Silencing effect of recombinant plasmids PPARgamma-pSUPER-EGFP on the expression of peroxisome proliferator-activated receptor gamma.
OBJECTIVE
To investigate the silencing effect of gene encoding peroxisome proliferator-activated receptor gamma (PPARgamma) on the expression of tumor necrosis factor alpha (TNFalpha) by constructing vectors for RNA interference in RAW264.7 cells.


METHODS
The pSUPER-EGFP vectors were used to transcribe functional small interfering RNA (siRNA). Four pairs of oligonucleotides (64 nt) targeting PPARgamma gene were inserted into the downstream of the H1 promotor, with their veracity confirmed by double digestion and sequencing. Western blotting and immunofluorescence assay were used to examine the silencing effect of PPARgamma gene in RAW264.7 cells. Meanwhile, the TNFalphalevel was determined by Sandwich ELISA.


RESULTS
Compared with other recombinant pSUPER-EGFP vectors (R-pSUPER.EGFP), R-pSUPER.EGFP2 induced the best silencing effect on the expression of PPARgamma in RAW264.7 cells, which played an obvious inhibitory role in down-regulating the TNFalphaexpression after the curcumin and lipopolysaccharide (LPS) stimulation.


CONCLUSIONS
PPARgamma-pSUPER-EGFP inducing a silencing effect on the expression of PPARgamma can efficiently play a negative role in controlling the inflammatory responses of RAW264.7 cells.
Yang
Ce
C
State Key Laboratory of Trauma, Burns and Combined Injury, Department 4, Institute of Surgery Research, Daping Hospital, Third Military Medical University, Chongqing 400042, China.
Wang
Zheng-Guo
ZG
Chen
Li-Yong
LY
Zhou
Jian
J
Zhu
Pei-Fang And Jiang Jian-Xin
PF
Jiang
Jian-Xin
JX