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


CONCLUSIONS
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.
Yan
Qitao
Q
Institute of Molecular Biology, Southern Medical University, Guangzhou, 510515, PR China. Yanqitao1979@126.com
Zhao
Rui
R
Zheng
Wenlin
W
Yin
Changxin
C
Zhang
Bao
B
Ma
Wenli
W
D000818:Animals; D001483:Base Sequence; D017173:Caenorhabditis elegans / Q000235:genetics*; D005786:Gene Expression Regulation; D015723:Gene Library; D017238:Genes, Helminth; D023281:Genomics / Q000379:methods*; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D018510:RNA, Helminth / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics
D018510:RNA, Helminth; D012333:RNA, Messenger