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 stable maintenance of large genomic inserts as human artificial episomal chromosomes (HAECs) in mammalian cells. The vector, essentially a hybrid BAC-HAEC, contains an F-based replication system as in a bacterial artificial chromosome (BAC) and the Epstein-Barr virus (EBV) latent origin 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 human cells and analyzed for episomal maintenance and expression of the beta-globin gene. FISH revealed an association of the vector with different human chromosomes but no integration. The beta-globin BAC-HAECs were present at an average copy number of 11-15 per nucleus in the stably transformed human cells. After 1 year of continuous in vitro cultivation, the HAECs persisted as structurally intact 200-kb episomes. While no beta-globin transcription could be detected in the parental D98/Raji cells, correctly spliced RT-PCR products were produced at significant levels in long-term cultures of the BAC-HAEC-transduced cells. The wide availability of BAC and PAC libraries, the ease in manipulating cloned DNA in bacteria, and the episomal stability of the pBH140 vector make this system ideal for studies on gene expression and other genomic functions in human cells. The potential significance of large, functionally active episomes for gene therapy is discussed.
Westphal
E M
EM
Lineberger Cancer Research Center, School of Medicine, University of North Carolina at Chapel Hill, 27599, USA.
Sierakowska
H
H
Livanos
E
E
Kole
R
R
Vos
J M
JM
D002461:Cell Line, Transformed; D002876:Chromosomes, Bacterial / Q000235:genetics; D002877:Chromosomes, Human; D003001:Cloning, Molecular / Q000379:methods*; D004261:DNA Replication; D019309:Epstein-Barr Virus Nuclear Antigens; D005111:Extrachromosomal Inheritance; D018628:Gene Dosage; D005786:Gene Expression Regulation; D005822:Genetic Vectors / Q000235:genetics*; D005914:Globins / Q000235:genetics*; D006801:Humans; D010957:Plasmids / Q000235:genetics*; D018741:Replication Origin / Q000235:genetics; D013997:Time Factors; D015533:Transcriptional Activation; D014162:Transfection
D019309:Epstein-Barr Virus Nuclear Antigens; D005914:Globins