SERUM-DEPENDENT AND CELL CYCLE-DEPENDENT EXPRESSION FROM A CYTOMEGALOVIRUS-BASED MAMMALIAN EXPRESSION VECTOR

Cytomegalovirus-based mammalian expression vectors are widely used to drive the expression of transfected genes in cultured cells. Immunoflu orescent staining of the WT1 protein in 3T3 and 293 cell clones, stabl y transfected with a cyomegalovirus (CMV) expression vector carrying a cDNA coding for the tumour suppressor protein WT1, showed extreme cel l to cell variation in the amount of recombinant protein expressed, in dicative of cell cycle dependence. This was investigated further by We stern blot and FACS analysis which showed that WT1 protein expression was highest in S phase and almost absent in G0/G1. Northern blot analy sis of cell clones expressing sense or antisense WT1 cDNAs regulated b y the CMV promoter/enhancer showed that RNA expression was also cell c ycle-dependent. Western blotting of cells expressing a luciferase repo rter gene driven by the CMV promoter/enhancer also showed apparent cel l cycle-dependent expression. We further demonstrated that the express ion of these gene constructs was serum responsive with a 10-fold incre ase in expression occurring 2 h after the addition of serum. These res ults show that the CMV promoter/enhancer system varied in its response to serum and the cell cycle state. Therefore, care must be taken when interpreting any phenotypic alterations (or lack of them) produced in cells transfected with CMV-based expression vectors. (C) 1997 Elsevie r Science B.V.