The pSV-β-Galactosidase Control Vector(a) is designed as a positive control vector for monitoring transfection efficiencies of mammalian cells. The SV40 early promoter and enhancer drive transcription of the bacterial lacZ gene, which in turn, is translated into the β-galactosidase enzyme. β-galactosidase is an excellent reporter enzyme (1,2), that can be assayed quickly and directly in cell extracts using spectrophotometric, fluorescent or chemiluminescent assays (3,4). This reporter enzyme is also widely used for in situ histochemical analysis using the substrate X-Gal (5).
The pSV-β-Galactosidase Control Vector can be co-transfected with your DNA of interest. For example, co-transfection with firefly luciferase gene vectors (pGL3 Vectors) provide cell extracts that can be assayed for both luciferase and β-galactosidase activities. In this manner, the pSV-β-Galactosidase Vector acts as an internal control for transient expression assays. A negative control extract, prepared from mock-transfected cells, should also be assayed for the presence of endogenous β-galactosidase activity in cultured cells (2). In addition, co-transfection with chloramphenicol acetyltransferse reporter gene vectors (pCAT?3 Vectors) permits assaying for both CAT and β-galactosidase activities.
The pSV-β-Galactosidase Vector is a modification of pRSV-βGAL (6) with SV40 and pUC18 sequences substituted for RSV and pBR322 sequences. The pSV-β- Galactosidase Vector will express β-galactosidase in E. coli due to the presence of the E. coli gpt promoter located upstream of the lacZ gene (1). Colonies of E. coli containing the pSV-β-Galactosidase Vector will appear blue when plated on media containing X-gal.