Department of Biology, University of North Carolina at Chapel Hill,
Chapel Hill, NC, USA.
The C. elegans che-12 gene is expressed in a subset of ciliated sensory neurons in the head and tail of the animal, including the amphid neurons with simple rod like cilia (Bacaj et al., 2008). CHE-12 localizes to the cilia in these neurons which form a bundle in the amphid channel. Previously, we observed CHE-12::GFP puncta along the amphid cilia suggesting coupling to intraflagellar transport machinery, however, the arrangement of amphid cilia in a bundle inside the amphid channel presented a challenge to follow a single fluorescent particle continuously from the cilia base to the distal tip (Das et al., 2015). In order to better visualize CHE-12 dynamics in a single amphid cilium, we devised a strategy to fluorescently tag the endogenous che-12 gene in a single amphid neuron, the ASER neuron.
Our cell-specific tagging cassette (CTC) includes a Stop codon and a strong terminator sequence (let-858 3’UTR) bounded at either end by two LoxP sites within short synthetic introns, followed by the coding sequence of any fluorescent protein (Figure 1A). We inserted a hygromycin resistance gene in the inter-genic region between the che-12 and B0024.4 genes for the selection of positive knock-in candidates. The CTC is introduced between the end of the coding sequence of the gene of interest and its stop codon using Cas9-targeted homologous recombination methods described in Dickinson et al., 2013. Animals bearing a correct insertion of the CTC at the selected genetic locus express the untagged version of the protein. A cell/tissue specific promoter is then used to drive the expression of Cre-recombinase in the specific cell/tissue where expression of the fluorescently tagged endogenous protein is desired. Cre-recombinase will excise out the region flanked by the LoxP sites in these specific cells, creating a fusion of the gene of interest and the fluorescent protein coding sequence, leading to cell/tissue-specific tagging of endogenous proteins. See Figure 1A an illustration of this strategy.
We introduced a CTC as outlined in Figure 1A at the 3’-end of the che-12 coding sequence using Cas9-targeted homologous recombination. We then used a ubiquitous or cell/tissue-specific promoter-driven Cre-recombinase, expressed from an extra-chromosomal array to allow selective recombination in those specific cells and fuse the che-12 coding sequence with mNeonGreen coding sequence. This strategy was successful in selectively labeling CHE-12 in a single neuron when we used gcy-5 promoter-driven Cre-recombinase (Figure 1B). In contrast, when we used a ubiquitous promoter such as Peft-3 to drive Cre-recombinase expression, we observed CHE-12::mNeonGreen signal in all amphid neurons (Figure 1C). The tagged CHE-12::mNeonGreen fusion protein localized correctly to the ASER cilium (Figure 1B and 1D).
This cell-specific tagging strategy is applicable for cell and tissue types for which strong cell/tissue-specific promoters have been characterized. This strategy can also be modified to delete domains within a protein or the entire coding sequence of a gene in a desired cell type by introducing synthetic introns containing embedded LoxP sites at the boundaries of a segment targeted for deletion.