Date Published: December 4, 2009
Publisher: Public Library of Science
Author(s): Felix Wussow, Helmut Fickenscher, B. Karsten Tischer, Brett Lindenbach. http://doi.org/10.1371/journal.pone.0008178
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, efficient, and easy-to-apply mini-F vector transposition procedure eliminates the last hurdle to perform virtually any kind of imaginable targeted BAC modifications in E. coli. The herpesviral terminal genomic junction was identified as an optimal mini-F vector integration site for the construction of an infectious BAC, which allows the rapid generation of mutant virus without any unwanted secondary genome alterations. The novel mini-F transposition technique can be a valuable tool to optimize, repair or restructure other established BACs as well and may facilitate the development of gene therapy or vaccine vectors.
Partial Text: Bacterial artificial chromosomes (BACs) are low-copy F-factor derived replicon units and well-established vectors to clone large DNA molecules in E. coli, . Regulatory elements of the mini-F replicon strictly maintain a BAC in one or maximally two copies per bacterial cell, reducing the potential for inter- and intramolecular homologous recombination between repeated sequences. Mini-F replicons can provide the stable propagation of complex DNA molecules up to 300 kb in recombination-deficient E. coli strains , . Consequently, BACs are widely used in sequencing projects and functional genomics of diverse organisms, and for the construction of targeting vectors and infectious viral DNA clones –. However, the chimeric constitution of prokaryotic and eukaryotic sequences in addition to the current localization of the mini-F vector within the cloned DNA limits the versatility of an established BAC clone.
In this work, we demonstrated that the low-copy F-factor sequences can be targeted and seamlessly transposed within BAC-cloned DNA in E. coli via Red recombination. We also demonstrated that the mini-F vector of an infectious herpesvirus BAC can be efficiently and seamlessly released from the genomic termini upon virus reconstitution in eukaryotic cells by inherent genome replication or maturation processes.