| description |
The Zebrafish is becoming a popular model organism for developmental biology and human disease. This increasing popularity has resulted in a demand for reverse genetics, which has traditionally been neglected in favour of forward genetic approaches because of the lack of an efficient gene knockout (KO) technology. Thousands of phenotypes have been identified using forward genetic approaches, but time consuming positional cloning has slowed the identification of the genes involved. Morpholino oligonucleotides have become the method of choice for gene knockdown in the Zebrafish, but are restricted to early processes, and insertional mutagenesis has also become a popular method for gene led investigations. However, this still does not allow the targeted knockdown of genes of choice. More recently, zinc finger nucleases have been shown to be effective for gene targeting, but this method can be expensive and labour intensive, and is not seen as practical for the KO of hundreds of genes.A project to sequence and finish (to 99.9% accuracy) the Zebrafish genome (one of only 3 vertebrate genomes to be completed to this accuracy, alongside human and mouse) has contributed to the effectiveness of a process termed TILLING, first described in arabadopsis and worm, to be used to create Zebrafish targeted alleles. This method traditionally uses cel-1 nuclease and fragment analysis to identify mutations, which is labour intensive and misses mutations, especially near SNPs, which are common. Here we show that by using re-sequencing, we are able to identify more mutations. Using this method we have screened 2 ENU mutagenised living libraries in 2 years, and identified ~200 KO. In total, the ZF Models Workpackage 4 collaboration has identified 243 Mutations in the Zebrafish, which will be publicly available via Ensembl, The Sanger Institute and ZIRC. In addition to identifying deleterious mutations in the Zebrafish, the Sanger Institiute has committed to describing the phenotype of all mutations created during this project. Both morphological and molecular (by microarray or transcript counting on the Illumina platform) phenotypes will be described and published via ensembl and Zfin. The advent of next generation sequencing technologies has significantly changed the amount of mutations that can be identified and has opened up the possibility of identifying a non-sense allele in every protein coding gene within a reasonable time frame and budget. We have designed a strategy that will allow us to amplify and sequence thousands of exonic fragments on a single lane of the Illumina sequencing platform. A primer design program has been written that selects 100bp exonic fragments that are most likely to give a non-sense allele by ENU mutagenesis. These fragments (up to 4000 exons per experiment) are amplified across DNA pools from 24 mutagenised Zebrafish, and each pool is run on one lane of the Illumina platform. Automated data analysis then searches the sequence for all potential non-sense alleles in the amplified fragments, and putative non-sense alleles are confirmed by capillary sequencing. Using this method the Zebrafish Mutation Resource aims to produce >1000 knockouts over the next 2 years, creating a rich resource for the Zebrafish research community. |