Long-term gene expression from autonomously replicating vectors in mammalian cells.
We tested the longevity of gene expression provided by autonomously replicating vectors. The vectors contain segments of human genomic DNA that provide efficient replication initiation and sequences derived from Epstein-Barr virus that provide nuclear retention. In order to monitor gene expression, the vectors also carry an expression cassette containing the chloramphenicol acetyl transferase gene. Replicating and control vectors were transfected into rapidly dividing human 293 cells, and gene expression and DNA retention were monitored. Gene expression decreased rapidly from a control vector lacking replication and retention functions, reaching near background levels by 10 days. Vectors having both replication and retention showed greatly prolonged gene expression, with vector DNA still detectable after 2 months. Autonomously replicating vectors also prolonged gene expression in rodent cells, in human lung epithelial cells, and in slowly dividing cell cultures. These results demonstrate the utility of this autonomous replication system for long-term retention and expression of introduced genes in mammalian cells. Such vectors could be useful for gene therapy, in combination with any of several methods for introduction of DNA.
Wohlgemuth
J G
JG
Department of Genetics, Stanford University School of Medicine, CA 94305, USA.
Kang
S H
SH
Bulboaca
G H
GH
Nawotka
K A
KA
Calos
M P
MP
D000818:Animals; D015139:Blotting, Southern; D002455:Cell Division; D002460:Cell Line; D015500:Chloramphenicol O-Acetyltransferase / Q000096:biosynthesis; D006224:Cricetinae; D019309:Epstein-Barr Virus Nuclear Antigens / Q000096:biosynthesis* / Q000235:genetics; D015870:Gene Expression; D017930:Genes, Reporter; D005822:Genetic Vectors; D004854:Herpesvirus 4, Human / Q000235:genetics / Q000502:physiology*; D006801:Humans; D007668:Kidney; D051379:Mice; D016133:Polymerase Chain Reaction / Q000379:methods; D018741:Replication Origin; D013728:Terminator Regions, Genetic; D014162:Transfection / Q000379:methods*; D014779:Virus Replication
D019309:Epstein-Barr Virus Nuclear Antigens; D015500:Chloramphenicol O-Acetyltransferase