Removal of Inserted BAC after linearizatiON (RIBON)—a novel strategy toexcise the mini-F sequences from viral BAC vectors

Journal of Veterinary Medical Science

Yukari ISHIHARA¹  Atsushi YASUDA¹  Motoyuki ESAKI¹ 

[1] Ceva Animal Health (Japan Campus), 1�?6 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa 230�?0045, Japan

关键词: bacterial artificial chromosome;    herpesvirus of turkeys;    mini-F sequences;   

DOI  :  10.1292/jvms.16-0038

学科分类：兽医学

来源: Japanese Society of Veterinary Science

【 摘 要 】

References(16)The bacterial artificial chromosome (BAC) technology has been a mainstay approach forgenerating recombinant viruses, and several methods for excision of the mini-F sequencesfrom the viral BAC vectors have been developed. However, these strategies either requirecomplicated procedures or leave scars of inserted sequences. To overcome these problems, anew method to excise the mini-F sequences from viral BAC vectors based on the Removal ofInserted BAC after linearizatiON (RIBON) strategy was developed in this study forherpesvirus of turkeys (HVT). Enhanced green fluorescent protein (eGFP) DNA and the mini-Fsequences were inserted into the gene encoding HVT thymidine kinase (TK) by homologousrecombination in chicken embryo fibroblasts (CEFs), and the constructed HVT-BAC vector wasused to transform Escherichia coli (pHVT-BAC). To remove the insertedeGFP and mini-F sequences, pHVT-BAC was linearized using a homing endonucleaseI-SceI and used to cotransfect CEFs together with a plasmid containingthe TK gene of HVT. The obtained viruses (44%) did not express eGFP, and DNA sequencing ofisolated clones revealed that they were completely free of the inserted BAC sequences.Moreover, growth kinetics and plaque morphology of reconstituted viruses were comparablewith those of the parental HVT. The results of this study demonstrate that the novel RIBONapproach to remove mini-F sequences from the viral genome is simple and effective.

【 授权许可】

Unknown