High-throughput isolation of Caenorhabditis elegans deletion mutants.
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.
Liu
L X
LX
Axys Pharmaceuticals, NemaPharm Group, South San Francisco, California 94080, USA. lliu@cambriabio.com
Spoerke
J M
JM
Mulligan
E L
EL
Chen
J
J
Reardon
B
B
Westlund
B
B
Sun
L
L
Abel
K
K
Armstrong
B
B
Hardiman
G
G
King
J
J
McCague
L
L
Basson
M
M
Clover
R
R
Johnson
C D
CD
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
D009153:Mutagens