Removal of Inserted BAC after linearizatiON (RIBON)-a novel strategy to excise the mini-F sequences from viral BAC...

The bacterial artificial chromosome (BAC) technology has been a mainstay approach for generating recombinant viruses, and several methods for excision of the mini-F sequences from the viral BAC vectors have been developed. However, these strategies either require complicated procedures or leave scars of inserted sequences. To overcome these problems, a new method to excise the mini-F sequences from viral BAC vectors based on the Removal of Inserted BAC after linearizatiON (RIBON) strategy was developed in this study for herpesvirus of turkeys (HVT). Enhanced green fluorescent protein (eGFP) DNA and the mini-F sequences were inserted into the gene encoding HVT thymidine kinase (TK) by homologous recombination in chicken embryo fibroblasts (CEFs), and the constructed HVT-BAC vector was used to transform Escherichia coli (pHVT-BAC). To remove the inserted eGFP and mini-F sequences, pHVT-BAC was linearized using a homing endonuclease I-SceI and used to cotransfect CEFs together with a plasmid containing the TK gene of HVT. The obtained viruses (44%) did not express eGFP, and DNA sequencing of isolated clones revealed that they were completely free of the inserted BAC sequences. Moreover, growth kinetics and plaque morphology of reconstituted viruses were comparable with those of the parental HVT. The results of this study demonstrate that the novel RIBON approach to remove mini-F sequences from the viral genome is simple and effective.