Direct genomic manipulation at a specific locus is still not feasible in most vertebrate model organisms. In vertebrate cell lines, genomic lesions at a specific site have been introduced using zinc-finger nucleases (ZFNs) . Here we adapt this technology to create targeted mutations in the zebrafish germ line. ZFNs were engineered that recognize sequences in the zebrafish ortholog of the vascular endothelial growth factor-2 receptor, kdr (also known as kdra ). Co-injection of mRNAs encoding these ZFNs into one-cell-stage zebrafish embryos led to mutagenic lesions at the target site that were 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. The ability to perform targeted genomic manipulation in the mouse using embryonic stem cell lines has established it as a central vertebrate model system . Similar manipulations in other vertebrate models have largely failed owing to the difficulty of generating embryonic stem cell lines. ZFNs, which are a chimeric fusion between a Cys 2 His 2 zinc-finger protein (ZFP) and the cleavage domain of
Nature Biotechnology – Springer Journals
Published: May 25, 2008
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