TILLING is the most established method of reverse genetics available in the zebrafish; however, new methodologies, reviewed in this volume, are becoming available. Insertional mutagenesis projects using pseudotyped retroviruses have produced large libraries of F1 fish carrying as many as 25 random heterozygous insertions that can be sequenced and mapped to produce an indexed library of mutant loci recoverable from cryopreserved sperm [32
] (see chapter by S. Burgess). Because retroviruses tend to integrate into the first introns of genes or into exons, many of these integrations are mutagenic as determined by a strong reduction in RNA levels. Zinc-finger nucleases, which can be designed to deliver frameshift mutations at defined target sites in the genome, have also recently been applied to zebrafish [33
] (see chapter by S. Amacher). To date, TILLING efforts have focused on identifying loss-of-function mutations, in the form of nonsense or splice alleles; however, as these other methods for generating loss-of-function mutations become more accessible and affordable, the unique ability of TILLING to generate allelic series, including a range of hypomorphic and even temperature-sensitive alleles caused by missense mutations, will become increasingly valuable. To this end, we and others continue to explore ways to improve the effectiveness and efficiency of TILLING methodologies.
Although current TILLING projects have produced loss-of-function alleles in over 150 zebrafish genes to date, the general approach is limited by the size of the available libraries and the low-to-medium throughput of the screening methodologies. To address the first of these, we have established a consortium of three labs (C. Moens, L. Solnica-Krezel and J. Postlethwait), who have independently established cryopreserved TILLING libraries and Cel1 screening methodology. Consortium-based screening will improve the likelihood of finding one or more loss-of-function alleles of genes submitted by the zebrafish community. Currently, the largest cryopreserved TILLING library of 8640 F1 fish has only a 58% chance of containing a nonsense mutation in any given 1 kb of coding sequence. This number climbs to 83% in the combined libraries of the consortium labs. By late 2008, members of the community will be able to submit requests to the consortium online. The zebrafish community will also continue to have access to the resequencing power of the Sanger Center with the ongoing Zebrafish Mutation Resource offered by the Stemple lab (http://www.sanger.ac.uk/Projects/D_rerio/mutres/
New screening methodologies are needed to address the issue of throughput. In our hands, Cel1 screening proceeds at a rate of about one fragment per week. Automated resequencing projects, such as the Sanger Center's, have a significantly higher throughput but remain labour and machine intensive. The advent of massively parallel sequencing platforms, that can generate more than half a billion base pairs of high-quality sequence per run, can be applied to TILLING. Here, the challenge is to uniquely identify genomic DNA templates from individual fish in large pooled populations, and to enrich pooled genomic DNAs for targets of interest to reduce the complexity of the sequencing template. By ligating oligonucleotide ‘barcodes’ to sheared genomic DNA from individuals or small pools, it may be possible to screen ‘superpools’ representing up to 8000 individuals in a single Solexa or equivalent sequencing run. Template DNA can be enriched for targets of interest by gene-specific PCR or by hybridization capture to custom microarrays [35
] or streptavidin beads loaded with biotinylated target-specific oligonucleotides. Using these tools it may be possible to screen as many as 50 500 bp targets regions in a single 8-lane Solexa run—a significant improvement in throughput and cost over either current methodology.
- TILLING is a method for identifying induced mutations in chemically mutagenized genomes.
- Zebrafish TILLING projects have identified over 150 loss-of-function mutations in genes that have not been identified in forward genetic screens.
- TILLING can be performed by mismatch detection using the enzyme Cel1 on pools of mutagenized F1 fish, or by direct resequencing of individual fish.
- TILLING is complementary to other reverse genetics approaches that are being developed in zebrafish.
- New approaches that will improve the effectiveness and efficiency of TILLING in zebrafish are being developed.