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 ∼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.
Footnotes
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Present addresses: 1Cambria Biosciences, Waltham, Massachusetts 02454 USA; 2Therion Biologics, Cambridge, Massachusetts 02138 USA; 3Phylos, Lexington, Massachusetts 02421 USA.
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↵4 Corresponding author.
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E-MAIL lliu{at}cambriabio.com; FAX (781) 736-3107.
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- Received May 18, 1999.
- Accepted July 15, 1999.
- Cold Spring Harbor Laboratory Press