Preliminary Research On Targeted Gene Disruption In Zebrafish Using Zinc Finger Nucleases

The Danio rerio (Zebrafish) is an excellent nonmammalian vertebrate model for research on vertebrate gene function due to its small size, easy feeding, rapid development, ectogenesis, fast reproduction, high reproduction rate, transparent embryos, and easy manipulation. At present, studies of gene function and disease'gene therapy are focusing on reverse genetics. While gene targeting can only be used in mammalian, such as mice, due to the facile embryonic stem cell line. And current reverse genetics on zebrafish and other nonmammalian vertebrate are limited to TILLING, RNAi and Morpholinos. However, heritable mutants can not be obtained using these methods.Fortunately, zinc finger nuclease (ZFN) has been developed to mediate directed mutagenesis in nonmammalian vertebrate. ZFNs are engineering restriction enzymes, which can cut the specific DNA sequence. ZFNs are fused with zinc finger protein (ZFP) and the cleavage domain of the restriction enzyme FokⅠ. The ZFP can bind the flanking sequences of cleavage sites, while the cleavage domains of Fok I form the heterodimer and create a double-strand break (DSB) in genome. DSB may be repaired by non-homologous end-joining (NHEJ), which may generate mutation in genome.We describe a protocol of ZFNs-mediated directed knockout on mitfa gene in zebrafish. At first, choose a targeted DNA segment between 2965bp and 2988bp of mitfa and design the animo acid sequences of zinc finger proteins (ZFPs). Secondly, the coding sequences for the ZFPs are constructed using DNAWorks and two-step gene synthesis. Thirdly, these coding sequences and the cleavage domain FokI are both linked into an expression vector to form ZFNs. Fourthly, ZFNs were expressed in DH5a Escherichia coli with 0.3mM IPTG at 22℃, purified by His-bind column. Fifthly, the cleavage capacity of ZFNs are detected at 25℃in vitro. Finally, the coding sequences of ZFNs were injected into single-cell embryos. Then a mutagenesis was found at target site of mitfa in zebrafish.A simple and rapid method for gene synthesis was used in this protocol. The soluble ZFNs were obtained using an optimization expression culture medium. The CMV promoter was performed to make ZFNs expressed widely in zebrafish. Hence, this protocol is much simpler than other reported protocols for directed mutagenesis in zebrafish.