar pads in a 2 µl drop of 1mM levamisole in phosphate buffered saline and imaged on an Olympus (Center Valley, PA) BX-60 microscope equipped with a Qimaging (Surrey, BC Canada) Retiga EXi monochrome CCD camera, a Lumencor AURA LED light source, Semrock (Rochester, NY) GFP-3035B and mCherry-A-000 filter sets, and a Tofra (Palo Alto, CA) focus drive, run using micro-manager 2.0ß software (Edelstein
et al.
, 2014) using a 40X air lens at 20% LED power with 50 ms exposures. PLM soma and ALM soma signals were quantified using the FIJI version of ImageJ software (Schindelin
et al.
, 2012) as described in Nonet (2020).
Plasmid constructions
Integration vectors were assembled using Golden Gate (GG) reactions as described in Nonet (2020). Other plasmids were constructed using standard cloning techniques.
The following were used:
NMp3055 DR274 U6
(Table S3 and Supplemental methods of Nonet, 2020)
NMp3401 DR274 CT linker
NMp3055 was digested with EcoRI and HindIII and the double stranded (ds) oligonucleotide NMo5948/49 was ligated into the vector.
NMp3403 DR274 CT-FP linker
NMp3055 was digested with EcoRI and HindIII and the ds oligonucleotide NMo5952/53 was ligated into the vector.
NMp3498 DR274 FP linker
NMp3403 was modified by DpnI-mediated mutagenesis using oligonucleotides NMo6120/21.
NMp3610 syn Gal4
SK
DB
(Table S3 and Supplemental methods of Nonet, 2020)
NMp3617 pSAP mec-4p GAL
SK
-VP64
(Table S3 and Supplemental methods of Nonet, 2020)
NMp3643 pLF3FShC
Available at Addgene. (Table S3 and Supplemental methods of Nonet, 2020)
NMp3735 DR274 5’arm- CT mec-4p
(Table S3 and Supplemental methods of Nonet, 2020)
NMp3736 DR274 TGG GGT mec-4p
(Table S3 and Supplemental methods of Nonet, 2020)
NMp3751 DR274 AAG GTA act-4 3’UTR
(Table S3 and Supplemental methods of Nonet, 2020)
NMp3777 DR274 3’arm tbb-2 UTR
(Table S3 and Supplemental methods of Nonet, 2020)
NMp3808 DR274 CT-NT linker-QF
AD
(Table S3 and Supplemental methods of Nonet, 2020)
NMp3821 DR274 FP tetR(iS)-L-QF
AD
(called DR274 FP tetR(iS)-L-QF in Table S3 and Supplemental methods of Nonet, 2020)
NMp3876 pLF3FShC mec-4p GAL4
SK
-L-QF
AD
NMp3736, NMp3610, NMp3808 and NMp3777 were co assembled into NMp3643 using a SapI GG reaction.
NMp4045 DR274 FP tetR-LL-QF
AD
NMp3821 was amplified with oligonucleotides NMo6867 and NMo7056, DpnI digested, purified, kinased, and religated.
NMp4048 pLF3FShC mec-4p tetR-LL-QF
AD
act-4
NMp3735, NMp4045, and NMp3751 were co assembled into NMp3643 using a SapI GG reaction.
NMp4049 pLF3FShC mec-4p GAL4
SK
-L4-VP64 tbb-2 3’UTR
NMp3736, NMp3610, NMp3401, NMp3498, NMp3777, the ds oligonucleotide NMo6866/67, and VP64 as a PCR product amplified from NMp3617 using oligonucleotides NMo6404/7057 were co assembled into NMp3643 using a SapI GG reaction.
