is a well-established model system (reviewed by Hodgkin [1
]) that is increasingly being used for genetic and molecular investigations into conserved biological processes, including those involved in human disease [2
]. Although simple in structure, C. elegans
is comparable to higher animals in development and forms most of the major tissue types that are important to vertebrate physiology. Indeed, in a comparison of 18,452 C. elegans
protein sequences against human EST databases, 83% (15,344 sequences) of the C. elegans
sequences were found to have human homologues [6
Because the sequence of the complete C. elegans
genome has been available since 1998, bioinformaticians have been presented with ample opportunity to mine the data, and a plethora of genomic and proteomic information is accessible to researchers wishing to build upon this information [7
]. Powerful in silico
techniques have also been developed for the analysis of genome sequence information and are used in the prediction of gene function, expression and interaction [5
]. Despite the exciting possibilities flowing from these studies, the testing of predictions made in silico
relies largely on the existence of efficient reverse genetic approaches that target specific genes or classes of genes in vivo. In vitro
techniques such as yeast two-hybrid analysis [10
] and microarray analysis [11
] have also been used to generate an abundance of valuable data about gene expression and protein interactions but, like the data generated in silico
, these data need to be verified in vivo
has approximately 19,800 protein-coding genes and 12,000 of these have been conserved over the 100 million years since this species has diverged from the related nematode Caenorhabditis briggsae
, indicating that they are likely important functional genes [12
]. In spite of this fact, however, only about 3,400 genes in C. elegans
have mutant alleles available for genetic and biochemical analysis to ascertain their function and importance [13
]. High-throughput reverse genetics is an ideal way of generating mutations in the remaining 16,400 genes and several such approaches have been developed for the nematode each of which has advantages and drawbacks that affect the applicability or efficiency of the technique as a tool for probing gene function on a genomic scale.
Currently, the most efficient and popular method to disrupt the activity of a gene in C. elegans
is the technique of RNA interference (RNAi) [14
]. Large-scale RNAi screens have demonstrated that the function of a diverse population of genes with roles in many biological processes can be disrupted by the injection of double-stranded RNA (dsRNA) directly into the gonad [15
], by soaking the nematodes in a dsRNA solution [16
], or by feeding the nematodes bacteria expressing dsRNA [17
]. These same studies, however, have also documented that the phenotypes resulting from the RNAi treatment often depend on the method of delivery. In addition, the RNAi technique cannot replace classical genetic analysis because the phenotypic effects are transient and not heritable, making classical genetic interaction studies impossible.
Another effective reverse-genetic technique that is being used successfully in C. elegans is mutagenesis with trimethylpsoralen and ultraviolet radiation (TMP/UV) followed by detection of gene knockouts by PCR. This is currently the method of choice for obtaining heritable loss-of-function mutations in C. elegans but there are also drawbacks to this approach. First, the limitations of the detection method necessitate using a high dosage of mutagen which requires multiple rounds of outcrossing to remove accompanying background mutations. In addition, missense alleles cannot be isolated and large deletion events may result in the loss of function from more than one locus simultaneously. Finally, although the reason for this is unclear, mutations in certain genes have been more difficult to obtain than in others.
Transposon-insertion mutagenesis is another tool that is available to the C. elegans
] but it shares many of the limitations discussed for the previous techniques in addition to some that are specific to this approach such as the fact that small genes are less likely to be targets of transposon insertion and certain regions of the genome may vary in the frequency at which transposons insert. The mutagenic effect of Tc1 insertions can also sometimes be circumvented by innate compensation mechanisms that allow spicing around the transposon.
A recently reported study of biolistic transformation in C. elegans
indicates that homologous recombination of introduced DNA is also possible in this species [21
] but, in spite of the potential of this technique to provide the long-sought ability to perform site-directed mutagenesis in C. elegans
, the low success rate and the fact that an elaborate microparticle bombardment set-up is required, make it unlikely that this procedure will soon become efficient enough for high-throughput reverse genetics.
As a result of drawbacks in currently used reverse genetic techniques, the pace of research into biological processes in C. elegans
is still largely dictated by the probability of obtaining a mutant of any given gene and, thus, new techniques are needed to complement those previously described. TILLING (T
enomes) is a relatively novel reverse genetics technique based on the use of a mismatch-specific enzyme that will identify mutations in any target gene through heteroduplex analysis [22
]. The technique involves PCR amplification of a target gene or region of DNA using fluorescently labelled primers, followed by digestion with an enzyme that specifically cleaves at the site of a mismatch such as that induced by ethylmethanesulfonate (EMS) mutagenesis (see Figure ). The sizes of the cleavage fragments resolved on polyacrylamide gels reveal the approximate position of the mutation within the amplicon. We report here on a pilot project to test the use of this technology in C. elegans
: we have constructed and arrayed a mutagenised population and used it to isolate mutations in 10 different genes. On the basis of these data we conclude that TILLING is as effective and cost-efficient in C. elegans
as it has been shown to be in other species in which it has been tested [23
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 (more ...)