DNA oligonucleotide-assisted genetic manipulation increases transformation and homologous recombination efficiencies:: Evidence from gene targeting of Dictyostelium discoideum
DNA oligonucleotide-assisted genetic manipulation increases transformation and homologous recombination efficiencies:: Evidence from gene targeting of Dictyostelium discoideum
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DOI：
10.1016/j.jbiotec.2007.11.002
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发表时间：
2008-02-29
影响因子：
4.1
通讯作者：
Ueda, Masahiro
中科院分区：
文献类型：
--
作者：
Kuwayama, Hidekazu;Yanagida, Toshio;Ueda, Masahiro
通过在活细胞中进行同源重组的人工基因改变，被称为基因靶向，提供了体内基因功能的基础和相当多的知识。原则上，这种方法可以应用于任何类型的基因和可转化细胞。然而，由于内源靶基因与外源转基因之间同源重组的频率较低，其成功受到限制。在这里，我们描述了一种通用的基因靶向方法，其中DNA寡核苷酸（寡聚物）的共转化可以显著提高同源重组频率和转化效率。低聚物被简单地设计成它们与目标结构的同源侧翼区域的两端相同。利用这种策略，二倍体细胞的两个目标等位基因在一次转化过程中同时被替换。因此，该方法的简单性和通用性适用于任何类型的细胞，可能会增加基因靶向的应用。(C) 2007 Elsevier B.V.版权所有
Artificial gene alteration by homologous recombination in living cells, termed gene targeting, presents fundamental and considerable knowledge of in vivo gene function. In principle, this method can possibly be applied to any type of genes and transformable cells. However, its success is limited due to a low frequency of homologous recombination between endogenous targeted gene and exogenous transgene. Here, we describe a general gene-targeting method in which co-transformation of DNA oligonucleotides (oligomers) could significantly increase the homologous recombination frequency and transformation efficiency. The oligomers were simply designed such that they were identical to both the ends of the homologous flanking regions of the targeting construct. Using this strategy, both targeted alleles of diploid cells were simultaneously replaced in a single transformation procedure. Thus, the simplicity and versatility of this method applicable to any type of cell may increase the application of gene targeting. (C) 2007 Elsevier B.V. All rights reserved.