Retrofitting BACs with G418 resistance, luciferase, and oriP and EBNA-1 - new vectors for in vitro and in vivo delivery

Background: Bacterial artificial chromosomes ( BACs) have been used
extensively for sequencing the human and mouse genomes and are thus
readily available for most genes. The large size of BACs means that
they can generally carry intact genes with all the long range
controlling elements that drive full levels of tissue-specific
expression. For gene expression studies and gene therapy
applications it is useful to be able to retrofit the BACs with
selectable genes such as G418 resistance, reporter genes such as
luciferase, and oriP/EBNA-1 from Epstein Barr virus which allows
long term episomal maintenance in mammalian cells. Results: We
describe a series of retrofitting plasmids and a protocol for in
vivo loxP/Cre recombination. The vector pRetroNeo carries a G418
resistance cassette, pRetroNeoLuc carries G418 resistance and a
luciferase expression cassette, pRetroNeoLucOE carries G418
resistance, luciferase and an oriP/EBNA-1 cassette and pRetroNeoOE
carries G418 resistance and oriP/EBNA-1. These vectors can be
efficiently retrofitted onto BACs without rearrangement of the BAC
clone. The luciferase cassette is expressed efficiently from the
retrofitting plasmids and from retrofitted BACs after transient
transfection of B16F10 cells in tissue culture and after
electroporation into muscles of BALB/c mice in vivo. We also show
that a BAC carrying GFP, oriP and EBNA-1 can be transfected into
B16F10 cells with Lipofectamine 2000 and can be rescued intact
after 5 weeks. Conclusion: The pRetro vectors allow efficient
retrofitting of BACs with G418 resistance, luciferase and/or
oriP/EBNA-1 using in vivo expression of Cre. The luciferase
reporter gene is expressed after transient transfection of
retrofitted BACs into cells in tissue culture and after
electroporation into mouse muscle in vivo. OriP/EBNA-1 allows
stable maintenance of a 150-kb BAC without rearrangement for at
least 5 weeks.