Creation of targeted mutations in the Zebrafish using zinc finger nucleases. Creation of targeted mutations in the Zebrafish using zinc finger nucleases

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 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 mutagenenesis 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 (ZFNs) have been shown to be effective for gene targeting. ZFNs are engineered that recognize sequences in Zebrafish genes. Co-injection of mRNAs encoding these ZFNs into one-cell-stage Zebrafish embryos lead to mutagenic lesions at the target site that are transmitted through the germ line with high frequency. The use of engineered ZFNs to introduce heritable mutations into a genome obviates the need for embryonic stem cell lines and should be applicable to most animal species for which early stage embryos are easily accessible. This project was created to identify lesions created by ZFNs in pools of Zebrafish embryos for rapid genotyping of ZFN injected embryos, to negate the need for cloning and re-sequencing.