Construction and application of eukaryotic expression vector of fragile X mental retardation 1 (FMR1) gene

: Objective To construct a eukaryotic expression vector of human fragile X mental retardation 1 (FMR1) gene and establish stably transfected HeLa cells. Methods The full-length FMR1 cDNA fragment was amplified by PCR and inserted into eukaryotic expression vector pEGFP-N2 using restriction enzyme. The recombinant plasmid pEGFP-N2-FMR1, after identified by restriction digestion and DNA sequencing, was transfected into HeLa cells by lipofectamine 2000. The stably transfected cell line was obtained by screening with G418. The expression and subcellular distribution of FMR protein was identified by Western blotting and immunofluorescence staining combined with laser-scanning confocal microscopy. Results Restriction digestion and DNA sequencing revealed that the eukaryotic expression plasmid of pEGFP-N2-FMR1 was successfully constructed. Besides, Western blotting and immunofluorescence staining showed that GFP-FMR protein was expressed in HeLa cells, which mainly was localized in the cytoplasm. Conclusion The recombinant eukaryotic expression vector of pEGFP-N2-FMR1 has been constructed successfully and stably expressed FMR protein in HeLa cells.