Highly efficient modification of bacterial artificial chromosomes (BACs) using novel shuttle vectors containing the R6Kγ origin of replication
Highly efficient modification of bacterial artificial chromosomes (BACs) using novel shuttle vectors containing the R6Kγ origin of replication
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DOI：
10.1101/gr.476202
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发表时间：
2002-12-01
期刊：
影响因子：
7
通讯作者：
Heintz, N
中科院分区：
文献类型：
--
作者：
Gong, SC;Yang, XW;Heintz, N
细菌人工染色体（BAC）介导的转基因已被证明是获得准确转基因表达的一种高度可靠的方法，用于基因表达和功能的体内研究。利用该技术表征大量基因的一个限制性步骤是，BACs可以在大肠杆菌中通过同源重组进行修饰。我们在此报告了一种高效修饰bac的方法，通过使用一组新的穿梭载体，其中包含用于DNA复制的R6Kgamma起源，用于重组的大肠杆菌RecA基因和用于负选择的SacB基因。这些新的载体极大地增加了克隆穿梭载体的便利性，以及筛选协整和分辨克隆。此外，我们将穿梭载体克隆简化为一步，通过结合“内置”分辨率盒快速去除不需要的载体序列。这个新系统已被用于修改十几个bac。它非常适合于高效生产用于各种体内研究的改性bac。
Bacterial artificial chromosome (BAC) mediated transgenesis has proven to be a highly reliable way to obtain accurate transgene expression for in vivo studies of gene expression and function. A rate-limiting step in use of this technology to characterize large numbers of genes has been the process with which BACs can be modified by homologous recombination in Escherichia coli. We report here a highly efficient method for modifying BACs by using a novel set of shuttle vectors that contain the R6Kgamma origin for DNA replication, the E. coli RecA gene for recombination, and the SacB gene for negative selection. These new vectors greatly increased the ease with which one can clone the shuttle vectors, as well as screen for co-integrated and resolved clones. Furthermore, we simplify the shuttle vector cloning to one step by incorporation of a "built-in" resolution cassette for rapid removal of the unwanted vector sequences. This new system has been used to modify a dozen BACs. It is well suited for efficient production of modified BACs for use in a variety of in vivo studies.