High-throughput isolation of caenorhabditis elegans deletion mutants

Leo X. Liu*, Jill M. Spoerke, Evan L. Mulligan, Jing Chen, Brian Reardon, Bethany Westlund, Lin Sun, Ken Abel, Barbara Armstrong, Gary Hardiman, Judith King, Lisa McCague, Michael Basson, Ralph Clover, Carl D. Johnson

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

143 Citations (Scopus)

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 ~ 1400 bp in size when using a ~ 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.

Original languageEnglish
Pages (from-to)859-867
Number of pages9
JournalGenome Research
Volume9
Issue number9
DOIs
Publication statusPublished - 01 Sep 1999

ASJC Scopus subject areas

  • Genetics
  • Genetics(clinical)

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