pECFP-Nuc encodes an enhanced cyan fluorescent variant of the Aequorea victoria green fluorescent protein (GFP) gene with three copies of the nuclear localization signal (NLS) of the simian virus 40 large T-antigen fused at its C-terminus (1, 2). The reiteration of the NLS sequence significantly increases the efficiency of translocation of ECFP into the nucleus of mammalian cells (3). The ECFP gene contains six amino acid substitutions. The Tyr-66 to Trp substitution gives ECFP fluorescence excitation (major peak at 433 nm and a minor peak at 453 nm) and emission (major peak at 475 nm and a minor peak at 501 nm) similar to other cyan emission variants (4-6). The five other substitutions enhance the brightness and solubility of the protein, primarily due to improved protein-folding properties and efficiency of chromophore formation (5, 7 & 8).

In addition to the chromophore mutations, ECFP contains >190 silent mutations that create an open reading frame comprised almost entirely of preferred human codons (9). Furthermore, upstream sequences flanking ECFP have been converted to a Kozak consensus translation initiation site (10). These changes increase the translational efficiency of the ECFP mRNA and consequently the expression of ECFP in mammalian and plant cells.

The vector contains an SV40 origin for replication and a neomycin resistance (Neo^r) gene for selection (using G418) in eukaryotic cells. A bacterial promoter (P) upstream of Neo^r expresses kanamycin resistance in E. coli. The vector backbone also provides a pUC19 origin of replication for propagation in E. coli and an f1 origin for single-stranded DNA production. The ECFP-Nuc vector can be transfected into mammalian cells using any standard transfection method. If desired, stable transfectants can be selected using G418 (11).

载体应用

pECFP-Nuc is used for the localized expression of ECFP in the nucleus of mammalian cells. It allows the visualization of the nucleus in living and fixed cells using fluorescence microscopy. ECFP can be used for dual-labeling experiments together with EYFP or EYFP fusion proteins using standard fluorescence microscopy when the appropriate filter sets are applied. Visit here for a complete listing of recommended filter sets. pECFP-Nuc is not meant to be used as a cloning vector. However, unique restriction sites at the 5' end of ECFP, between ECFP and the three copies of the NLS, and at the 3' end of the fusion protein, allow excision or insertion of DNA.