[Construction and expression of fusion gene eukaryotic expression plasmid of pEGFP-C(3)-insig2 and its influence to downstream genes].
OBJECTIVE
To construct the eukaryotic expression plasmid of insig2 gene and detect the expression of downstream gene adiponectin and adipocyte fatty acid-binding protein 2 (AP2) after the transfection of 3T3-L1 cells.


METHODS
Insig2 gene of the mouse was amplified by RT-PCR and then cloned into the eukaryon expression vector pEGFP-C(3), After confirmed by double restriction enzyme digestion analysis and DNA sequencing, pEGFP-C(3)-insig2 was transfected into 3T3-L1 cells by lipofectamine 2000. The expression of insig2 and downstream gene in the 3T3-L1 cells were detected by RT-PCR and fluorescence microscope.


RESULTS
The eukaryotic expression plasmid of pEGFP-C(3)-insig2 was constructed. The expression of fusion protein in the endochylema was confirmed. The transcription of adiponectin mRNA and AP2 mRNA was down-regulated after transfection for 24 h and 72 h.


CONCLUSIONS
The eukaryotic expression plasmid of pEGFP-C(3)-insig2 is successfully constructed. The transfected insig2 may have an effect on fat metabolism of 3T3-L1 cells.
Chen
Ke
K
Department of Endocrinology, Third Xiangya Hospital of Central South University, Changsha 410013, China. easd04mzh@126.com
Mo
Zhao-Hui
ZH
Xing
Xiao-Wei
XW
Hu
Ping-An
PA
Yang
You-Bo
YB
Xie
Yan-Hong
YH
D041721:3T3-L1 Cells; D052242:Adiponectin / Q000235:genetics; D000818:Animals; D050556:Fatty Acid-Binding Proteins / Q000235:genetics; D005786:Gene Expression Regulation / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000235:genetics*; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism; D051379:Mice; D008856:Microscopy, Fluorescence; D010957:Plasmids / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014158:Transcription, Genetic / Q000235:genetics; D014162:Transfection
D052242:Adiponectin; C493411:Fabp4 protein, mouse; D050556:Fatty Acid-Binding Proteins; C506120:Insig2 protein, mouse; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins