High-throughput isolation of Caenorhabditis elegans deletion mutants

Citation
Lx. Liu et al., High-throughput isolation of Caenorhabditis elegans deletion mutants, GENOME RES, 9(9), 1999, pp. 859-867
Citations number
35
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Molecular Biology & Genetics
Journal title
GENOME RESEARCH
ISSN journal
1088-9051 → ACNP
Volume
9
Issue
9
Year of publication
1999
Pages
859 - 867
Database
ISI
SICI code
1088-9051(199909)9:9<859:HIOCED>2.0.ZU;2-K
Abstract
The nematode Caenorhabditis elegans is the First animal whose genome is com pletely sequenced, providing a rich source of gene information relevant to metazoan biology and human disease. This abundant sequence information perm its a broad-based gene inactivation approach in C. elegans, in which chemic ally mutagenized nematode populations are screened by PCR for deletion muta tions in a specific targeted gene. By handling mutagenized worm growth, gen omic DNA templates, PCR screens, and mutant recovery ail in 96-well microti ter plates, we have scaled up this approach to isolate deletion mutations i n >100 genes to date. Four chemical mutagens, including ethyl methane sulfo nate, ethlynitrosourea, diepoxyoctane, and ultraviolet-activated trimethylp soralen, induced detectable deletions at comparable Frequencies. The deleti ons averaged similar to 1400 bp in size when using a -3 kb screening window . The vast majority of detected deletions removed portions of one or more e xons, likely resulting in loss of gene function. This approach requires onl y the knowledge of a target gene sequence and a suitable mutagen, and thus provides a scalable systematic approach to gene inactivation for any organi sm that can be handled in high density arrays.