A novel in vitro Caenorhabditis elegans transcription system

Abstract

Caenorhabditis elegans is an excellent model organism for biological research, but its contributions to biochemical elucidation of eukaryotic transcription mechanisms have been limited. One of the biggest obstacles for biochemical studies of C. elegans is the high difficulty of preparing functionally active nuclear extract due to its thick surrounding cuticle. By employing Balch homogenization, we have achieved effective disruption of larval and adult worms and have obtained functionally active nuclear extract through subcellular fractionation. In vitro transcription reactions were successfully re-constituted using such nuclear extract. Furthermore, two non-radioactive detection methods, PCR and qRT-PCR, have been adapted into our system to qualitatively and quantitatively detect transcription, respectively. Using this system to assess how pathogen infection affects C. elegans transcription revealed that Pseudomonas aeruginosa infection increased transcription activity. Our in vitro system is useful for biochemically studying C. elegans transcription mechanisms and gene expression regulations. The effective preparation of functionally active nuclear extract in our system fills a technical gap in biochemical studies of C. elegans and will expand the usefulness of this model organism in addressing many biological questions beyond transcription.