Targeted gene inactivation in zebrafish using engineered zinc-finger nucleases.
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.
Meng
Xiangdong
X
Program in Gene Function and Expression, University of Massachusetts Medical School, Lazare Research Building, 6th Floor, 364 Plantation St., Worcester, Massachusetts 01605, USA.
Noyes
Marcus B
MB
Zhu
Lihua J
LJ
Lawson
Nathan D
ND
Wolfe
Scot A
SA
D000818:Animals; D030801:Animals, Genetically Modified / Q000502:physiology*; D003851:Deoxyribonucleases / Q000235:genetics; D020868:Gene Silencing; D018390:Gene Targeting / Q000379:methods*; D005818:Genetic Engineering / Q000379:methods*; D016297:Mutagenesis, Site-Directed / Q000379:methods; D015202:Protein Engineering / Q000379:methods; D015027:Zebrafish / Q000235:genetics*; D029961:Zebrafish Proteins / Q000235:genetics*; D016335:Zinc Fingers / Q000235:genetics*
D029961:Zebrafish Proteins; D003851:Deoxyribonucleases