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Sequence-Validated and Expression-Tested Human cDNA in a Dual Expression,,,Vector

The GeneConnection clones contain features designed for constitutive high-level protein expression in mammalian cells.3 The vector contains the promoter and enhancer region of the human cytomegalovirus (CMV)### immediate early gene for constitutive expression of the pDual GC clones in either transiently or stably transfected mammalian cells.

To determine the protein expression level of the pDual GC vector, firefly luciferase activity was measured in transiently transfected Chinese hamster ovary (CHO) cells. Luciferase was chosen because it can be assayed both enzymatically and immunologically. Results of the enzymatic luciferase assays (Figure 2) demonstrate that the fusion protein is biologically active. Control transfections, with the reagents alone or the pDual GC vector without an insert, show low background levels.

Fig.2

To demonstrate that it is easy to detect the luciferase fusion protein, we performed Western blot analyses with anti-c-myc antibody using cell lysates prepared from transfected cells. The cells were transfected with pDual GC with a cDNA insert encoding luciferase, pCMV-Script (with an insert encoding luciferase containing a carboxy terminal c-myc epitope) or the pDual GC vector without an insert. To verify that the luciferase fusion protein was being detected, we also performed Western blot analyses with an antiluciferase antibody. Results indicate that the luciferase fusion protein is easily detected in both Western blot analyses (Figure 3).

Fig.3

Prior to commercial release, all expression-tested clones are tested by Western blot analysis with the anti-c-myc antibody to veri
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