( site upstream of three contiguous copie...)New Mammalian Expression Vectors Employ Stable, High-Level Fluorescence ,,, Humanized Renilla GFP Reporter

site upstream of three contiguous copies of either the FLAG or hemagglutin (HA) epitope tag sequence, respectively. In both vectors, duplicate stop codons follow the final epitope tag sequence to ensure termination of the inserted gene transcript and expression of an epitope-tagged native protein. The stop codons are followed by the coding region of an internal ribosome entry site (IRES) for reentry of the ribosome and protein production of the downstream hrGFP gene. In these bicistronic vectors, translation of two open reading frames from one mRNA allows expression of a gene of interest to be monitored at the single-cell level by virtue of expression of hrGFP on the same transcript. Expression of hrGFP can be detected visually, and the FLAG- or HA-tagged fusion product is easily detected by Western blot analysis.

We used a Vitality bicistronic vector to monitor expression of the luciferase gene, cloned into the multiple cloning site, and hrGFP. We transfected HeLa cells with the pIRES-hrGFP-1a vector carrying the luciferase gene. As can be seen by fluorescent microscopy, hrGFP expression is vivid (Figure 2). Western blot analysis clearly indicates expression of the luciferase protein (Figure 2).

Fig.2

New Expression Vectors Generate hrGFP Fusion Proteins

The newest Vitality vectors are the phrGFP-N1 and phrGFP-C protein fusion vectors (Figure 1). These vectors are powerful tools for localizing recombinant protein expression in cells. With the phrGFP-N1 vector, insertion of a gene of interest into the multiple cloning site results in expression of hrGFP fused to the
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