Reducing Induced Protein Expression In The Metallothionein Promoter

The metallothionein (pMT) vector is an essential tool for the transfection and induced expression of DNA in cultured cells. The promoter sequence of the pMT vector is unique in that transcription initiation is stimulated readily in the presence of a metal, such as copper. Although the pMT vector is extremely efficient in expressing a gene of interest, the high rates of expression make it difficult to study issues related to protein polarity, localization and activity in the realm of cancer research. In this project, I either truncated or mutated DNA elements such as the metal regulatory region, E-Box and STAT binding site in the pMT promoter. This was done because these elements are responsible for the recruitment of transcription machinery or expression of high levels of mRNA. These alterations were performed by the use of Quickchange Mutagenesis and PCR. Next, polarity proteins such as atypical protein kinase C and Miranda were ligated into each type of mutated pMT vector. The regular protocol was carried out for the transfection and induction of these vectors into Drosophila Schneider 2 cells (S2 cells). Degrees of protein expression were recorded via antibody staining,  immunofluorescence and confocal microscopy. Degrees of protein expression were analyzed with the computer programs ImageJ and Prism. These data conclude that induced protein expression is reduced upon these changes in the pMT promoter. This vector may now be used in S2 cell experiments that require an expression sensitive background, such as in the case of protein polarity, cellular differentiation and cancer.