Scientia Agricultura Sinica ›› 2013, Vol. 46 ›› Issue (24): 5196-5204.doi: 10.3864/j.issn.0578-1752.2013.24.014

• ANIMAL SCIENCE·VETERINARY SCIENCERE·SOURCE INSECT • Previous Articles     Next Articles

Construction of siRNA Expression Plasmids Targeting ChREBP Gene and Its Effect on Lipogenesis in Primary Cultured Porcine Adipocytes

 LU  Jian-Xiong, ZHANG  Guo-Hua, LI  Chang-Hui, CHEN  Yan, HUO  Sheng-Dong, GUO  Peng-Hui, CAI  Yong   

  1. College of Life Science and Engineering, Northwest University for Nationalities, Lanzhou 730030
  • Received:2013-05-13 Online:2013-12-16 Published:2013-07-19

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Abstract: 【Objective】To study the role of ChREBP in the regulation of lipogenesis by glucose in porcine adipocytes, siRNA expression plasmids targeting ChREBP gene were constructed and transfectd into primary cultured porcine adipocytes to silence its expression.【Method】The 4 pairs of oligonucleotides targeting ChREBP gene were synthesized and respectively inserted into linearized pcDNA™6.2-GW/EmGFP vector to construct siRNA expression plasmids. The plasmids were identified by sequencing, and then transfected into the adipocytes from subcutaneous adipose tissue obtained from 3-day-old piglets. Silencing efficiency was determined by fluorescence real-time PCR. The transfected adipocytes were exposed to 0-20 mmol·L-1 glucose for 48 h and lipogenesis and expression of lipogenic genes were measured.【Result】One siRNA expression plasmid with high transfection efficiency and 85% silencing efficiency for ChREBP gene was successfully constructed. Lipogenesis and expression of ACC1 and FAS mRNA were significantly decreased in the transfected adipocytes compared with in negative-siRNA plasmid transfected and non-transfected adipocytes (P＜0.05), and the glucose levels had no effect on this decrease of lipogenesis (P＞0.05).【Conclusion】The constructed siRNA expression plasmid could effectively interfere ChREBP expression in porcine adipocytes, and ChREBP mediates the regulation of lipogenesis by glucose in the cells.

Key words: ChREBP , siRNA , pig , adipocyte , lipogenesis

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