Objective        To construct a multi-gene eukaryote expression vector in which co-expression of multiple genes was achieved and their products were located in different organelles in a single cell under control of the same promoter.       Methods        Sequences of polypeptide including two different ‘2A’ peptides and several restriction sites were synthesized to form a single open reading frame using the presence of different   ‘2A’ peptide sequences and inserted into the eukaryotic expression vector pcDNA3.1(-) Myc/His(B). ERFP with membrane location signal

EGFP with nuclear location signal and cytoplasmic EYFP were respectively inserted into the sites between the ‘2A’ peptides for the construction of eukaryotic expression vector pcDNA3.1 RGY. The vector was transfected to NIH3T3 cells or 293 cells by Liposome

and expression levels of the fluorescent proteins were measured and their sub-cellular locations were detected by flow cytometry

fluorescence and confocal microscopy

respectively.       Results        The expression levels of ERFP and EGFP were measured by flow cytometry in 24 h after the transfection

and the expression rates of the two proteins were quite similar. The green and red fluorescences were observed under fluorescence microscope and green

red and yellow fluorescences in a single cell were observed under confocal fluorescence microscope. ERFP

EGFP and EYFP were located on the cytomembrane

in the nuclei and the cytoplasm

respectively.       Conclusion       Expression of multiple genes can be achieved under control of the single promoter by inserting several 2A peptides and multiple genes into the same open reading frame. In addition

different sub-cellular locations can be observed in a single cell by adding signal peptide into the gene that makes it target the corresponding organelles.