Retrofitting bacterial artificial chromosomes by homologous recombination into enhanced BAC vectors for functional studies

摘要

The very nature of genome research demands regular improvement and development of innovative molecular tools and technologies for biotechno-logical and therapeutic applications. Current bacterial artificial chromosome (BAC) vector technology has enabled the construction of BAC libraries from a large number of species, including the human RPCI-11 and the mouse RPCI-23 libraries, which were used in the corresponding public sequencing genome projects. Such libraries are invaluable for an initial understanding of the genome sequence and structure of each organism and for the isolation of clones carrying most genomic loci as intact functional units. However, the majority of these libraries were made in BAC cloning vectors that do not facilitate functional studies in eukary-otic cells. To overcome this limitation, retrofitting genomic inserts as Notl fragments into novel BAC vectors has been reported. Retrofitting strategies based on targeting the insertion of a variety of cassettes into the loxP site of PI artificial chromosome (PAC) and BAC vectors by Cre recombinase have also been reported. Such site-specific modifications invariably depend on the insertion of a second antibiotic selection marker on the vector backbone. They also require the prior removal or inactivation of multicopy origins of replication on the retrofitting plasmids and are limited to sequences recognized by Cre recombinase. It is, therefore, not possible with these approaches to transfer genomic inserts from first generation PAC or BAC vectors into novel BAC vectors that are optimized for functional studies in yeast and higher organisms.