High-throughput target-selected gene inactivation in zebrafish.

There is an increasing requirement for efficient reverse genetics in the zebrafish, Here we describe a method that takes advantage of conventional mutagenized libraries (identical to ones used in forward screens) and re-sequencing to identify ENU-induced mutations in genes of interest. The efficiency of TILLING (Targeting Induced Local Legions IN Genomes) depends on the rate of mutagenesis in the library being screened, the amount of base pairs screened, and the ability to effectively identify and retrieve mutations on interest. Here we show that by improving the mutagenesis protocol, using in silico methods to predict codon changes for target selection, efficient PCR and re-sequencing, and accurate mutation detection we can vastly improve current TILLING protocols. Importantly it is also possible to use this method for screening for splice and mis-sense mutations, and with even a relatively small library, there is a high chance of identifying mutations across any given gene.

Single nucleotide polymorphism (SNP) panels for rapid positional cloning in zebrafish.

Despite considerable genetic and genomic resources the positional cloning of forward mutations remains a slow and manually intensive task, typically using gel based genotyping and sequential rounds of mapping. We have used the latest genetic resources and genotyping technologies to develop two commercially available SNP panels of thousands of markers that can be used to speed up positional cloning.