title: A precise excision of the complete Epstein-Barr virus genome in a plasmid based on a bacterial artificial chromosome.
abstract: 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.
authors: Cancer Research Institute, Central South University, Changsha, Hunan 410078, China.
authors: Zhengyuan
authors: Z
authors: Yu
authors: Jianhong
authors: J
authors: Lu
authors: Haibo
authors: H
authors: Yu
authors: Qijia
authors: Q
authors: Yan
authors: Lielian
authors: L
authors: Zuo
authors: Guiyuan
authors: G
authors: Li
mesh_terms: D000818:Animals; D010582:Bacteriophage lambda / Q000201:enzymology; D002460:Cell Line; D022202:Chromosomes, Artificial, Bacterial / Q000235:genetics*; D005818:Genetic Engineering / Q000379:methods; D005822:Genetic Vectors; D016679:Genome, Viral / Q000235:genetics*; D004854:Herpesvirus 4, Human / Q000235:genetics*; D059765:Homologous Recombination; D006801:Humans; D051379:Mice; D008819:Mice, Nude; D010957:Plasmids; D017384:Sequence Deletion; D014162:Transfection