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Vol. 9, Issue 9, 859-867, September 1999

METHODS
High-Throughput Isolation of Caenorhabditis elegans Deletion Mutants

Leo X. Liu,1,4 Jill M. Spoerke, Evan L. Mulligan, Jing Chen,2 Brian Reardon, Bethany Westlund, Lin Sun,3 Ken Abel, Barbara Armstrong, Gary Hardiman, Judith King, Lisa McCague, Michael Basson, Ralph Clover, and Carl D. Johnson

Axys Pharmaceuticals, NemaPharm Group, South San Francisco, California 94080 and La Jolla, California USA

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.

   Present addresses: 1Cambria Biosciences, Waltham, Massachusetts 02454 USA; 2Therion Biologics, Cambridge, Massachusetts 02138 USA; 3Phylos, Lexington, Massachusetts 02421 USA.
4   Corresponding author.

9:859-867 ©1999 by Cold Spring Harbor Laboratory Press  ISSN 1088-9051/99 $5.00
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