title: High-throughput isolation of Caenorhabditis elegans deletion mutants.
abstract: The nematode Caenorhabditis elegans is the first animal whose genome is completely sequenced, providing a rich source of gene information relevant to metazoan biology and human disease. This abundant sequence information permits a broad-based gene inactivation approach in C. elegans, in which chemically mutagenized nematode populations are screened by PCR for deletion mutations in a specific targeted gene. By handling mutagenized worm growth, genomic DNA templates, PCR screens, and mutant recovery all in 96-well microtiter plates, we have scaled up this approach to isolate deletion mutations in >100 genes to date. Four chemical mutagens, including ethyl methane sulfonate, ethlynitrosourea, diepoxyoctane, and ultraviolet-activated trimethylpsoralen, induced detectable deletions at comparable frequencies. The deletions averaged approximately 1400 bp in size when using a approximately 3 kb screening window. The vast majority of detected deletions removed portions of one or more exons, likely resulting in loss of gene function. This approach requires only the knowledge of a target gene sequence and a suitable mutagen, and thus provides a scalable systematic approach to gene inactivation for any organism that can be handled in high density arrays.
authors.affiliation: Axys Pharmaceuticals, NemaPharm Group, South San Francisco, California 94080, USA. lliu@cambriabio.com
authors.forename: L X
authors.initials: LX
authors.lastname: Liu
authors.forename: J M
authors.initials: JM
authors.lastname: Spoerke
authors.forename: E L
authors.initials: EL
authors.lastname: Mulligan
authors.forename: J
authors.initials: J
authors.lastname: Chen
authors.forename: B
authors.initials: B
authors.lastname: Reardon
authors.forename: B
authors.initials: B
authors.lastname: Westlund
authors.forename: L
authors.initials: L
authors.lastname: Sun
authors.forename: K
authors.initials: K
authors.lastname: Abel
authors.forename: B
authors.initials: B
authors.lastname: Armstrong
authors.forename: G
authors.initials: G
authors.lastname: Hardiman
authors.forename: J
authors.initials: J
authors.lastname: King
authors.forename: L
authors.initials: L
authors.lastname: McCague
authors.forename: M
authors.initials: M
authors.lastname: Basson
authors.forename: R
authors.initials: R
authors.lastname: Clover
authors.forename: C D
authors.initials: CD
authors.lastname: Johnson
mesh_terms: D000465:Algorithms; D000818:Animals; D017173:Caenorhabditis elegans / Q000235:genetics*; D017353:Gene Deletion; D015723:Gene Library; D008957:Models, Genetic; D016296:Mutagenesis; D009153:Mutagens / Q000494:pharmacology; D020411:Oligonucleotide Array Sequence Analysis; D016133:Polymerase Chain Reaction / Q000379:methods*; D017422:Sequence Analysis, DNA / Q000379:methods; D013696:Temperature
chemical_list: D009153:Mutagens