Gene Activation Using FLP Recombinase in C. elegans

The FLP enzyme catalyzes recombination between specific target sequences in DNA. Here we use FLP to temporally and spatially control gene expression in the nematode C. elegans. Transcription is blocked by the presence of an “off cassette” between the promoter and the coding region of the desired product. The “off cassette” is composed of a transcriptional terminator flanked by FLP recognition targets (FRT). This sequence can be excised by FLP recombinase to bring together the promoter and the coding region. We have introduced two fluorescent reporters into the system: a red reporter for promoter activity prior to FLP expression and a green reporter for expression of the gene of interest after FLP expression. The constructs are designed using the multisite Gateway system, so that promoters and coding regions can be quickly mixed and matched. We demonstrate that heat-shock-driven FLP recombinase adds temporal control on top of tissue specific expression provided by the transgene promoter. In addition, the temporal switch is permanent, rather than acute, as is usually the case for heat-shock driven transgenes. Finally, FLP expression can be driven by a tissue specific promoter to provide expression in a subset of cells that can only be addressed as the intersection of two available promoters. As a test of the system, we have driven the light chain of tetanus toxin, a protease that cleaves the synaptic vesicle protein synaptobrevin. We show that we can use this to inactivate synaptic transmission in all neurons or a subset of neurons in a FLP-dependent manner.


Introduction
Every widely-used genetic model organism can be manipulated to express genes introduced by the experimenter. In C. elegans it is simple to create a transgene by injecting DNA containing a complete genomic region. In most cases the transgene will be expressed in its native temporal and spatial pattern and will rescue the mutant phenotype. However, if the researcher would like to test the function of a gene at a specific time or in a specific tissue of the worm, one would need a very specific promoter. Due to a limited set of promoters available, it is often impossible to express a gene of interest in a specific cell. More importantly, there is only one temporally inducible promoter available -the heat-shock promoter. This promoter has been a workhorse of the field, but has a major drawback because it is expressed ubiquitously. Three techniques have been developed that provide more precise temporal and spatial control by making gene expression dependent on two independently controllable events.
The Chalfie laboratory has developed one solution by expressing the gene product as two complementary halves. In this case, the full gene product is reconstituted only in the cells that express both promoters. When expressed under the control of two different overlapping promoters, the complete protein is only reconstituted in a small number of cells. They have demonstrated that a two-part GFP can be used to label specific cells [1] and that a two-part caspase can be used to kill specific cells [2]. Their technique can also be applied to the temporal control of gene expression. If one of the promoters is a heat-shock inducible promoter, specific cells can be killed on command. The limitation of the two-part system is that it requires gene products that can reconstitute activity from two halves.
A second combinatorial control technique relies on temperature-dependent degradation of the mRNA of the target gene. This method was independently proposed by several groups, but made practical by Getz, Xu and Fire (A. Fire personal communication) [3].The nonsense mediated decay (NMD) pathway specifically degrades mRNAs with long 39 untranslated regions containing many introns. Transgenes can be engineered with long 39 UTRs so that their mRNAs are targeted for degradation. Strains with temperature-sensitive mutations in NMD components cannot degrade these mRNAs at the restrictive temperature. Thus, the transgene is more strongly expressed at the restrictive temperature (when the NMD system is not functioning) than at the permissive temperature (when NMD is actively degrading aberrant mRNAs). This system gives some degree of control over expression levels, although there is a moderate background level of expression even in the ''off'' state, which has limited its use. Bacaj and Shaham have developed a method to add heat-shock control to a transgene expressed under a tissue-specific promoter [4]. The method uses a mutant background in which the heatshock response is defective. By rescuing the mutant defect with tissue-specific promoters, a tissue-specific heat shock response is generated. This method requires using the hsf-1 mutant background and gene expression from the heatshock promoter is still acute. Ideally, a combinatorial expression system would be in the wild-type strain and provide a permanent change in the genotype of the cells of interest.
Here we describe a method that uses FLP recombinase to control transgene expression in C. elegans. In this configuration, the transgene is expressed at the combinatorial intersection of two different promoters: either two spatially restricted promoters, or a spatially restricted and temporally controlled promoter.

