Kotzamanis George, Kotsinas Athanassios
Department of Histology and Embryology, School of Medicine, University of Athens, 75 Mikras Asias Str., Athens, 11527, Greece,
Methods Mol Biol. 2015;1227:123-39. doi: 10.1007/978-1-4939-1652-8_6.
BAC clones containing the entire genomic region of a gene including the long-range regulatory elements are very useful for gene functional analysis. However, large genes often span more than the insert of a BAC clone, and single BACs covering the entire region of interest are not available. Here, we describe a general system for linking two or more overlapping BACs into a single clone. Two rounds of homologous recombination are used. In the first, the BAC inserts are subcloned into the pBACLink vectors. In the second, the two BACs are combined together. Multiple BACs in a contig can be combined by alternating use of the pBACLInk vectors, resulting in several BAC clones containing as much of the genomic region of a gene as required. Such BACs can then be used in gene expression studies and/or gene therapy applications.
包含基因整个基因组区域（包括远距离调控元件）的BAC克隆对于基因功能分析非常有用。然而，大基因通常跨越超过一个BAC克隆的插入片段，并且没有覆盖整个感兴趣区域的单个BAC。在此，我们描述了一种将两个或更多重叠BAC连接成单个克隆的通用系统。使用两轮同源重组。在第一轮中，将BAC插入片段亚克隆到pBACLink载体中。在第二轮中，将两个BAC组合在一起。重叠群中的多个BAC可以通过交替使用pBACLink载体进行组合，从而产生几个包含所需基因基因组区域的BAC克隆。然后，这样的BAC可用于基因表达研究和/或基因治疗应用。
