Chinese Journal of Tissue Engineering Research ›› 2011, Vol. 15 ›› Issue (24): 4489-4492.doi: 10.3969/j.issn.1673-8225.2011.24.029

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Construction and identification of small interfering RNA expression plasmid target to differentiated embryo-chondrocyte expressed gene 1  

Wei Yan1, Jia Yan-fei2, Zheng Yan2, Ma Xiao-li2, Wang Yun-shan1, 2   

  1. 1Institute of Basic Medicine, Shandong Academy of Medical Sciences, Jinan  250062, Shandong Province, China
    2Jinan Central Hospital, Shandong University, Jinan  250013, Shandong Province, China
  • Received:2010-12-08 Revised:2011-01-07 Online:2011-06-11 Published:2011-06-11
  • Contact: Wang Yun-shan, Doctor, Professor, Doctoral supervisor, Institute of Basic Medicine, Shandong Academy of Medical Sciences, Jinan 250062, Shandong Province, China; Jinan Central Hospital, Shandong University, Jinan 250013, Shandong Province, China sdjnwys@163.com
  • About author:Wei Yan★, Studying for master’s degree, Institute of Basic Medicine, Shandong Academy of Medical Sciences, Jinan 250062, Shandong Province, China weiyan985@126.com
  • Supported by:

    the Plan of Science and Technology Bureau of Jinan City, No. 200905045-1*; the National Natural Science Foundation of China, No.81000869*

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Abstract:

BACKGROUND: Differentiated embryo-chondrocyte expressed gene 1 (DEC1) is a basic helix-loop-helix transcription factor, which is closely associated with some malignant cancers.
OBJECTIVE: To construct small interfering RNA (siRNA) expression plasmid target to DEC1.  
METHODS: The mRNA sequence of DEC1 gene was searched from NCBI. Utilize of Katahdin siRNA technology, DEC1-siRNA oligonucletides were inserted into pGreenPuro™ shRNA Cloning and Expression Lentivector, after annealing, then transformed into JM-109. The recombinant plasmid was identified by Agarose gel electrophoresis analysis, ultraviolet spectrophotometer analysis, PCR and DNA sequencing.  
RESULTS AND CONCLUSION: The recombinant plasmid pGreenPuro™ shRNA Cloning and Expression Lentivector-DEC1 was obtained by connecting 25 bp segment containing DEC1 sequence to pGreenPuro™ shRNA Cloning and Expression Lentivector. Agarose gel electrophorsis analysis and ultraviolet spectrophotometer analysis confirmed that plasmid DNA had higher purity. DNA sequencing showed Targeting siRNA oligonucleotides were correctly inserted into the eukaryotic expression vector pGreenPuro™ shRNA Cloning and Expression Lentivector without base mutation. The interference vector pGreenPuro™ shRNA cloning and expression lentivector-DEC1 was successfully constructed.

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