Construct Design
The site specific recombinases Cre and FLP have been used in many systems to control gene structure and expression [5][6][7][8][9][10]. These enzymes align tandem copies of the target sequence, perform site-specific recombination, and remove the sequence between the targets as a circular DNA molecule. If the intervening sequence disrupts expression, removal by recombination will allow the transgene to be activated. We designed an ''off cassette'', composed of a putative transcriptional terminator, that could be placed between a promoter and a coding region to disrupt expression of the coding region ( Figure 1). Expression of FLP recombinase will excise the cassette as a circular DNA molecule. This rearrangement will place the promoter adjacent to the downstream coding regions, converting the transgene to the ''on'' state. Thus, expression is dependent on both the promoter driving the coding region and the promoter driving expression of FLP.
The constructs are designed to provide a fluorescent readout in either the 'off' or 'on' state. The FRT-flanked ''off cassette'' contains the mCherry coding sequence followed by the 39 genomic region from the let-858 gene. The red-fluorescent mCherry protein acts as a reporter for the promoter activity of the transgene,

Author Summary
Genes turn on and off as a natural part of development. The nematode C. elegans has been an important model system for studying the roles of genes in animal development and physiology. However, worm researchers have had a limited toolkit for controlling gene activation. These drawbacks have been particularly restrictive when studying the function of a gene that has different roles in several cell types or at different times in development.
Here we describe a way to turn any gene on at a specific time in specific cells. We provide a set of mix-and-match reagents that give researchers a way to quickly build new combinations of regulatory elements. These reagents will allow researchers to express a single gene in a wide array of temporal or spatial patterns, or to serially express many genes in a single cell. As a proof of principle, we made an artificial worm gene composed of a neurotoxin that would block neurotransmission. When we activated the gene in a small number of neurons in adult animals, these cells ceased to function. We anticipate that this new technique will find a wide variety of uses by the C. elegans community. verifying that the transgene is present and expressing in the expected cell types prior to FLP-induced recombination. The let-858 39 genomic region provides the poly-adenylation signal for mCherry mRNA as well as a putative transcriptional terminator, preventing expression of downstream sequences. GFP acts as a reporter for transcriptional read-through or reinitiation of transcription in the let-858 genomic region. Recombination of the FRT sequences removes the mCherry coding sequence and the terminator and brings a GFP coding region under control of the promoter, to indicate that the FLP reaction was successful. The coding region for any gene of interest can be fused in frame to the 39 end of the GFP sequence, thus providing FLP-inducible expression of that protein.

Modular Design
To speed assembly of constructs and to make use of genome reagents generated by other laboratories, we based our constructs on the Multisite Gateway TM in vitro recombination system from Invitrogen [11,12]. These vectors allow rapid, modular construction of plasmids using a site-specific recombinase from the bacteriophage lambda ( Figure 2A). The recombinase target sequences are designed to allow pairwise recombination between specific DNA sequences. The standard multisite system uses three libraries of entry vectors: a promoter library, a cDNA library, and transcriptional terminator library (to be recombined into slots 1, 2 or 3, respectively). Individual components from these libraries can be selected and mixed with a destination vector to generate a desired combination of promoter, cDNA and terminator in a single reaction. Many reagents are already available for the construction of C. elegans expression constructs using this system. The C. elegans promoterome consortium has cloned the 59 regulatory regions of approximately 6,000 different C. elegans genes into Gateway 'entry' vectors compatible with the slot 1 entry vectors [13]. The ORFeome project from the Vidal laboratory has cloned cDNAs from approximately 11,000 genes into slot 2 entry vectors [14].
Because the promoterome constructs are designed to recombine directly to ORFeome constructs, it was not possible to introduce the off-cassette between them. Instead, we built vectors that have the off-cassette in either the promoter slot (slot 1) ( Figure 2B) or in the cDNA slot (slot 2) ( Figure 2C). The former arrangement requires construction of a promoter-FRT construct, but is compatible with the existing ORFeome library. The latter requires placing the ORF to the third slot, but is compatible with the existing promoterome library.
The ORFeome-compatible format requires that the ''offcassette'' be cloned into entry vectors containing various promoters. We generated several potentially useful promoter-''off-cassette'' entry clones ( Figure 2B, Table 1). These plasmids can then be recombined with any ORFeome clone to produce a large number of different open reading frames under the same FLP-inducible promoter sequence. This arrangement is particularly useful when one is primarily interested in expressing a number of different proteins in a particular cell or tissue.
