Studies on glc-3, a potential target of Ivermectin in parasitic nematodes

The free-living nematode Caenorhabditis elegans has been used for many years as an expression system for genes from parasitic species. We wished to further develop and improve this system by using CRISPR/Cas9 to delete specific genes from C. elegans and replace them with single copies of orthologous genes from the parasite, Haemonchus contortus. Initial experiments focussed on glc-3 which encodes a subunit of the glutamate-gated chloride channels, the target of the avermectin/milbemycin family of anthelmintics. We cloned the promoters from the glc-3 genes of both species and compared the expression patterns of mCherry under the control of both promoters. The C. elegans glc-3 promoter drove expression in a subset of head interneurons, as previously reported whereas the H. contortus promoter drove expression in a pharyngeal motoneuron, M4. We were able to generate heterozygous worms in which one copy of glc-3 was deleted, but we could never obtain homozygous knock-outs. Further investigation of the mRNAs encoded by glc-3 revealed a novel transcript, glc-3T, which encodes a severely truncated form of GLC-3. The presence of such truncated transcripts may explain the unexpected difficulties encountered in attempting to knock out ion channel genes in C. elegans.