AN ENGINEERED PGK PROMOTER AND LAC OPERATOR-REPRESSOR SYSTEM FOR THE REGULATION OF GENE-EXPRESSION IN MAMMALIAN-CELLS

Previous reports have demonstrated that the Escherichia coli lac repressor can operate effectively in mammalian cells to repress expression of genes driven by modified viral or metallothionein (MT) promoters. We have developed a more general expression system using the promoter from the PGK1 gene (encoding murine 3-phosphoglycerate kinase) which is widely expressed in almost all cell types, including early embryonic and ES (embryonic stem) cells. Firstly, we engineered the lac repressor to include a nuclear localisation signal and placed it under control of the PGK1 promoter. Efficient nuclear localisation of the repressor was demonstrated by mobility-shift assays and immunofluorescence detection. For the target vectors, we modified the wild-type (wt) PGK1 promoter to include lac operator (lac0) sites for binding of the lac repressor and compared a number of different lac0 positions and arrangements based on proximity to the native start points for transcription (tsp) and translation. In the absence of repressor, we observed reduced expression of the neo reporter gene for some placements of the lac0, but wt expression for placements near the tsp. When both target and repressor were present in the cells, we observed that the expression of neo could be strongly suppressed and reversibly regulated by induction with IPTG. In particular, for a promoter which contained two spaced lac0 replacing native sequence around the major tsp, we observed 90-95% repression by the lac repressor for the neo reporter gene and up to 98% repression for the cat reporter gene. Efficient derepression by IPTG was observed in both cases. This modified promoter has been incorporated into a general expression vector named pPOP which has appropriate cloning sites for insertion and regulated expression of other genes.