For the promoterome-compatible constructs, we placed the offcassette into the position usually occupied by the ORF of interest. We then used standard cloning methods to place several ORFs of interest into slot 3 entry vectors ( Figure 2C, Table 2). Any of the set of promoterome clones can be recombined with these clones to express a single ORF in a wide array of tissues in a FLP-dependent manner. This arrangement is particularly useful when one is determining the focus of gene rescue or inactivating different groups of cells.

FLP-Mediated Recombination
As a proof of principle, we produced two FLP-inducible constructs that would express GFP-tagged histone in different muscle cells. The first construct was in the ORFeome compatible configuration described in Figure 2B. We used a pharyngeal muscle promoter (Pmyo-2) followed by the ''off-cassette'' in the first Gateway slot. We added the HIS-11 open reading frame in the second slot and an unc-54 39 polyadenylation site in the third slot ( Figure 3A-D). We injected this plasmid together with a plasmid encoding FLP-recombinase under the control of the hsp-16.48 heat shock promoter [15] and a lin-15(+) co-injection marker to produce a line of transgenic worms. As expected, these worms expressed diffuse red fluorescence in their pharyngeal muscles with no apparent GFP fluorescence ( Figure 3A, B). The lack of GFP fluorescence confirms that the let-858 39 genomic region functions as expected to prevent read-through into the downstream gene. We then exposed the worms to a one hour, 34u heat shock and imaged the worms 2 hours, 3 hours and 15 hours later. Although no GFP was apparent at 2 hours, the heat shock-induced expression of the GFP::HIS-11 fusion protein was visible at three hours ( Figure S1) and was strong at 15 hours ( Figure 3C, D). From  Applications One important application for the FLP-on method is to silence neurotransmission in specific neurons in a temporally-controlled manner. The tetanus toxin light chain is a highly specific protease that recognizes and cleaves synaptobrevin [17]. Since synaptobrevin is one of the three SNARE-class proteins required for calcium-mediated release of neurotransmitter [18], expressing tetanus toxin eliminates the ability of a neuron to signal through chemical synapses. The high specificity of tetanus toxin preserves all other functions of the neuron, including electrical coupling through gap junctions. Temporal control of tetanus toxin expression is important for two reasons. First, synaptic transmission is essential for development. Animals lacking acetylcholine neurotransmission (in cha-1 mutants) or all synaptic neurotransmission (in unc-13 deletion alleles) are arrested in the first larval stage. Thus, loss of synaptic transmission in at least some neurons may lead to broad developmental defects that blind the investigator to functions for a neuron in the adult. Using the FLP-on method, expression of tetanus toxin can be activated after the developmental requirement for a neuron. Conversely, early loss of neuronal function can lead to developmental compensation in the nervous system. For example, when particular sensory neurons were ablated in males in the L3 stage the nervous system compensated for their loss; by contrast ablation of these same neurons in the L4 stage led to behavioral abnormalities in the adult [19]. The ability to silence these neurons in adulthood allows one to assay the function of the synaptic connectivity of a neuron in an existing developmentally-normal circuit.
To test if tetanus toxin induction could inactivate neurotransmission, we designed an ORFeome-compatible tetanus toxin expression construct. The tetanus toxin sequence was inserted into slot 2, and the FRT-flanked terminator was placed in slot 1 after the GABAspecific neuron promoter Punc-47. unc-47 encodes the vesicular GABA transporter required to fill synaptic vesicles with GABA, and is expressed in all GABA neurons. We chose this promoter because loss of GABA function produces two distinct phenotypes: a locomotory phenotype and a defecation motor program defect.
Animals lacking GABA neurotransmission exhibit a distinctive locomotory defect [20]. These animals cannot back when touched on the head but rather execute an accordion-like ''shrinking'' response. This symmetrical contraction of the body muscles occurs due to the lack of contralateral inhibitory inputs from the GABA motor neurons. Prior to heat shock, transgenic worms exhibited normal movement. Animals were exposed to heat shock at 34u for one hour. 24 hours later the animals exhibited a clear shrinking phenotype (Video S1). The structure of the GABA neurons was not affected by toxin expression (Figure S2), suggesting that the toxin was simply silencing synaptic transmission in these neurons. As expected the shrinking phenotype was associated with the presence of the transgene: 99 of 100 shrinker animals carried the Pmyo-2::GFP extrachromosomal array marker. The presence of nonshrinking animals in the population was due to loss of the extrachromosomal array: 29 of the 30 non-shrinker animals had lost the Pmyo-2::GFP transgene marker. The one non-shrinking animal carrying the array lacked the mCherry marker in the VD and DD motor neurons, demonstrating that this animal was a somatic mosaic which lacked the array in the motor neurons.
