A SYSTEM FOR SHUTTLING 200-KB BAC PAC CLONES INTO HUMAN-CELLS - STABLE EXTRACHROMOSOMAL PERSISTENCE AND LONG-TERM ECTOPIC GENE ACTIVATION/

A novel shuttle vector, pBH140, has been constructed that allows stabl e maintenance of large genomic inserts as human artificial episomal ch romosomes (HAECs) in mammalian cells. The vector, essentially a hybrid BAC-HAEC, contains an F-based replication system as in a bacterial ar tificial chromosome (BAC) and the Epstein-Barr virus (EBV) latent orig in of replication system, oriP, for replication in human cells. A 185- kb DNA insert containing the entire human beta-globin locus, including its locus control region (LCR), was retrofitted into this vector. The resulting beta-globin BAC-HAEC clone, p148BH, was transfected into hu man cells and analyzed for episomal maintenance and expression of the beta-globin gene. FISH revealed an association of the vector with diff erent human chromosomes but no integration. The beta-globin BAC-HAECs were present at an average copy number of 11-15 per nucleus in the sta bly transformed human cells. After 1 year of continuous in vitro culti vation, the HAECs persisted as structurally intact 200-kb episomes. Wh ile no beta-globin transcription could be detected in the parental D98 /Raji cells, correctly spliced RT-PCR products were produced at signif icant levels in long-term cultures of the BAC-HAEC-transduced cells. T he wide availability of BAC and PAC libraries, the ease in manipulatin g cloned DNA in bacteria, and the episomal stability of the pBH140 vec tor make this system ideal for studies on gene expression and other ge nomic functions in human cells. The potential significance of large, f unctionally active episomes for gene therapy is discussed.