Abstract
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)1,2,3. 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.
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Acknowledgements
We would like to thank Keith Joung for helpful discussions and for pST1374. We thank Charles Sagerstrom and Arndt Siekmann for critical reading of this manuscript. We are grateful to Mike Kacergis for valuable assistance in fish care and genotyping. We are grateful to Claude Gazin, Ellen Kittler and Maria Zapp for providing MPSS protocols and technical advice in support of this work. We also thank the UMass Deep Sequencing Core for MSPP analysis service and David LaPointe for providing bioinformatic and computational support. X.M., M.B.N. and S.A.W. and their work were supported by 1R01GM068110 from National Institute of General Medical Sciences. N.D.L. and his work was supported by R01HL079266 from National Heart, Lung, and Blood Institute.
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X.M., N.D.L. and S.A.W. designed experiments and analyzed data. X.M. and N.D.L. carried out experiments. M.B.N. developed the omega-based bacterial one-hybrid system. S.A.W. developed the algorithm and PERL script to identify preferred recognition elements. L.J.Z. analyzed Solexa sequencing data. X.M., N.D.L. and S.A.W. wrote the paper.
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Meng, X., Noyes, M., Zhu, L. et al. Targeted gene inactivation in zebrafish using engineered zinc-finger nucleases. Nat Biotechnol 26, 695–701 (2008). https://doi.org/10.1038/nbt1398
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DOI: https://doi.org/10.1038/nbt1398
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