Development of a gene capture method to rescue a large deletion mutant of human cytomegalovirus.

Abstract Human cytomegalovirus (HCMV) is an opportunistic human pathogen that causes serious clinical illness in immunocompromised individuals. A major breakthrough in the progression of HCMV genetics studies is the development of bacterial artificial chromosome (BAC) clones of the viral genome. Recently, a luciferase reporter gene was inserted in the BAC clone (BAC luc ) which facilitates monitoring of the virus growth both in vitro and in vivo . The virus made from the BAC luc grew with the similar kinetics as the wild-type strain indicating that the luciferase gene insertion does not interfere with the virus growth. Although the construction of the BAC clone has eased genetic studies of herpesviruses tremendously, there are still difficulties in cloning large DNA fragments of the virus to rescue mutations with large deletions. This paper describes a novel method termed “gene capture”, which allows easier cloning of large pieces of DNA. As an application of this method, a 15-kb fragment that was deleted from the HCMV genome was rescued back into the viral genome. A mutant HCMV clone with the 15-kb region deletion was generated first using the λ prophage recombination system in E. coli . Utilizing the new rescue method, the deleted fragment was then rescued in two steps: the 15-kb region was captured into a vector by homologous recombination; and the captured DNA fragment from the vector was inserted back into its native site in the mutant viral BAC by second homologous recombination. This method will be useful particularly for cloning large DNA fragments from any genome without introducing undesired mutations by traditional PCR-based approaches.