Takken Frank L W, Van Wijk Ringo, Michielse Caroline B, Houterman Petra M, Ram Arthur F J, Cornelissen Ben J C
Swammerdam Institute for Life Sciences, Plant Pathology, University of Amsterdam, Kruislaan 318, 1098 SM, Amsterdam, The Netherlands.
Curr Genet. 2004 Apr;45(4):242-8. doi: 10.1007/s00294-003-0481-5. Epub 2004 Jan 24.
Bacterial artificial chromosomes (BACs) are widely used for the construction of physical maps, positional-cloning and whole-genome sequencing strategies. Unfortunately, their use for functional genomics is limited, as currently there is no efficient method to use BACs directly for complementation. We describe a novel strategy for one-step conversion of any BAC into a binary BAC (BIBAC). Using Agrobacterium tumefaciens, these BIBACs can be efficiently transformed to virtually all organisms, including plants, fungi, yeasts and human cells. As the strategy is based on in vivo recombineering and does not depend on restriction sites, it is applicable to any vector. To show the feasibility of the method five BACs, containing 0-75 kb of fungal DNA, were converted into BIBACs. These were subsequently transformed to the plant pathogenic fungus Fusarium oxysporum f.sp. lycopersici and to Aspergillus awamori, a filamentous fungus often used for large-scale protein production. Molecular characterisation of the transformants showed that the BIBACs were efficiently transferred to the fungi and stably integrated into their genomes.
细菌人工染色体（BAC）被广泛用于构建物理图谱、定位克隆和全基因组测序策略。不幸的是，它们在功能基因组学中的应用受到限制，因为目前尚无直接使用BAC进行互补的有效方法。我们描述了一种将任何BAC一步转化为二元BAC（BIBAC）的新策略。利用根癌农杆菌，这些BIBAC可以有效地转化到几乎所有生物体中，包括植物、真菌、酵母和人类细胞。由于该策略基于体内重组工程且不依赖于限制酶切位点，因此适用于任何载体。为了证明该方法的可行性，将五个含有0 - 75 kb真菌DNA的BAC转化为BIBAC。随后将这些BIBAC转化到植物病原真菌尖孢镰刀菌番茄专化型以及泡盛曲霉中，泡盛曲霉是一种常用于大规模蛋白质生产的丝状真菌。转化体的分子特征表明，BIBAC已有效地转移到真菌中并稳定整合到它们的基因组中。
