High-throughput isolation of Caenorhabditis elegans deletion mutants - PubMed (original) (raw)

J M Spoerke, E L Mulligan, J Chen, B Reardon, B Westlund, L Sun, K Abel, B Armstrong, G Hardiman, J King, L McCague, M Basson, R Clover, C D Johnson

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High-throughput isolation of Caenorhabditis elegans deletion mutants

L X Liu et al. Genome Res. 1999 Sep.

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 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.

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Figures

Figure 1

Figure 1

Schematic of mutant library construction and screening. F1 larvae of chemically mutagenized P0 animals are cultured in microtiter arrays to produce F2 larvae, constituting the worm library. An aliquot of worms from each well is removed and processed into a corresponding genomic DNA array; simultaneously, the 96 samples from each plate are pooled and processed to produce genomic DNA plate pools. The DNA plate pools are screened by nested PCR in 96-well plates for deletion amplicons, which identify a positive worm library plate Y. After confirmation by quadruplicate PCR (not illustrated), the genomic DNA array for that specific library plate is similarly screened to identify the specific well. The corresponding well is then retrieved from the frozen mutant worm library and thawed. Individual live worms are allowed to have self-progeny in microtiter wells, and aliquots of each well are tested by PCR to determine which of the thawed worms contain the deletion (see Methods for details).

Figure 2

Figure 2

Deletion size by mutagen, depicting those deletions selected with a PCR screening window of 2800–3400 bp. Not shown are a small number of larger (>3000 bp) deletions identified with PCR primers placed 4–5 kb apart in the target region, as well as smaller (<500 bp) deletions fortuitously detected on screening of specific library plates. Horizontal bar, average deletion size for each mutagen.

Figure 3

Figure 3

Schematics of representative deletions. (a) The deletion of daf-18 PTEN removes a portion of exon 3, which encodes the distal portion of the tensin homology domain and the catalytic phosphatase domain known to be required for lipid phosphatase activity (Gil et al. 1999). (b) The sel-12 deletion removes exons 3–6, which encode predicted transmembrane domains necessary for proper protein topology (Westlund 1998). (c) The cdc-25 deletion removes the entire ORF.

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