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.

Results: To determine whether TILLING represents an effective reverse genetic strategy for C. elegans we generated an EMS-mutagenised population of approximately 1500 individuals and screened for mutations in 10 genes. A total of 71 mutations were identified by TILLING, providing multiple mutant alleles for every gene tested. Some of the mutations identified are predicted to be silent, either because they are in non-coding DNA or because they affect the third bp of a codon which does not change the amino acid encoded by that codon. However, 59% of the mutations identified are missense alleles resulting in a change in one of the amino acids in the protein product of the gene, and 3% are putative null alleles which are predicted to eliminate gene function. We compared the types of mutation identified by TILLING with those previously reported from forward EMS screens and found that 96% of TILLING mutations were G/C-to-A/T transitions, a rate significantly higher than that found in forward genetic screens where transversions and deletions were also observed. The mutation rate we achieved was 1/293 kb, which is comparable to the mutation rate observed for TILLING in other organisms.

Conclusion: We conclude that TILLING is an effective and cost-efficient reverse genetics tool in C. elegans. It complements other reverse genetic techniques in this organism, can provide an allelic series of mutations for any locus and does not appear to have any bias in terms of gene size or location. For eight of the 10 target genes screened, TILLING has provided the first genetically heritable mutations which can be used to study their functions in vivo.

Figure 1

Overview of the TILLING procedure. Pooled DNA is amplified using fluorescently tagged, gene-specific primers. The forward and reverse primers are labelled with different fluorophors that label both ends of the fragment. The amplified products are denatured by heating and then allowed to cool slowly so that they randomly re-anneal. Heteroduplex molecules form when mutant and wild-type PCR products anneal together, and these then become targets for a single-strand-specific nuclease found in Celery Juice Extract (CJE). The nuclease cleaves these heteroduplex fragments at one of the two strands, 3' to the site of the mismatch in the DNA. The PCR products that retain one of the labelled primers can then be detected on polyacrylamide denaturing LI-COR gels. Individuals with a mutation in the gene of interest are identified by the smaller cleavage fragment seen on the gel as well as the wild-type product. Because the nuclease cleaves either of the two strands randomly, cleavage products can be detected in both the IRD700 and IRD800 channels of the gel image. The position of the mutation within the PCR amplicon can be calculated from the size of the two fragments carrying the forward, IRD700-labeled primer, and the reverse, IRD800-labeled primer. Grey bands on the gel are thought to result from partial PCR products and aid in sizing of mutant bands.

Figure 2

Outline of C. elegans TILLING procedure. Animals are mutagenised with EMS, picked individually to plates, and allowed to self. One third of the worms are used for DNA and the remaining two thirds are frozen for future analysis. DNA is pooled 8-fold to reduce time and expense. TILLING is performed in order to determine which individuals carry mutations in the gene of interest. Mutations are sequenced and individuals from lines carrying mutations that have an effect on the gene product are thawed and genotyped to isolate heterozygous or homozygous mutants.

Figure 3

Gene models depicting the distribution of different types of mutations within the genes. The figure was designed from PARSESNP [42] output files. Blue lines indicate the extent of the amplified region that was used for TILLING. Orange open boxes denote exons. Purple up arrows indicate a change in the DNA sequence that does not affect the amino acid product. Purple down arrows indicate a change in non-coding DNA. Black up arrows indicate a change that induces a missense mutation in the predicted protein product. Red up arrows indicate a premature stop codon or splice junction error.

Figure 4

Restriction enzyme digests of DNA from heterozygous and homozygous mutants. A) CAPS analysis of sibling lines for CN646 htp-3(vc1) using the restriction enzyme Taq1. The lanes labelled N2 are wildtype controls. Lane marked 4 exhibits additional bands when digested with this enzyme indicating this line is heterozygous for the vc1 mutation. B) CAPS analysis of sibling lines for CN711 mdf-2(vc15), using the restriction enzyme Hinf1. The lanes labelled N2 are wildtype controls. Lanes marked 4, 5 and 6 show additional cleavage bands and are missing the wildtype band indicating that they are homozygous for the vc15 mutation.