Supplementary Movie S1. GFP is expressed in transgenic worms carrying Pmyo-3:RFP-IRES-GFP:let858.3 micrometers. Scale bar, 500 micrometers.
Supplementary Movie S2. RFP is expressed in transgenic worms carrying Pmyo-3:RFP-IRES-GFP:let858.3micrometers. Scale bar, 500 micrometers.
We did not closely examine the relative expression efficiency of the IRES-driven GFP compared with RFP in Pmyo-3:RFP-IRES-GFP:let858.3′ transgenic worms, making further studies necessary to determine how IRES affects relative expression efficiency.
In this study, Western blot analysis and microscopy observations demonstrated that the RFP and GFP genes, driven the by myo-3 promoter, can be co-expressed in muscle tissue from a bicistronic vector containing a predicted C. elegans IRES derived from Hsp-3 (Bip) gene. The major advantage of the RFP-IRES-GFP plasmid is that it can solve the problem of gene silencing due to the interference between two plasmids. Furthermore, it avoids the use of multiple promoters in vectors of limited size. This vector can be used for the establishment of stable worm lines, and is applicable to biolistic bombardment protocols. Its ability to co-express genes can be used for specific applications, such as protein-protein interaction studies and immunization via co-expressed antigens and co-stimulatory proteins. Bicistronic vectors in C. elegans, based on the use of a spliced leader 2 sequence and trans-splicing, have also recently been described (12,13). However, IRES-dependent initiation of translation is potentially less complex than spliceosome-mediated RNA trans-splicing for bicistronic expression. Eukaryotic ribosomes have the intrinsic ability to bind mRNA in the absence of initiation factors and ATP (14). IRESs are descendants of spliceosomal introns; therefore, IRES elements may be more efficient. In addition, RNA trans-splicing is site specific. An extrachromosomal array is formed following transformation in C. elegans, and the efficiency of trans-splicing may be different in each transformation because of the difference of the copy numbers and transmission rates among transgenic strains. Thus, our C. elegans IRES-dependent bicistronic vector offers a novel, powerful tool for C. elegans research.
We gratefully acknowledge support from the program for Chang Jiang Scholars, Innovative Research Teams in Chinese Universities (No.IRT0866) and the earmarked fund for Modern Agro-industry Technology Research System.
The authors declare no competing interests.
Address correspondence to Ming Wang, College of Veterinary Medicine, China Agriculture University, No.2 Yuanmingyuan West Road, Haidian District, Beijing 100193, China. Email: