Clone and Express PCR Products in Mammalian Cells
The pTargeT™ Mammalian Expression Vector System is a convenient system for cloning PCR products and for expressing cloned PCR products in mammalian cells. The vector is prepared by digestion with EcoRV followed by addition of a 3´ terminal thymidine to each end. These single 3´-T overhangs at the insertion site greatly improve the efficiency of ligation of a PCR product into the plasmid in two ways. First, the overhangs prevent recircularization of the vector; second, they provide a compatible overhang for PCR products generated by thermostable polymerases that add a single deoxyadenosine, in a template-independent fashion, to the 3´-ends of amplified fragments. The pTargeT™ Vector also contains a modified coding sequence of the α-peptide of β-galactosidase, which allows recombinants to be selected using blue/white screening.
The pTargeT™ Mammalian Expression Vector System includes JM109 High Efficiency Competent Cells for transformation of ligation reactions. Recombinant clones can be efficiently selected using Blue/White Screening on indicator plates. The insert DNA can easily be removed for sub-cloning using an EcoRI single digest.
The pTargeT™ Vector uses the human cytomegalovirus (CMV) immediate-early enhancer/promoter region to promote strong, constitutive expression of cloned DNA inserts in mammalian cells. In transgenic mice, expression of the chloramphenicol acetyltransferase (CAT) gene under the regulation of the CMV enhancer/promoter was observed in 24 of the 28 tissues examined. The vector is maintained as an episome in cells expressing the SV40 large T antigen, leading to even higher levels of expression.
References
- Mezei, L.M. and Storts, D.R. (1994) In: PCR Technology: Current Innovations, Griffin, H.G. and Griffin, A.M., eds., CRC Press, Boca Raton, FL, 21.
- Robles, J. and Doers, M. (1994) Promega Notes 45, 19–20.
- Clark, J.M. (1988) Nucl. Acids Res. 16, 9677–86.
- Newton, C.R. and Graham, A. (1994) In: PCR, BIOS Scientific Publishers, Ltd., Oxford, UK, 13.
- Schmidt, E.V. et al. (1990) Mol. Cell. Biol. 10, 4406–11.