TILLING is an effective reverse genetics technique for Caenorhabditis elegans

Abstract

Background: TILLING (Targeting Induced Local Lesions in Genomes) is a reverse genetic technique based on the use of a mismatch-specific enzyme that identifies mutations in a target gene through heteroduplex analysis. We tested this technique in Caenorhabditis elegans, a model organism in which genomics tools have been well developed, but limitations in reverse genetics have restricted the number of heritable mutations that have been identified.

Forward and reverse mutagenesis in C. elegans

“…While this method has only been used once to isolate point mutation in a gene of interest in C. elegans , it has been used more in other organisms. Mutations in a population of C. elegans are generated using EMS or ENU [94]. PCR targeting a gene of interest is performed on genomic DNA derived from many pools of animals (Figure 4).…”

“…CJE cuts heteroduplex DNA where a mismatch or indel creates a single-stranded bulge. Samples are run on denaturing LI-COR gels, and the gels are examined in both fluorescent channels to determine in which pool a mismatch occurs [94]. In a screen for ten genes (ranging from 788 bp to 9.1 kb in size), 71 mutations were identified.…”

“…In a screen for ten genes (ranging from 788 bp to 9.1 kb in size), 71 mutations were identified. Of these, 59% constituted missense alleles, and 3% were nonsense alleles [94]. …”

“…PCR primers are designed over an area of 1.5 kb, mainly encompassing exonic regions. Primers can be designed using the program CODDLE to select for regions high in G/C content, which are most prone to mutation by EMS [94]. …”

A rich TILLING resource for studying gene function in Brassica rapa

“…[8-17]). For plants, large mutant populations are generated by the treatment of seed or pollen with a chemical mutagen - most commonly ethyl methane sulfonate (EMS) - that can induce point mutations at a very high density, sufficient to establish a series of allelic mutations in all genes.…”