Pharmaceutical Science Division, King's College London, London SE1 9NH, UK.
Nucleic Acids Res. 2010 Sep;38(16):e166. doi: 10.1093/nar/gkq596. Epub 2010 Jul 9.
Recombineering is employed to modify large DNA clones such as fosmids, BACs and PACs. Subtle and seamless modifications can be achieved using counter-selection strategies in which a donor cassette carrying both positive and negative markers inserted in the target clone is replaced by the desired sequence change. We are applying counter-selection recombineering to modify bacmid bMON14272, a recombinant baculoviral genome, as we wish to engineer the virus into a therapeutically useful gene delivery vector with cell targeting characteristics. Initial attempts to replace gp64 with Fusion (F) genes from other baculoviruses resulted in many rearranged clones in which the counter-selection cassette had been deleted. Bacmid bMON14272 contains nine highly homologous regions (hrs) and deletions were mapped to recombination between hr pairs. Recombineering modifications were attempted to decrease intramolecular recombination and/or increase recombineering efficiency. Of these only the use of longer homology arms on the donor molecule proved effective permitting seamless modification. bMON14272, because of the presence of the hr sequences, can be considered equivalent to a highly repetitive BAC and, as such, the optimized method detailed here should prove useful to others applying counter-selection recombineering to modify BACs or PACs containing similar regions of significant repeating homologies.
基因重排被用于修饰大型 DNA 克隆,如 fosmid、BAC 和 PAC。通过使用反选择策略,可以实现微妙且无缝的修饰,其中携带正选择和负选择标记的供体盒被插入目标克隆中,然后被所需的序列变化所取代。我们正在应用反选择基因重排来修饰 bacmid bMON14272,这是一种重组杆状病毒基因组,因为我们希望将病毒工程改造为具有细胞靶向特性的治疗性有用基因传递载体。最初尝试用来自其他杆状病毒的融合(F)基因替代 gp64,但导致许多重排克隆中,反选择盒已被删除。bacmid bMON14272 包含九个高度同源区域(hrs),缺失发生在 hr 对之间的重组。尝试了基因重排修饰以减少分子内重组和/或提高基因重排效率。其中,只有供体分子上使用更长的同源臂证明是有效的,可以实现无缝修饰。由于存在 hrs 序列,bMON14272 可以被认为等同于高度重复的 BAC,因此,此处详细介绍的优化方法应该对其他应用反选择基因重排修饰含有相似高度重复同源区域的 BAC 或 PAC 的人有用。