[Construction of eukaryotic expression vector of murine chemokine (C-C motif) receptor 2 (Ccr2) and establishment of its stably transfected RAW264.7 cell line].
OBJECTIVE
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/Ccr2was 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.


CONCLUSIONS
The recombinant pEGFP-N1/Ccr2 has been constructed successfully and has been stably expressed in RAW264.7 cells.
Department of Biochemistry and Molecular Biology, Basic Medical Science College of Ningxia Medical University; Key Laboratory of Reproduction, Yinchuan 750004, China. vivian2056@163.com
Wei
W
Zhao
Xiao-Hong
XH
Lu
Xiu-Ying
XY
Pei
D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D005434:Flow Cytometry; D005822:Genetic Vectors / Q000235:genetics*; D051379:Mice; D021381:Protein Transport; D054390:Receptors, CCR2 / Q000235:genetics* / Q000378:metabolism; D015183:Restriction Mapping; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection / Q000379:methods*
C515335:Ccr2 protein, mouse; D054390:Receptors, CCR2