Mini-λ:: a tractable system for chromosome and BAC engineering
Mini-λ:: a tractable system for chromosome and BAC engineering
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DOI：
10.1016/s0378-1119(03)00728-5
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发表时间：
2003-10-02
期刊：
影响因子：
3.5
通讯作者：
Sharan, SK
中科院分区：
文献类型：
--
作者：
Court, DL;Swaminathan, S;Sharan, SK
噬菌体lambda （lambda）重组系统Red已被用于将大片段DNA克隆到P1和细菌人工染色体（BAC或PAC）载体上。到目前为止，这种重组系统已被用于将BAC或PAC克隆转移到含有有缺陷的lambda噬菌体的细菌细胞中。在这里，我们描述了一种迷你λ DNA的产生，它可以提供红色重组功能，并且可以很容易地通过电穿孔引入任何大肠杆菌菌株，包括携带dh10b的BACs或PACs。迷你DNA作为有缺陷的前噬菌体整合到细菌染色体中。此外，由于它保留了附着位点，它可以被切除以治愈噬菌体DNA的细胞。我们在这里描述了通过在BAC克隆的载体序列中引入一个可选择的标记物来修饰BAC的mini-lambda重组系统的使用。此外，使用mini-lambda，我们在没有使用任何可选择标记的情况下，在BAC中克隆的人类BRCA2基因中创建了单个错义突变。这种非常有效地产生重组的能力证明了mini-h作为一种非常简单的移动系统的有用性，可以通过同源重组进行体内基因组工程，这一过程被称为重组。(C) 2003 Elsevier B.V.版权所有
The bacteriophage lambda (lambda) recombination system Red has been used for engineering large DNA fragments cloned into P1 and bacterial artificial chromosomes (BAC or PAC) vectors. So far, this recombination system has been utilized by transferring the BAC or PAC clones into bacterial cells that harbor a defective lambda prophage. Here we describe the generation of a mini-lambda DNA that can provide the Red recombination functions and can be easily introduced by electroporation into any E. coli strain, including the DH10B-carrying BACs or PACs. The mini-lambda DNA integrates into the bacterial chromosome as a defective prophage. In addition, since it retains attachment sites, it can be excised out to cure the cells of the phage DNA. We describe here the use of the mini-lambda recombination system for BAC modification by introducing a selectable marker into the vector sequence of a BAC clone. In addition, using the mini-lambda, we create a single missense mutation in the human BRCA2 gene cloned in a BAC without the use of any selectable marker. The ability to generate recombinants very efficiently demonstrates the usefulness of the mini-h as a very simple mobile system for in vivo genome engineering by homologous recombination, a process named recombineering. (C) 2003 Elsevier B.V. All rights reserved.