GABA function is also required for the motor program of the defecation cycle [21,22]. The defecation motor program requires the AVL and DVB GABA neurons to stimulate contraction of the enteric muscles via a GABA-gated cation channel [23]. Prior to heat shock, transgenic worms expressing mCherry in the AVL and DVB GABA neurons had wild-type enteric muscle contractions during the defecation cycle (n = 10 worms, 10 cycles per worm, Figure 4). After heat shock, transgenic animals lacked the enteric muscle contractions during the defecation cycle (Figure 4), as expected for loss of GABA neurotransmission. Because AVL and DVB are partially redundant, these data suggest that FLP function must be greater than 95% effective. In addition, we were able to see induction of GFP-tagged tetanus toxin by confocal microscopy (not shown). Tetanus toxin expression is continuous using this FLP-on construct; thus, unlike direct heatshock-induced expression of the toxin, the behavioral change is permanent.

Discussion
FLP-dependent gene expression will have two general uses in C. elegans: to provide spatial specificity and temporal specificity for gene expression. Because it provides expression within the spatial overlap of two promoters, the method essentially squares the number of transgene expression patterns now available. In many cases, especially in the nervous system, this can restrict expression to single cells of the worm.
FLP recombinase can also be used to provide temporal specificity for gene expression. For most purposes temporal control is best provided by activation of the heatshock promoter. Thus, heatshock-driven expression of FLP recombinase will confer temporal specificity to any promoter. This will be particularly useful for expression of dominant negative or constitutively activated gene products that may kill cells before their effects can be assayed. Moreover, acute expression can outflank the criticism most often thrown at genetic analysis -that homeostatic mechanisms will compensate for chronic genetic changes. In this way the heatshock FLP-on method and the cell-specific rescue of hsf-1 method developed by the Shaham laboratory are similar. In the FLP-on method, expression is permanently activated, whereas in the hsf-1 rescue method, expression is acute and depends on the length of the heatshock response. Depending on the circumstances and the gene product being expressed, one method may be more advantageous than the other.
For the neuroscientist, FLP-on constructs provide a method for analyzing the role of a neuron in a circuit by killing a specific cell, inactivating the cell, or activating the cell. By combining our system with the Chalfie split caspase system (Table 1), cells can be killed at the intersection of three promoters. These three promoters could each provide a spatial component, giving extremely tight spatial control to potentially address the few single neuron types that have escaped the two-promoter system. Alternatively, one of the three could provide temporal inducibility, adding heat shock control onto many two-promoter single-cell killing experiments. Silencing the cell can allow specific dissection of the chemical synapses present in the system while leaving the gap junction connections in the network intact. In addition, leaving the cell in place will minimize any developmental perturbations of the circuit that might be caused by removing a neuron by the cell death pathway. In addition to eliminating a cell entirely, or silencing the chemical neurotransmission in a cell, single cells can be electrically silenced or activated on command by expressing halorhodopsin, a light activated chloride pump protein [24], or channelrhodopsin, a light activated cation channel protein [25], at the intersection of two spatial promoters.
For the developmental biologist, FLP-on constructs can provide temporal control of gene expression so that the role of a gene during different developmental periods can be evaluated. This application is limited by the time delay required for FLP expression, recombination and gene expression. We observed a three hour delay from the end of heat shock to the expression of Pmyo-2::GFP. FLP regulation will also be useful for analyzing promoter expression patterns by permanently marking descen- A GFP-tetanus toxin fusion product was expressed from the GABA neuron specific unc-47 promoter in a FLP-dependent manner. C. elegans adult hermaphrodites undergo a defecation cycle every 50 seconds. Wild-type worms execute enteric muscle contractions (EMCs) during 90% of defecation cycles. Prior to heat shock, the FLP-inducible GFP-tetanus toxin animals are not significantly different from the wild type (95%). However, after heat shock they exhibit enteric muscle contractions in only 10% of defecation cycles, significantly less than the wild type and not significantly different from unc-25 mutants. unc-25 encodes the biosynthetic enzyme for GABA, glutamic acid decarboxylase. Similar to animals in which tetanus toxin is blocking synaptic transmission from the GABA neurons, unc-25 mutants exhibit enteric muscle contractions in only 11% of defecation cycles. Neither wild-type nor unc-25 worms are significantly affected 24 hours after heat shock (95% and 10% EMC/defecation cycle). Ten defecation cycles were scored for each of ten worms for each condition. Counts were done between 23 and 27 hours after heat shock. Error bars represent the standard error of the mean. doi:10.1371/journal.pgen.1000028.g004 dents of cells that have expressed a promoter. Combined with the known cell lineage of C. elegans, the expression pattern can quickly pinpoint the expression pattern of a transgene that may come on very briefly and in only a few cells in the embryo. Using traditional GFP reporters, such expression might be missed entirely, or if it is detected it might be very difficult to unambiguously identify the expressing cell among the dividing cells of the embryo. This technique could be combined with forward genetic screens. A FLP-dependent histone GFP reporter (Table 1) will easily identify mutant backgrounds in which a gene is transiently misexpressed during development.
