A “FLP-Out” System for Controlled Gene Expression in Caenorhabditis elegans

Abstract

We present a two-part system for conditional FLP-out of FRT-flanked sequences in Caenorhabditis elegans to control gene activity in a spatially and/or temporally regulated manner. Using reporters, we assess the system for efficacy and demonstrate its use as a cell lineage marking tool. In addition, we construct and test a dominant-negative form of hlh-12, a gene that encodes a basic helix-loop-helix (bHLH) transcription factor required for proper distal tip cell (DTC) migration. We show that this allele can be conditionally expressed from a heat-inducible FLP recombinase and can interfere with DTC migration. Using the same DTC assay, we conditionally express an hlh-12 RNAi-hairpin and induce the DTC migration defect. Finally, we introduce a set of traditional and Gateway-compatible vectors to facilitate construction of plasmids for this technology using any promoter, reporter, and gene/hairpin of interest.T WO-COMPONENT gene expression systems are indispensable tools to probe molecular mechanisms underlying development. Because control can be exerted by each component independently, exquisite temporal and spatial control of gene activation or repression can be achieved. Using different promoter combinations to drive each component of these systems, additional control can be obtained beyond that afforded by heat-inducible or tissue-specific promoters alone. Site-specific recombination systems such as the FLP/FRT system have been used to control gene expression by ''FLP-out'': a recombinase-catalyzed intramolecular excision of spacer DNA that lies between tandemly oriented FRT sites. The spacer includes a transcriptional stop so that prior to activation of the FLP recombinase (and subsequent FLP-out) the gene downstream of the spacer is not transcribed (Golic and Lindquist 1989;Struhl and Basler 1993; Figure 1A). After the FRT-containing cassette is excised by the FLP recombinase, the downstream gene is brought into proximity to the promoter and is expressed (reporter 2 in Figure 1A). This system and related systems have proven quite powerful and flexible in model organisms including Drosophila and mouse (see Branda and Dymecki 2004, for review;McGuire et al. 2004). However, prior to our study presented here, and a recently published study (Davis et al. 2008), these systems had not been developed for use in Caenorhabditis elegans.An ideal FLP-out system provides the means to generate both loss-and gain-of-function effects in a spatially and temporally controlled manner. In addition, wild-type gene expression can be turned on in particular cells at particular times in an otherwise mutant background. In organisms where transgenes can be reliably inserted in single copy, FLP-out can also be used to eliminate wild-type gene expression by excision of an FRT-flanked wild-type cassette in a mutant background. In C. elegans, the most common methods for generating transgenic C. elegans introduce multiple copies of transgenes on extrachromosomal arrays (Stinchcomb et al. 1985;Mello et al. 1991;Kelly et al. 1997). ...

Repurposing an endogenous degradation system for rapid and targeted depletion ofC. elegansproteins

“…We have shown that ZF1-tagged proteins can be degraded in all examined embryonic and larval somatic cell types, and by altering the promoter used to express ZIF-1, we have demonstrated both spatial (tissue-specific promoter) and temporal (heat-shock promoter) control of targeted protein destruction. By combining ZF1 tagging with existing conditional gene expression technologies, such as the FLP-FRT system and tissue-specific heat-shock methods Voutev and Hubbard, 2008;Bacaj and Shaham, 2007;Churgin et al, 2013), it should be feasible to achieve simultaneous spatial and temporal control of gene product function. Using recently developed genome editing techniques, we have shown that sequences encoding the ZF1 tag can be inserted directly into endogenous loci, greatly facilitating genetic analysis by ZF1-tagging and obviating the need for a loss-of-function mutation.…”

Precise and Heritable Genome Editing in Evolutionarily Diverse Nematodes Using TALENs and CRISPR/Cas9 to Engineer Insertions and Deletions

“…rex-32 was excised from animals containing both FRT sites, using FLP recombinase, which was introduced into the gonad by injecting FLP-encoding RNA at concentration of 1500 ng/ml. RNA was made by in vitro transcription from a PCR fragment containing the T7 promoter, the FLP recombinase sequence, derived from pGC92 (Voutev and Hubbard 2008), and 59-and 39-UTRs appropriate for germline translation of C. elegans mRNAs: 59-UTR, ATT TAG GTG ACA CTATAG AAT ACA CGG AAT TCT AGA TGA TCC CCG CGT ACC GAG CT CAGA AAAA; 39-UTR, GCC TGA GCT CAC GTC GAC CGG GGC CCT GAG ATC TGC TGC AG.…”

The transcriptional repressor CTBP-1 functions in the nervous system of Caenorhabditis elegans to regulate lifespan

“…ctbp-1a::V5 was amplified from the resulting plasmid pMSCVpuro ctbp-1a::V5 with primers containing attachment sites attB4 and attB3. This amplicon was incorporated into the pGC188 (Jane Hubbard: Addgene plasmid 19619) (Voutev and Hubbard, 2008) plasmid in a BP reaction using the Gateway® Cloning system (Life Technologies, Australia). For expression in the nervous system, the panneuronal promoter of the aex-3 gene (1333 bp) was amplified from N2 genomic DNA and inserted into pENTR™/D-TOPO® plasmid (Life Technologies) according to the manufacturer's instructions.…”

“…For expression in the nervous system, the panneuronal promoter of the aex-3 gene (1333 bp) was amplified from N2 genomic DNA and inserted into pENTR™/D-TOPO® plasmid (Life Technologies) according to the manufacturer's instructions. A LR reaction was performed with the following plasmids: pENTR p aex-3, pGC188 ctbp-1a::V5 and the destination plasmid, pGC247 (Jane Hubbard: Addgene plasmid 19634) (Voutev and Hubbard, 2008). For constitutive expression of ctbp-1a::V5, the gfp sequence was flipped out of the plasmid pAER10 p aex-3::FRT-gfp-FRT::ctbp-1a::V5 following co-transformation with the pMS40 plasmid (Snaith et al, 1996) in BL21(DE3) cells (Invitrogen) and IPTG (isopropyl-beta-D-thiogalactopyranoside) induction of FLP recombinase.~8 μg of the resulting plasmid pAER2 p aex-3::ctbp-1a::V5 which also contains the unc-119(+) sequence in the backbone was linearised by digestion with EcoRV for biolistic transformation into unc-119(ed3) mutants.…”