Construction of a bicistronic vector for the co-expression of two genes in Caenorhabditis elegans using a newly identified IRES

Abstract

The nematode Caenorhabditis elegans is an important model animal for biological research. Currently, transgenic C. elegans strains are mainly generated by injecting DNA encoding a gene of interest, in combination with a reporter gene, into the gonad. With this approach, the interpretation of negative results, such as the failure to observe reporter expression, is frequently required. Single, selectable vectors are urgently required. Internal ribosome entry site (IRES) elements are known to bind the eukaryotic ribosomal translation initiation complex and independently promote translation initiation. Bioinformatic analysis predicted an IRES motif upstream of the start codon of the C. elegans Hsp-3 gene. While this sequence has a Y-shaped double-hairpin secondary structure characteristic of IRES elements, it was unclear if it could function as an IRES. In the present study, this predicted Hsp-3 IRES was incorporated into a bicistronic vector driven by the myo-3 promoter, which allowed co-expression of RFP and GFP genes in the muscle tissue of C. elegans and thereby demonstrated that this IRES element is functional. This vector provides a novel, powerful tool for C. elegans research.