载体编号：P0064
载体名称：pSV-beta-gal
载体抗性：Amp
载体大小：6820bp
载体宿主：mammalian cells
载体特点：The pSV-β-Galactosidase Control Vector is designed as a positive control vector for monitoring transfection efficiencies of mammalian cells. The SV40 early romoter 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 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 (e.g., 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.
1. Hall, C.V. et al. (1983) Expression and regulation of Escherichia coli lacZ gene fusions in mammalian cells. J. Mol. Appl. Genet. 2, 101.9.
2. Rosenthal, N. (1987) Identification of regulatory elements of cloned genes with functional assays. Methods Enzymol. 152, 704.20.
3. Silhavy, T. et al. (1984) In: Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 266.
4. Schenborn, E. and Groskreutz, D. (1999) Reporter gene vectors and assays. Mol. Biotechnol. 13, 29.44.
5. Lim, K. and Chae, C.B. (1989) A simple assay for DNA transfection by incubation of the cells in culture dishes with substrates for β-galactosidase. Biotechniques 7, 576.9.
6. Edlund, T. et al. (1985) Cell-specific expression of the rat insulin gene: Evidence for role of two distinct 5′ flanking elements. Science 230, 912.6.
7. Protocols and Applications Guide, Online Edition. (2006) Promega Corporation.
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