Regulated expression of nuclear genes by T3 RNA polymerase and lac repressor, using recombinant vaccinia virus vectors.
Recombinant vaccinia viruses that express the bacteriophage T3 RNA polymerase (VV-T3pol) or the Escherichia coli lac repressor (VV-lacI) under control of the early-late vaccinia promoter P7.5 were constructed. To determine whether phage polymerase and lac repressor can function in the nucleus of mammalian cells, the bacterial chloramphenicol acetyltransferase (CAT) gene was cloned downstream of a T3 promoter (PT3-CAT) or downstream of a T3 promoter-lac operator fusion element (PT3Olac-CAT), and these reporter gene cassettes were introduced stably into NIH 3T3 or Ltk- cells. Infection of 3T3/PT3-CAT or Ltk-/PT3-CAT cells by VV-T3pol led to rapid expression of CAT (greater than 20 ng of CAT protein per 10(6) cells). The presence of hydroxyurea (which blocks virus DNA replication) did not prevent CAT production. When 3T3/PT3Olac-CAT cells were infected with both VV-T3pol and VV-lacI (multiplicities of infection of 2.5 and 10, respectively), greater than 30-fold repression of CAT gene activity by lac repressor was observed. This could be reversed to unrepressed levels by the presence of 10 mM o-nitrophenyl-beta-D-galactoside (IPTG) in the medium. Regulated expression of the target gene was observed with cell lines that had been maintained for over 1 year (greater than 50 passages in culture), and Southern blot analysis revealed the presence of the CAT gene only in the nuclear fraction in these cells, demonstrating the stability of the target gene. These results indicate that vaccinia virus-encoded proteins can function in the mammalian nucleus and provide the basis for a genetic system in which essential vaccinia virus genes, placed in the chromosome of a cell, can be used to complement defective virus particles. This approach may prove useful for other virus systems.
Department of Biochemistry, State University of New York-Health Science Center, Brooklyn 11203-2098.
D
D
Rodriguez
Y W
YW
Zhou
J R
JR
Rodriguez
R K
RK
Durbin
V
V
Jimenez
W T
WT
McAllister
M
M
Esteban
D000818:Animals; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism*; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D005822:Genetic Vectors; D007700:Kinetics; D007739:L Cells / Q000378:metabolism; D051379:Mice; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics* / Q000378:metabolism; D013604:T-Phages / Q000201:enzymology / Q000235:genetics*; D014157:Transcription Factors / Q000235:genetics*; D014616:Vaccinia virus / Q000235:genetics*
D011994:Recombinant Proteins; D012097:Repressor Proteins; D014157:Transcription Factors; D015500:Chloramphenicol O-Acetyltransferase; D012321:DNA-Directed RNA Polymerases