Oligonucleotides
NMo number
|
Sequence
|
5948 |
AATTGCTCTTCgGCGGGCAGCGGTGGCAGTGGAGGTACCGGCGGAAGCGGTATGcGAAG |
5949 |
AGCTCTTCgCATACCGCTTCCGCCGGTACCTCCACTGCCACCGCTGCCCGCcGAAGAGC |
5952 |
AATTGCTCTTCgGGTGGCAGCGCTGGAGGTACCGGCGGTAGTGCCGGAGGCACGcGAAG |
5953 |
AGCTCTTCgCGTGCCTCCGGCACTACCGCCGGTACCTCCAGCGCTGCCACCcGAAGAGC |
6120 |
GCTCTTCAATGGCCGGCTCCGCCGGCTCT |
6121 |
CGGAGCCGGCCATTGAAGAGCAATTCAAAAATCATACC |
6404 |
CAAGCTCTTCGCGTTTAGTTAATCAGCATGTCCAGG |
6866 |
AAGGGAGGAGCGGGTTCTGGATCTGGATCTGGAGGTTCC |
6867 |
ACCGGAACCTCCAGATCCAGATCCAGAACCCGCTCCTCC |
7056 |
GGTGGCAGCGCTGGAGGTACCGGCGGTAGTGCCGGAACGggtcgtcaacttga |
7057 |
AATGCTCTTCaGGTGGCAGCGCTGGAGGTACCGGCGGTTCTGGTGGCGGAGGG |
 
Transgenes
Name
|
Description
|
Full Designation
|
Comments
|
jsTi1493 IV
|
Chr IV landing site
|
jsTi1493 [mosL loxP mex-5p FLP sl2 mNeonGreen rpl-28p FRT GFP-HIS-58 FRT3 mosR] IV
|
Nonet, 2020 |
jsSi1515 IV
|
mec-4p::GAL4
SK
-QF
AD
|
jsTi1493 jsSi1515 [mosL loxP mec-4p GAL4
SK
-QF
AD
tbb-2 3′ FRT3 mosR] IV
|
Nonet, 2020 |
jsSi1516 IV
|
mec-4p::tetR-QF
AD
|
jsTi1490 jsSi1516 [mosL loxP mec-4p tetR-QF
AD
tbb-2 3′ FRT3 mosR] IV
|
Nonet, 2020 |
jsSi1518 I
|
11X UAS::GFP
|
jsTi1453 jsSi1518 [mosL loxP 11X UAS ∆pes-10p GFP-C1 tbb-2 3′ FRT3 mosR] I
|
Nonet, 2020 |
jsSi1519 I
|
7X tetO ∆pes-10::GFP
|
jsTi1453 jsSi1519 [mosL loxP 7X tetO ∆pes-10p GFP-C1 tbb-2 3′ FRT3 mosR] I
|
Nonet, 2020 |
jsSi1525 IV
|
mec-4p::GAL4
SK
-VP64
|
jsTi1493 jsSi1525 [mosL loxP mec-4p GAL4
SK
-VP64 tbb-2 ‘3 FRT3 mosR] IV
|
Nonet, 2020 |
jsSi1543 I
|
7X tetO ∆mec-7p::GFP
|
jsTi1453 jsSi1543 [mosL loxP tetO 7X ∆mec-7p GFP-C1 tbb-2 3′ FRT3 mosR] I
|
Nonet, 2020 |
jsSi1560 IV
|
mec-4p::tetR-L-QF
AD
|
jsTi1493 jsSi1560 [mosL loxP mec-4p tetR-L-QF
AD
act-4 3′ FRT3 mosR] IV
|
Nonet, 2020 |
jsSi1588 IV
|
mec-4p::GAL4
SK
-L-QF
AD
|
jsTi1493 jsSi1588 [mosL loxP mec-4p GAL4
SK
-L-QF
AD
act-4 3′ FRT3 mosR] IV
|
RMCE insertion of NMp3876 into
jsTi1493
|
jsSi1661 IV
|
mec-4p::tetR-L-QF
AD
|
jsTi1493 jsSi1661 [mosL loxP mec-4p tetR-LL-QF
AD
act-4 3′ FRT3 mosR] IV
|
RMCE insertion of NMp4048 into
