Chen Qingwen, Narayanan Kumaran
School of Science, Monash University Malaysia, Bandar Sunway, Malaysia.
Methods Mol Biol. 2015;1227:27-54. doi: 10.1007/978-1-4939-1652-8_2.
Recombineering is a powerful genetic engineering technique based on homologous recombination that can be used to accurately modify DNA independent of its sequence or size. One novel application of recombineering is the assembly of linear BACs in E. coli that can replicate autonomously as linear plasmids. A circular BAC is inserted with a short telomeric sequence from phage N15, which is subsequently cut and rejoined by the phage protelomerase enzyme to generate a linear BAC with terminal hairpin telomeres. Telomere-capped linear BACs are protected against exonuclease attack both in vitro and in vivo in E. coli cells and can replicate stably. Here we describe step-by-step protocols to linearize any BAC clone by recombineering, including inserting and screening for presence of the N15 telomeric sequence, linearizing BACs in vivo in E. coli, extracting linear BACs, and verifying the presence of hairpin telomere structures. Linear BACs may be useful for functional expression of genomic loci in cells, maintenance of linear viral genomes in their natural conformation, and for constructing innovative artificial chromosome structures for applications in mammalian and plant cells.
重组工程是一种基于同源重组的强大基因工程技术，可用于独立于DNA序列或大小对其进行精确修饰。重组工程的一个新应用是在大肠杆菌中组装线性细菌人工染色体（BAC），其可作为线性质粒自主复制。将来自噬菌体N15的短端粒序列插入环状BAC中，随后通过噬菌体原端粒酶切割并重新连接，以产生具有末端发夹端粒的线性BAC。端粒封端的线性BAC在体外和大肠杆菌细胞体内均受到核酸外切酶攻击的保护，并且可以稳定复制。在这里，我们描述了通过重组工程将任何BAC克隆线性化的分步方案，包括插入和筛选N15端粒序列的存在、在大肠杆菌体内将BAC线性化、提取线性BAC以及验证发夹端粒结构的存在。线性BAC可用于细胞中基因组位点的功能表达、以天然构象维持线性病毒基因组，以及构建用于哺乳动物和植物细胞应用的创新人工染色体结构。
