PMID- 1709496
OWN - NLM
STAT- MEDLINE
DCOM- 19910619
LR  - 20190501
IS  - 0305-1048 (Print)
IS  - 1362-4962 (Electronic)
IS  - 0305-1048 (Linking)
VI  - 19
IP  - 8
DP  - 1991 Apr 25
TI  - A novel BK virus-based episomal vector for expression of foreign genes in 
      mammalian cells.
PG  - 1925-31
AB  - A composite mammalian cell-E. coli shuttle vector was developed based on the 
      human papova virus BK and pSV-neo. The vector contains a dioxin-responsive 
      enhancer (DRE) controlling a mouse mammary tumor virus (MMTV) promoter for the 
      inducible expression of inserted genes. In human cells the vector replicates 
      episomally, presumably utilizing the BKV rather than the SV40 origin, and 
      expresses the BK T/t antigens. A deletion in the late BK region precludes the 
      expression of the core/capsid proteins VP1, VP2, and VP3, thereby preventing the 
      infectious lytic cycle. HeLa cells which were transfected with this vector and 
      selected for resistance to the antibiotic G418 maintained the construct primarily 
      in episomal form during more than one year of continuous culture, with little or 
      no integration into the host genome. Transformed cells cultured in higher 
      concentrations of G418 contained higher copy numbers of the vector. This permits 
      one to vary the dosage of an inserted gene easily and reversibly without the need 
      of conventional amplification techniques and clonal analysis. Using a 
      chloramphenicol acetyl transferase (CAT) reporter gene inserted downstream of the 
      MMTV promoter, we found that CAT expression was greater in clones with higher 
      vector copy number. CAT expression was inducible with 
      2,3,7,8-tetrachlorodibenzo-p-dioxin, but inducibility was found to be inversely 
      proportional to the copy number. Transformation of bacteria with plasmid 
      molecules retrieved from the mammalian host was efficient, making this vector 
      well adapted for the screening of cDNA libraries for the ability to express a 
      phenotype in mammalian cells. Moreover, DNA sequences were stable during 
      long-term passage in mammalian cells; vector passaged continuously for more than 
      one year retained fully functional bacterial genes for resistance to 
      chloramphenicol and ampicillin.
FAU - De Benedetti, A
AU  - De Benedetti A
AD  - Department of Biochemistry, University of Kentucky College of Medicine, Lexington 
      40536.
FAU - Rhoads, R E
AU  - Rhoads RE
LA  - eng
GR  - GM20818/GM/NIGMS NIH HHS/United States
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PT  - Research Support, U.S. Gov't, P.H.S.
PL  - England
TA  - Nucleic Acids Res
JT  - Nucleic acids research
JID - 0411011
RN  - 0 (Gentamicins)
RN  - 63231-63-0 (RNA)
RN  - 9007-49-2 (DNA)
RN  - A08F5XTI6G (antibiotic G 418)
RN  - EC 2.3.1.28 (Chloramphenicol O-Acetyltransferase)
SB  - IM
MH  - BK Virus/*genetics
MH  - Blotting, Northern
MH  - Blotting, Southern
MH  - Chloramphenicol O-Acetyltransferase/genetics
MH  - Chromatography, Affinity
MH  - Cloning, Molecular
MH  - DNA/isolation & purification
MH  - Drug Resistance/genetics
MH  - Gene Amplification
MH  - *Genetic Vectors
MH  - Gentamicins/pharmacology
MH  - HeLa Cells
MH  - Humans
MH  - *Plasmids
MH  - RNA/isolation & purification
MH  - Simian virus 40/genetics
MH  - Transfection
MH  - Transformation, Bacterial
MH  - Transformation, Genetic
PMC - PMC328125
EDAT- 1991/04/25 00:00
MHDA- 1991/04/25 00:01
CRDT- 1991/04/25 00:00
PHST- 1991/04/25 00:00 [pubmed]
PHST- 1991/04/25 00:01 [medline]
PHST- 1991/04/25 00:00 [entrez]
AID - 10.1093/nar/19.8.1925 [doi]
PST - ppublish
SO  - Nucleic Acids Res. 1991 Apr 25;19(8):1925-31. doi: 10.1093/nar/19.8.1925.