Long-term stability of large insert genomic DNA episomal shuttle vectors in human cells

We have constructed an episomal shuttle vector which can transfer large (>1 00 kb) human genomic DNA inserts back and forth between bacteria and human cells and which can be tracked in rapidly dividing human cells using a live cell assay. The vector (p5170) is based on the F factor-derived bacterial artificial chromosome cloning vector used in Escherichia coli, with the add ition of the family of repeats element from the Epstein-Barr virus (EBV) la tent origin of replication. This element provides nuclear retention in cell s expressing the EBV protein EBNA-1. We have subcloned a series of genomic DNA inserts into p5170 and transfected the constructs into an EBNA-1(+) hum an cell line. Episomal mitotic stability was quantitatively analysed using flow cytometry. The episomes were also tracked by time course photography o f expanding colonies. A 117 kb episome was retained at similar to 2 copies/ cell and could be shuttled unrearranged from the human cells into bacterial cells after 15 months of continuous cell growth. Furthermore, the episome could still be rescued from human cells cultured in the absence of selectio n for 198 days. Such a trackable E.coli/human cell line shuttle vector syst em capable of carrying >100 kb of genomic DNA in human cells could prove a valuable tool in gene expression studies.