An efficient method for high-fidelity BAC/PAC retrofitting with a selectable marker for mammalian cell transfection
An efficient method for high-fidelity BAC/PAC retrofitting with a selectable marker for mammalian cell transfection
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DOI：
10.1101/gr.159001
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发表时间：
2001-01-01
期刊：
影响因子：
7
通讯作者：
Storb, U
中科院分区：
文献类型：
--
作者：
Wang, ZD;Engler, P;Storb, U
大规模的基因组测序计划已经提供了许多基因的DNA序列信息，但大多数基因的生物学功能只能通过功能研究才能知道。细菌人工染色体（Bacterial artificial chromosomes，BAC）和Pi衍生的人工染色体（Pi-derived artificial chromosomes，PAC）是稳定地保存在细菌中的大的基因组克隆，并且由于它们的大尺寸和稳定性，在通过转染进行功能研究中是非常重要的。因为大多数BAC或PAC载体不具有哺乳动物选择标记，所以用克隆在BAC或PAC中的基因转染哺乳动物细胞需要将哺乳动物选择标记插入BAC/PAC中。然而，目前可用的程序是不令人满意的效率和保真度。我们描述了一个非常简单和有效的程序，允许一个改造几十个BAQ在一天内没有检测到的缺失或不必要的重组。我们使用BAC/PAC改造载体，其在转化成感受态BAC或PAC菌株时，将通过体内cre/loxP位点特异性重组催化自身特异性插入BAC/PAC载体。
Large-scale genomic sequencing projects have provided DNA sequence information for many genes, but the biological Functions for most of them will only be known through functional studies. Bacterial artificial chromosomes (BACs) and Pi-derived artificial chromosomes (PACs] are large genomic clones stably maintained in bacteria and are very important in functional studies through transfection because of their large size and stability. Because most BAC or PAC vectors do not have a mammalian selection marker, transfecting mammalian cells with genes cloned in BACs or PACs requires the insertion into the BAC/PAC of a mammalian selectable marker. However, currently available procedures are not satisfactory in efficiency and fidelity. We describe a very simple and efficient procedure that allows one to retrofit dozens of BAQ in a day with no detectable deletions or unwanted recombination. We use a BAC/PAC retrofitting vector that, on transformation into competent BAC or PAC strains, will catalyze the specific insertion of itself into BAC/PAC vectors through in vivo cre/loxP site-specific recombination.