Scientia Agricultura Sinica ›› 2013, Vol. 46 ›› Issue (23): 5026-5036.doi: 10.3864/j.issn.0578-1752.2013.23.020

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

Selection of Effective SREBP1 shRNA in Cattle and the Construction of Recombinant Adenovirus Vector

 FU  Chang-Zhen-1, ZAN  Lin-Sen-12, WANG  Hong-1, CHENG  Gong-12, WANG  Hong-Bao-12, LI  Yao-Kun-1, JIANG  Bi-Jie-1, GAO  Jian-Bin-1, YANG  Ning-1   

  1. 1.College of Animal Science and Technology, Northwest A&F University, Yangling 712100, Shaanxi
    2.National Beef Cattle Improvement Center of Northwest A&F University, Yangling 712100, Shaanxi
  • Received:2013-04-25 Online:2013-12-01 Published:2013-09-30

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Abstract: 【Objective】 The aim of this study was to construct recombinant adenovirus carrying effective small hairpin RNA (shRNA) which can exclusively interfere NotI bovine sterol-regulatory-element-binding protein 1 (SREBP1) gene expression, thus providing a basis for studying the function and mechanism of SREBP1 gene at cellular level.【Method】According to the coding sequence (CDS) region of SREBP1 gene, six pairs of inhibition shRNA and one pairs of negative control shRNA were designed and further inserted into pENTR-U6 to construct pENTR-U6-shRNA expression vector. The cotransfection of expression vector psiCHECK-Ⅱcarrying SREBP1 and the obtained pENTR-U6-shRNA was carried out in 293 A cell lines to select efficient shRNA. The efficient shRNA and shRNA-NC were connected to pAD/BL-DEST to construct the recombinant plasmid, respectively. The obtained recombinant adenovirus vectors were transfected into 293A cells to package. Then, the adenovirus were amplified and harvested. Real-time PCR (qRT-PCR) was used to detect and confirm the interference effect of the harvested adenovirus on the target gene SREBP1 in bovine pre-adipocytes. The viral titer was determined by GFP labeling method. 【Result】Results showed that shRNA-1053 significantly decreased the expression of SREBP1 by 87.4%. The linearized recombinant adenovirus vector carrying shRNA-1053 and shRNA-NC transfected 293A cells, and further packaged and amplified high-titer recombinant adenovirus Ad-1053 and Ad-NC (7×108 GFU/mL and 9×108 GFU/mL). qRT-PCR results elucidated Ad-1053 significantly down-regulated mRNA expression level of SREBP1 gene in bovine pre-adipocytes (Interfering efficiency ＞85%), while Ad-NC did not.【Conclusion】In this study, the recombinant adenovirus, carrying efficient shRNA designed for RNA interference study of bovine SREBP1 gene, was constructed successfully.

Key words: cattle , SREBP1 , shRNA , recombinant adenovirus

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