Gene activation using FLP recombinase in C. elegans

Abstract

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.

Figure 1. FLP Recombinase Strategy.

A FLP inducible transgene in the “off” configuration expresses the mCherry reporter under the control of a promoter sequence. The let-858 transcriptional terminator prevents transcription into the downstream elements. FLP expression leads to the looping out of the mCherry::let-858 fragment. This brings the GFP and the in-frame open reading frame (ORF) from the gene of interest under control of the promoter.

Figure 2. The Basic Design of FLP-Dependent Transgenes Using Gateway.

A standard multisite Gateway construct (A) is composed of a promoter, open reading frame (ORF) and 3′ untranslated region (UTR) that terminates transcription. These can be made FLP inducible by adding the FRT flanked cassette in slot 1 flanked by the attB4 and attB1 lambda recombination targets(B), or in slot 2 flanked by the attB1 and attB4 lambda recombination sites (C). These two formats allow compatibility with the already constructed promoterome or ORFeome clones. The red and green arrows represent mCherry and GFP coding regions, as in Figure 1. Red octagons represent the polyadenylation site and transcription terminator from the let-858 3′ genomic DNA or the polyadenylation site from the unc-54 3′ genomic region.

Figure 3. FLP-Dependent GFP-Histone Expression.

Before heat induction, the myo-2 promoter (A,B in strain EG4866 pWD200) or myo-3 promoter (E,F in strain EG4859 pWD198) drive mCherry expression in the pharyngeal or body muscle, respectively. FLP recombinase is induced by activation of the heatshock promoter; FLP removes the mCherry terminator. 15 hours after heat induction, these transgenes produce nuclear-localized GFP-histone fusion protein (C,D,G, H).

Figure 4. FLP-Dependent Tetanus Toxin Expression.

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.