red-mediated transposition and final release of the mini-f vector of a cloned infectious herpesvirus genomered-mediated换位和最终释放mini-f向量的克隆感染疱疹病毒基因组.pdfVIP

Red-Mediated Transposition and Final Release of the Mini-F Vector of a Cloned Infectious Herpesvirus Genome 1 1 1,2 Felix Wussow , Helmut Fickenscher *, B. Karsten Tischer * 1 Institute for Infection Medicine, Christian-Albrecht University of Kiel and University Medical Center Schleswig-Holstein, Kiel, Germany, 2 Institute of Virology, Freie ¨ Universitat Berlin, Berlin, Germany Abstract Bacterial artificial chromosomes (BACs) are well-established cloning vehicles for functional genomics and for constructing targeting vectors and infectious viral DNA clones. Red-recombination-based mutagenesis techniques have enabled the manipulation of BACs in Escherichia coli without any remaining operational sequences. Here, we describe that the F-factor- derived vector sequences can be inserted into a novel position and seamlessly removed from the present location of the BAC-cloned DNA via synchronous Red-recombination in E. coli in an en passant mutagenesis-based procedure. Using this technique, the mini-F elements of a cloned infectious varicella zoster virus (VZV) genome were specifically transposed into novel positions distributed over the viral DNA to generate six different BAC variants. In comparison to the other constructs, a BAC variant with mini-F sequences directly inserted into the junction of the genomic termini resulted in highly efficient viral DNA replication-mediated spontaneous vector excision upon virus reconstitution in transfected VZV-permissive eukaryotic cells. Moreover, the derived vector-free recombinant progeny exhibited virtually indistinguishable genome properties and replication kinetics to the wild-type virus. Thus, a sequence-independent, efficie