| Resumo: |
To obtain an optimised form of BFP for use as a reporter gene in mammalian cells, the brightest available GFP form, EGFP, was mutated at 5 different positions, yielding 8 different mutagenised forms of BFP. The intensity of the fluorescent signals attained in mammalian cells with all these various versions of BFP was analysed by flow cytometry of transiently transfected COS 7 cells. The best mutant obtained can be detected readily both by flow cytometry and fluorescence microscopy, even when expressed together with GFP. To explore whether cellular localization could enhance the fluorescence signals any further, plasmid constructs were made to target optimised versions of GFP and BFP to the nucleus, the endoplasmic reticulum (ER) and the cell surface. Expression in the nucleus or ER increased the fluorescence signal by ca. 50%, whereas cell surface expression resulted in a five-fold decrease compared to the ER and nuclear forms. Co-expression of GFP and BFP in the same cellular compartment did not result in any significant absorption of the blue fluorescence by GFP. Thus, targeting of GFP and BFP to various cellular compartments adds even further versatility to this convenient dual reporter-gene system.
|
