Ends-out, or replacement, gene targeting in Drosphila
Ends-out, or replacement, gene targeting in Drosphila
复制标题
DOI：
10.1073/pnas.0535280100
复制
发表时间：
2003-03-04
影响因子：
11.1
通讯作者：
Golic, KG
中科院分区：
文献类型：
--
作者：
Gong, WJ;Golic, KG
末端向内和末端向外是指可用于基因靶向的供体DNA的两种排列。两者都已用于靶向诱变，但需要不同设计的供体。末端外靶向更常用于小鼠和酵母，因为它提供了一种直接的途径来替换或删除靶位点。虽然末端进入靶向在果蝇属中是成功的，但末端出去靶向的尝试失败了。为了测试末端外靶向是否可以用于果蝇，我们应用了两种策略在果蝇的内源性黄色（y）基因座进行末端外基因置换。首先，以约1/800配子的速率用一个8-kb的y（+）DNA片段替换突变等位基因。第二，野生型基因在外显子1中插入标记基因，以约1/380配子的比例被破坏。单独的I-SceI内切核酸酶组分不足以靶向：还需要FLP重组酶来产生染色体外供体。当使用这两种组分时，我们发现末端外靶向可以近似地与末端内靶向一样有效，并且可能通常用于果蝇基因靶向。
Ends-in and ends-out refer to the two arrangements of donor DNA that can be used for gene targeting. Both have been used for targeted mutagenesis, but require donors of differing design. Ends-out targeting is more frequently used in mice and yeast because it gives a straightforward route to replace or delete a target locus. Although ends-in targeting has been successful in Drosophiia, an attempt at ends-out targeting failed. To test whether ends-out targeting could be used in Drosophila, we applied two strategies for ends-out gene replacement at the endogenous yellow (y) locus in Drosophila. First, a mutant allele was rescued by replacement with an 8-kb y(+) DNA fragment at a rate of approximate to1/800 gametes. Second, a wild-type gene was disrupted by the insertion of a marker gene in exon 1 at a rate of approximate to1/380 gametes. The I-SceI endonuclease component alone is not sufficient for targeting: the FLP recombinase is also needed to generate the extrachromosomal donor. When both components are used we find that ends-out targeting can be approximately as efficient as ends-in targeting, and is likely to be generally useful for Drosophila gene targeting.