large-scale screening for targeted knockouts in the Caenorhabditis elegans genome

Abstract

The nematode Caenorhabditis elegans is a powerful model system to study contemporary biological problems. This system would be even more useful if we had mutations in all the genes of this multicellular metazoan. The combined efforts of the C. elegans Deletion Mutant Consortium and individuals within the worm community are moving us ever closer to this goal. At present, of the 20,377 protein-coding genes in this organism, 6764 genes with associated molecular lesions are either deletions or null mutations (WormBase WS220). Our three laboratories have contributed the majority of mutated genes, 6841 mutations in 6013 genes. The principal method we used to detect deletion mutations in the nematode utilizes polymerase chain reaction (PCR). More recently, we have used array comparative genome hybridization (aCGH) to detect deletions across the entire coding part of the genome and massively parallel short-read sequencing to identify nonsense, splicing, and missense defects in open reading frames. As deletion strains can be frozen and then thawed when needed, these strains will be an enduring community resource. Our combined molecular screening strategies have improved the overall throughput of our gene-knockout facilities and have broadened the types of mutations that we and others can identify. These multiple strategies should enable us to eventually identify a mutation in every gene in this multicellular organism. This knowledge will usher in a new age of metazoan genetics in which the contribution to any biological process can be assessed for all genes.

Keywords: deletion mutations; genomics; knockouts; multi-gene families.

Figure 1

PCR/deletion screening. Worms were grown in liquid culture in arrays of 96. A portion of each well was harvested for DNA preparation while the rest was frozen for later retrieval of animals. DNA was pooled into a grid of rows and columns, which could later be used to address individual wells. Nested PCR primers were designed for amplification of target region. Primers were designed using the program Aceprimer (McKay and Jones 2002). Shown are four slab gels stained with SYBR Gold used to identify deletions (faster running bands) from the wild-type DNA (slower bands). The four panels are (A) the initial screen, (B) first sib, (C) second sib, and (D) third sib. The WT band for this primer set is 2099 bp (long arrow), and the marker is a 100-bp molecular weight ladder with strong intensity bands at 1 kb and 3 kb (marked by horizontal bars on right hand side of image). The screening image (A) shows two different hits (in duplicate screening, short arrows), one at just over 1 kb (18^th set of four lanes, in first two lanes of the four), and one at about 1.6 kb (23^rd set of four lanes, in first two lanes). The one at 1 kb passed addressing but was not recovered in sib selection, so the remaining images (B–D) are for the 1.6 kb candidate. Through rounds of sib selection, one enriches for animals segregating the deletion band. This enrichment proceeds from initial detection in (A), a mix of hundreds to thousands of animals, to first-round sibbing in (B), tens of animals, and finally single animal picks in sib2 (C) and sib3 (D), where one has single animals segregating the deletion band. For sib3 (D), there are 4 × 24 single-worm populations, 1–4. Populations 1 and 2 are in the first half of the comb, and 3 and 4 are in the second half. Set 3 is actually 24/24 positive, whereas the others are less than that, so set 3 was picked to go forward as it was homozygous at that point. The example shown is gk3287 in the gene F11E6.1 (gba-3).

Figure 2

WormBase view of image and annotation for deletions. (A) Screenshot of four genes on chromosome V. Red bars denote deletions, and the length of the bar indicates size of the deletion. (B) The red bars in (A) are hot links to text describing the deletions in greater detail. Besides the details on deletion breakpoints, primers used to amplify the deletion region are listed. Depicted here is the link for deletion tm1530.

Figure 3

Comparison of distribution of all the mutations (black) and only the lethal mutations (red) throughout the whole genome. This figure is based on 6764 total genes and 1436 essential genes (WS220).