Generation of an external guide sequence library for a reverse genetic screen in Caenorhabditis elegans

Abstract

Background: A method for inhibiting the expression of particular genes using external guide sequences (EGSs) has been developed in bacteria, mammalian cells and maize cells. Results: To examine whether EGS technology can be used to down-regulate gene expression in Caenorhabditis elegans (C. elegans), we generated EGS-Ngfp-lacZ and EGS-Mtgfp that are targeted against Ngfp-lacZ and Mtgfp mRNA, respectively. These EGSs were introduced, both separately and together, into the C. elegans strain PD4251, which contains Ngfp-lacZ and Mtgfp. Consequently, the expression levels of Ngfp-lacZ and Mtgfp were affected by EGS-Ngfp-lacZ and EGS-Mtgfp, respectively. We further generated an EGS library that contains a randomized antisense domain of tRNA-derived EGS ("3/4 EGS"). Examination of the composition of the EGS library showed that there was no obvious bias in the cloning of certain EGSs. A subset of EGSs was randomly chosen for screening in the C. elegans strain N2. About 6% of these EGSs induced abnormal phenotypes such as P0 slow postembryonic growth, P0 larval arrest, P0 larval lethality and P0 sterility. Of these, EGS-35 and EGS-83 caused the greatest phenotype changes, and their target mRNAs were identified as ZK858.7 mRNA and Lin-13 mRNA, respectively. Conclusion: EGS technology can be used to down-regulate gene expression in C. elegans. The EGS library is a research tool for reverse genetic screening in C. elegans. These observations are potentially of great importance to further our understanding and use of C. elegans genomics. © 2009 Yan et al; licensee BioMed Central Ltd.