A highly efficient Escherichia coli-based chromosome engineering system adapted for recombinogenic targeting and subcloning of BAC DNA
A highly efficient Escherichia coli-based chromosome engineering system adapted for recombinogenic targeting and subcloning of BAC DNA
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DOI：
10.1006/geno.2000.6451
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发表时间：
2001-04-01
期刊：
影响因子：
4.4
通讯作者：
Copeland, NG
中科院分区：
文献类型：
--
作者：
Lee, EC;Yu, DG;Copeland, NG
最近，描述了用于大肠杆菌中染色体工程的高效重组系统，其使用缺陷性A原噬菌体来提供保护线性DNA靶向盒并使其与其底物序列重组的功能（Yu et al.，2000，Proc. Natl. Acad. Sci. USA 97，5978-5983）。重要的是，重组精通短至30-50 bp的DNA同源性，使得可以使用PCR扩增的片段作为靶向盒。在这里，我们适应这种原噬菌体系统用于细菌人工染色体（BAC）工程，将其转移到DH 1 OB细胞，BAC宿主菌株。此外，将阿拉伯糖诱导的cre和flpe基因引入这些细胞中以促进使用loxP和FRT位点的BAC修饰。接下来，我们通过使用该重组系统将Cre靶向到250-kb BAG上携带的小鼠神经元特异性烯醇化酶（Eno 2）基因的3 '末端来证明该重组系统的实用性，这使得有可能产生在所有成熟神经元中特异性表达Cre的BAC转基因小鼠。此外，我们表明，大至80 kb的片段可以亚克隆从BAC间隙修复使用这种重组系统，避免了对限制性内切酶或DNA连接酶的需要。最后，我们表明，BAC可以修改与此重组系统中的药物选择的情况下。精确修饰或亚克隆基因组DNA大片段的能力将有助于许多以前难以或不可能进行的基因组实验，并有助于后基因组时代的基因功能研究。(C)北京：科学出版社.
Recently, a highly efficient recombination system for chromosome engineering in Escherichia coli was described that uses a defective A prophage to supply functions that protect and recombine a linear DNA targeting cassette with its substrate sequence (Yu ct al., 2000, Proc. Natl. Acad. Sci. USA 97, 5978-5983). Importantly, the recombination is proficient with DNA homologies as short as 30-50 bp, making it possible to use PCR-amplified fragments as the targeting cassette. Here, we adapt this prophage system for use in bacterial artificial chromosome (BAC) engineering by transferring it to DH1OB cells, a BAC host strain. In addition, arabinose inducible cre and flpe genes are introduced into these cells to facilitate BAC modification using loxP and FRT sites. Next, we demonstrate the utility of this recombination system by using it to target cre to the 3 ' end of the mouse neuron-specific enolase (Eno2) gene carried on a 250-kb BAG, which made it possible to generate BAC transgenic mice that specifically express Cre in all mature neurons. In addition, we show that fragments as large as 80 kb can be subcloned from BACs by gap repair using this recombination system, obviating the need for restriction enzymes or DNA Ligases. Finally, we show that BACs can be modified with this recombination system in the absence of drug selection. The ability to modify or subclone large fragments of genomic DNA with precision should facilitate many kinds of genomic experiments that were difficult or impossible to perform previously and aid in studies of gene function in the postgenomic era. (C) 2001 Academic Press.