Cancer Research Institute, Central South University, Changsha, Hunan 410078, China.
J Virol Methods. 2011 Sep;176(1-2):103-7. doi: 10.1016/j.jviromet.2011.06.015. Epub 2011 Jun 23.
The Maxi-EBV is a bacterial artificial chromosome (BAC)-based plasmid that contains the complete Epstein-Barr virus (EBV) genome of 172 kb. This plasmid also carries an additional cassette of 11.5 kb in size for the expression of a mini F factor, selection markers and GFP. In the intracellular study of EBV infection based on the Maxi-EBV system, a parallel control that only contains this 11.5 kb vector is desirable but unavailable. In order to construct the vector in this approach, a clean deletion of the complete EBV genome from the Maxi-EBV was performed. This was achieved by homologous recombination using the bacteriophage λ Red system. Initially, an FRT-flanked kanamycin-resistance (kan) fragment of 1.4 kb with 61 bp homologies on the ends was introduced into the Maxi-EBV plasmid, replacing the 172-kb EBV genome. The kan gene was then removed by Flp/FRT excision. The results of identification demonstrated that the mutation was generated precisely. The results highlight the feasibility for a genome as large as 172 kb to be replaced by a greatly shorter fragment and for a much smaller vector backbone to be retrieved. Cell lines derived from the transfection of the vector will subsequently be appropriate controls in the related study.
Maxi-EBV 是一种基于细菌人工染色体 (BAC) 的质粒，包含完整的 172kb 大小的 Epstein-Barr 病毒 (EBV) 基因组。该质粒还携带一个额外的 11.5kb 大小的盒，用于表达微型 F 因子、选择标记和 GFP。在基于 Maxi-EBV 系统的 EBV 感染的细胞内研究中，需要但缺乏仅包含此 11.5kb 载体的平行对照。为了在这种方法中构建载体，使用噬菌体 λ Red 系统进行了从 Maxi-EBV 中对完整 EBV 基因组的同源重组缺失。最初，在 Maxi-EBV 质粒中引入了一个 1.4kb 的 FRT 侧翼卡那霉素抗性 (kan) 片段，其末端有 61bp 的同源性，取代了 172kb 的 EBV 基因组。然后通过 Flp/FRT 切除去除了 kan 基因。鉴定结果表明突变是精确产生的。结果突出表明，对于大小为 172kb 的基因组，可以用大大缩短的片段替换，并且可以回收更小的载体骨架。随后，从转染载体衍生的细胞系将成为相关研究中的适当对照。