jsTi1493
|
jsSi1664 IV
|
mec-4p::GAL4
SK
-L4-VP64
|
jsTi1493 jsSi1664 [mosL loxP mec-4p GAL4
SK
-L4-VP64 tbb-2 3’ FRT3 mosR] IV
|
RMCE insertion of NMp4049 into
jsTi1493
|
Worm Strains
NM strain
|
Genotype
|
Source
|
5179 |
jsTi1493 [mosL loxP mex-5p FLP sl2 mNeonGreen rpl-28p FRT GFP-HIS-58 FRT3 mosR] IV
|
Nonet, 2020; CGC |
5213 |
jsTi1453 jsSi1518 [mosL loxP 11X UAS ∆pes-10p GFP-C1 tbb-2 3′ FRT3 mosR] I; him-8(e1489) IV
|
Nonet, 2020 |
5214 |
jsTi1453 jsSi1519 [mosL loxP 7X tetO ∆pes-10p GFP-C1 tbb-2 3′ FRT3 mosR] I; him-8(e1489) IV
|
Nonet, 2020 |
5225 |
jsTi1453 jsSi1519 [mosL loxP tetO 7X ∆pes-10p GFP-C1 tbb-2 3′ FRT3 mosR] I; jsTi1490 jsSi1516 [mosL loxP mec-4p tetR-QF
AD
tbb-2 3′ FRT3 mosR] IV
|
This work |
5233 |
jsTi1453 jsSi1518 loxP UAS 11X ∆pes-10p GFP-C1 tbb-2 3′ FRT3 mosR] I; jsTi1493 jsSi1515 [mosL loxP mec-4p GAL4
SK
-QF
AD
tbb-2 3’ FRT3 mosR] IV
|
Nonet, 2020; CGC |
5264 |
jsTi1453 jsSi1543 [mosL loxP tetO 7X ∆mec-7p GFP-C1 tbb-2 3′ FRT3 mosR] I; him-8(e1489) IV
|
Nonet, 2020 |
5295 |
jsTi1493 jsSi1560 [mosL loxP mec-4p tetR-L-QF
AD
act-4 ‘3 FRT3 mosR] IV
|
Nonet, 2020 |
5301 |
jsTi1453 jsSi1518 [mosL loxP UAS 11X ∆pes-10p GFP-C1 tbb-2 3′ FRT3 mosR] I; jsTi1493 jsSi1525 [mosL loxP mec-4p GAL4
SK
-VP64 tbb-2 3’ FRT3 mosR] IV
|
Nonet, 2020 |
5353 |
jsTi1493 jsSi1588 [mosL loxP mec-4p GAL4
SK
-L-QF
AD
act-4 3′ FRT3 MosR] IV
|
This work |
5362 |
jsTi1453 jsSi1518 [mosL loxP UAS 11X ∆pes-10 GFP-C1 tbb-2 3′ FRT3 mosR] I; jsTi1493 jsSi1588 [mosL loxP mec-4p GAL4
SK
-L-QF
AD
act-4 3′ FRT3 mosR] IV
|
This work |
5467 |
jsTi1453 jsSi1543 [mosL loxP tetO 7X ∆mec-7p GFP-C1 tbb-2 3′ FRT3 mosR] I; jsTi1493 jsSi1560 [mosL loxP mec-4p tetR-L-QF
AD
act-4 3’ FRT3 mosR] IV
|
This work |
5468 |
jsTi1453 jsSi1543 [mosL loxP tetO 7X ∆mec-7p GFP-C1 tbb-2 3′ FRT3 mosR] I; jsTi1493 jsSi1661 [mosL loxP mec-4p tetR-LL-QF
AD
act-4 3′ FRT3 mosR 3′ ] IV
|
This work |
5470 |
jsTi1453 jsSi1518 [mosL loxP UAS 11X ∆pes-10 GFP-C1 tbb-2 3′ FRT3 mosR] I; jsTi1493 jsSi1664 [mosL loxP mec-4p GAL4
SK
-L4–QF
AD
tbb-2 3’ FRT3 mosR] IV
|
This work |