Rapid and efficient in vitro excision of BAC sequences from herpesvirus genomes using Cre-mediated recombination

Abstract Cre-mediated recombination is a widely used technique for the re-arrangement of DNA sequences that are bracketed by loxP recognition sites. This bacteriophage P1 enzyme is commonly used to excise the bacterial artificial chromosome (BAC) sequence, a vector sequence used for large herpesvirus genomes for the purposes of propagation and manipulation in Escherichia coli . Most methods utilize cell lines that can be induced for the expression of Cre enzyme to facilitate this excision. In addition, methods have been developed that express Cre from the virus genome and enable auto-excision of the BAC plasmid. We report a versatile and rapid in vitro method based on purified Cre enzyme to carry out the same process in a test tube and does not require cell line generation or cloning into the virus genome. This method greatly increases the repertoire of methods available to modify the genome prior to reconstitution of virus infectivity in a mammalian host.