Efficient episomal expression vector for human transitional carcinoma cells.
To develop an efficient expression vector for human transitional carcinoma cells, we evaluated the replication activity of episomal vectors derived from the BK virus (BKV) and the Epstein-Barr virus (EBV) in HT-1376 bladder carcinoma cells. Southern blot analysis of transient transfectants indicated that the BKV-derived episome replicated extrachromosomally whereas the EBV replicon did not appear to be functional in these cells. HT-1376 cells were stably transfected with BKV-derived episomes containing the neomycin resistance gene as a selectable marker. Southern analysis demonstrated that these stable transfectants contained approximately 150 copies of the BKV episome per cell. There was no evidence of integration of the BKV episome into genomic DNA following selection with G418 for 10 weeks. These stable episomal transfectants had approximately 20-fold higher levels of expression of neomycin resistance gene mRNA than clones of HT-1376 cells transfected with pSV2NEO which contained five integrated copies of this gene. In soft agar cloning experiments, BKV episomes were efficiently transferred to the progeny of these transfectants during cell division over multiple generations. Additionally, BKV episomal copy number is maintained in stable transfectants after withdrawal of selection pressure for over 2 months. These data demonstrate that BKV-derived episomes replicate efficiently in bladder carcinoma cells, yielding stable transfectants having a high episomal copy number and expressing encoded genes at high levels. BKV-derived episomes may be useful in gene therapy strategies to modulate the growth of bladder carcinoma cells.
Cooper
M J
MJ
Department of Medicine, University Hospitals of Cleveland, Case Western Reserve University School of Medicine, OH 44106.
Miron
S
S
D001739:BK Virus / Q000235:genetics*; D015139:Blotting, Southern; D002295:Carcinoma, Transitional Cell / Q000235:genetics / Q000628:therapy; D004279:DNA, Viral / Q000096:biosynthesis / Q000235:genetics; D005822:Genetic Vectors; D004854:Herpesvirus 4, Human / Q000235:genetics*; D006801:Humans; D010957:Plasmids; D014158:Transcription, Genetic; D014162:Transfection; D014407:Tumor Cells, Cultured; D001749:Urinary Bladder Neoplasms / Q000235:genetics / Q000628:therapy
D004279:DNA, Viral