Construction of eukaryotic expression vector of murine chemokine(C-C motif) receptor 2(Ccr2) and establishment of its stably transfected RAW264.7 cell line

AIM: To construction of eukaryotic expression vector of murine Ccr2 and establishment of its stable transfected RAW264.7 cell line.METHODS: The whole coding region of murine Ccr2 mRNA was amplified by PCR and was inserted into a vector pEGFP-N1.The recombinant pEGFP-N1/Ccr2 was transfected into RAW264.7 cells by Lipofectamine 2000 reagent.The stable transfected RAW264.7 cells were screened by G418-media.The expression of Ccr2 on the membrane of the stable transfected cells was identified by RT-PCR,Western blot and flow cytometry.The fusion protein CCR2-EGFP was located by a converted fluorescence microscope.RESULTS: The recombinant plasmid pEGFP-N1/Ccr2 was successfully constructed and the stably transfected RAW264.7 cells was established.Both RT-PCR and Western blot revealed the higher expression of Ccr2 in the stably transfected RAW264.7 cells.Under the fluorescence microscope,the CCR2 was located on the membrane of RAW264.7 cells.CONCLUSION: The recombinant pEGFP-N1/Ccr2 has been constructed successfully and has been stably expressed in RAW264.7 cells.