Shuttling of integrated vectors from mammalian cells toE. coli is mediated by head-to-tail multimeric inserts

Abstract

With the aim of producing nonviral shuttle vectors for mammalian cells, we have constructed mouse mitochondrial DNA derivatives comprising the xanthine-guanine phosphoribosyltransferase gene as a selectable marker. Complete or subcomplete mitochondrial genomes were inserted into the plasmid pBB3 and transferred into hepatoma cells in order to generate, in vivo, new recombinant molecules. A second- and a third-generation vector, p12.2b and pδ respectively, were thus isolated for their ability to shuttle from mammalian cells to recA⁺ E. coli. Transfection of rodent fibroblasts and hepatoma cells showed that, contrary to our expectations, p12.2b and pδ are not self-replicating episomes; their shuttling from mammalian cells torecA ⁺ E. coli is mediated by tandem integrated copies. The relevant property of p12.2b and pδ is a ubiquitous propensity to form head-to-tail multimeric structures when they integrate into mammalian host chromosomes. This ability is missing in pBB3 and appears only following the insertion of various mitochondrial or nuclear DNA fragments into the plasmid. These data are discussed in terms of homologous recombination and shuttling of integrated vectors.