In conclusion, FLP-dependent excision of a transcriptional terminator provides a simple way to make expression of a transgene dependent on the activity of two promoters. Depending on the promoters used, FLP-on constructs can confer combinatorial spatial or temporal control of gene expression in C. elegans. We anticipate that the combination of the wide availability of the Gateway reagents and the imagination of the C. elegans community will yield many new applications.

Materials and Methods
Plasmids pWD157 (slot 2 TeTx): Tetanus toxin light chain was PCR amplified from CMV-LC-Tx (Heiner Niemann) using primers containing attB1 and attB2 tails GGGGACAAGTTTGTACAA-AAAAGCAGGCTTAATGCCGATCACCATCAACAACTTC and GGGGACCACTTTGTACAAGAAAGCTGGGTTTAAG-CGGTACGGTTGTACAGGTT and recombined with the attP1 and attP2 sites in the slot 2 donor vector pDONR221 (Invitrogen) using the BP recombination reaction. pWD170 (slot 3 TeTx): Tetanus toxin light chain was PCR amplified from CMV-LC-Tx using primers GTATGCCGATC-ACCATCAACAAC and TTAAGCGGTACGGTTGTACAGG and cloned as a blunt fragment into pMH472 using SrfI. pMH472 is a slot 3 entry vector containing a SrfI site followed by two stop codons and then the unc-54 39 UTR.
pPD119FRTRFPGFP: A fragment of mRFP1 was PCR amplified and cloned between the SalI and SmaI sites in pPD118.33. pPD118.33 is a Pmyo-2::GFP plasmid (gift of Andrew Fire). Tandem FRT sites [8] were cloned at the junctions as SalI-BamHI and MluI-SmaI double-stranded oligonucleotides.
pWD79-2RV (Phsp-16-48:FLP): A PCR fragment containing the FLP coding sequence from pOG44 (Stratagene) was cloned as an MluI-NheI fragment into pJL44 (J.L. Bessereau). pJL44 contains the Phsp-16-48 heat-shock promoter and the glh-2 39 UTR. The FLP coding sequence in pOG44 contains a point mutation which was repaired using a PCR fragment from the FLP coding sequence cloned into pBR322 (Makkuni Jayaram). An artificial intron was introduced into the FLP coding sequence at the EcoRV site using a double-stranded oligo: GTAAGTTTAAACATATATACTAAC-TAACCCTGATTATTTAAATTTTCAG.
pWD173 (slot 2 FLP-no stop): The FLP ORF was PCR amplified from pWD79-2RV using primers containing attB2 and attB3 tails: GGGGACAAGTTTGTACAAAAAAGCAGGCTT-AATGCCACAATTTGGTATATTATGT and GGGGACCA-CTTTGTACAAGAAAGCTGGGTATATGCGTCTATTTAT-GTAGGATG and BP cloned into pDONR221 (Invitrogen). This version of FLP does not contain a stop codon, and can be used to make C-terminally tagged proteins.
Gateway BP and LR in vitro recombination reactions were carried out according to manufacturer instructions.
'FTF' symbolizes the off cassette composed of FRT-mCherryterminator-FRT. Figure S1 FLP-Dependent GFP-Histone Expression. Before heat induction, the myo-2 promoter drives mCherry expression in the pharyngeal muscle. FLP recombinase is induced by a 34u heat shock for one hour. Three hours after heat induction, the transgene produces nuclear-localized GFP-histone fusion protein.