High-Throughput Isolation of Caenorhabditis elegans Deletion Mutants

AUTOR(ES)

Liu, Leo X.

FONTE

Cold Spring Harbor Laboratory Press

RESUMO

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.

Documentos Relacionados

• High-Throughput Gene Mapping in Caenorhabditis elegans
• High-throughput reverse genetics: RNAi screens in Caenorhabditis elegans
• High-Throughput Expression of C. elegans Proteins
• Staphylococcus aureus Virulence Factors Identified by Using a High-Throughput Caenorhabditis elegans-Killing Model
• High-Throughput Method for Detecting Genomic-Deletion Polymorphisms