Chen Ke, Mo Zhao-Hui, Xing Xiao-Wei, Hu Ping-An, Yang You-Bo, Xie Yan-Hong
Department of Endocrinology, Third Xiangya Hospital of Central South University, Changsha 410013, China.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2007 Apr;23(4):309-11.
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.
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.
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.
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.
构建insig2基因真核表达质粒，并检测其转染3T3-L1细胞后下游基因脂联素和脂肪细胞脂肪酸结合蛋白2（AP2）的表达。
通过RT-PCR扩增小鼠的Insig2基因，然后克隆至真核表达载体pEGFP-C(3)。经双酶切分析及DNA测序鉴定后，采用脂质体2000将pEGFP-C(3)-insig2转染至3T3-L1细胞。通过RT-PCR及荧光显微镜检测3T3-L1细胞中insig2及下游基因的表达。
构建了pEGFP-C(3)-insig2真核表达质粒，证实了融合蛋白在细胞内的表达。转染24 h和72 h后，脂联素mRNA和AP2 mRNA的转录下调。
成功构建了pEGFP-C(3)-insig2真核表达质粒。转染的insig2可能对3T3-L1细胞的脂肪代谢有影响。
