An inducible eukaryotic host-vector expression system: amplification of genes under the control of the polyoma late promoter in a cell line producing a thermolabile large T antigen.
We have taken advantage of the inherent instability of integrated polyoma (Py) DNA sequences in the presence of a functional viral large T antigen (LT) to develop a eukaryotic host-vector system where copy number is controlled by temperature. A mouse cell line WOP32-4, that constitutively expresses a temperature sensitive (ts) LT, was transfected with plasmids containing the Py origin of DNA replication (ori) and either a neomycin-resistance gene (neo) or chloramphenicol acetyl transferase gene (cat) linked to the Py late promoter. Stable transformants were selected at 39 degrees C, the non-permissive temperature for the ts LT function. Upon shift to 33 degrees C, the resident Py sequences present in the WOP32-4 cells cannot excise due to an ori deletion. However, excision of the transfected plasmid molecules and subsequent extrachromosomal replication occur at high rates leading in some cases to the production of 1000-2000 copies per cell (average) of the plasmid. Proportional increases in either neo-specific mRNA or CAT activity were also observed. In situ hybridization for one cell line indicated that about 20% of temperature-shifted cells contained amplified plasmid DNA.
D000818:Animals; D000957:Antigens, Viral, Tumor / Q000235:genetics*; D004261:DNA Replication; D005784:Gene Amplification; D005786:Gene Expression Regulation; D005822:Genetic Vectors; D051379:Mice; D010957:Plasmids; D011120:Polyomavirus / Q000235:genetics*; D011401:Promoter Regions, Genetic; D014176:Protein Biosynthesis; D013696:Temperature; D014158:Transcription, Genetic
D000957:Antigens, Viral, Tumor