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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Efficient Gene Targeting In Drosophila By Direct Embryo Injection With Zinc-finger Nucleases
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Efficient Gene Targeting In Drosophila By Direct Embryo Injection With Zinc-finger Nucleases

机译：锌指核酸酶直接胚胎注射在果蝇中的高效基因靶向。

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摘要

We report very high gene targeting frequencies in Drosophila by direct embryo injection of mRNAs encoding specific zinc-finger nucleases (ZFNs). Both local mutagenesis via nonhomologous end joining (NHEJ) and targeted gene replacement via homologous recombination (HR) have been achieved in up to 10% of all targets at a given locus. In embryos that are wild type for DNA repair, the products are dominated by NHEJ mutations. In recipients deficient in the NHEJ component, DNA ligase IV, the majority of products arise by HR with a coinjected donor DNA, with no loss of overall efficiency in target modification. We describe the application of the ZFN injection procedure to mutagenesis by NHEJ of 2 new genes in Drosophila melanogaster. coil and pask. Pairs of novel ZFNs designed for targets within those genes led to the production of null mutations at each locus. The injection procedure is much more rapid than earlier approaches and makes possible the generation and recovery of targeted gene alterations at essentially any locus within 2 fly generations.
机译：我们通过直接胚胎注射编码特定锌指核酸酶（ZFNs）的mRNA报告果蝇中很高的基因靶向频率。通过非同源末端连接（NHEJ）进行的局部诱变和通过同源重组（HR）进行的靶基因替代都在给定基因座的所有靶标中达到了10％。在野生型的DNA修复胚胎中，产物以NHEJ突变为主。在NHEJ成分DNA连接酶IV缺乏的受体中，大多数产物是由HR与共注射的供体DNA共同产生的，而不会影响靶标修饰的整体效率。我们描述了ZFN注射程序在果蝇中2个新基因的NHEJ诱变中的应用。卷和掌。针对那些基因中的靶标设计的成对的新型ZFN导致在每个基因座处产生无效突变。注射过程比以前的方法快得多，并使得在2个蝇代内的任何基因座上靶基因改变的产生和恢复成为可能。

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