Meng Xiangdong, Noyes Marcus B, Zhu Lihua J, Lawson Nathan D, Wolfe Scot A
Program in Gene Function and Expression, University of Massachusetts Medical School, Lazare Research Building, 6th Floor, 364 Plantation St., Worcester, Massachusetts 01605, USA.
Nat Biotechnol. 2008 Jun;26(6):695-701. doi: 10.1038/nbt1398. Epub 2008 May 25.
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.
在大多数脊椎动物模型生物中，在特定基因座进行直接基因组操作仍然不可行。在脊椎动物细胞系中，已使用锌指核酸酶（ZFN）在特定位点引入基因组损伤。在这里，我们采用这项技术在斑马鱼生殖系中产生靶向突变。我们设计了能识别斑马鱼血管内皮生长因子-2受体kdr（也称为kdra）直系同源物序列的ZFN。将编码这些ZFN的mRNA共同注射到单细胞期斑马鱼胚胎中，导致靶位点出现诱变损伤，并以高频率通过生殖系传递。使用工程化ZFN将可遗传突变引入基因组，无需胚胎干细胞系，并且应该适用于大多数易于获取早期胚胎的动物物种。
