Chinese Journal of Tissue Engineering Research ›› 2010, Vol. 14 ›› Issue (11): 1951-1954.doi: 10.3969/j.issn.1673-8225.2010.11.013

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Construction and identification of small interfering RNA expression plasmid target to angiotensinogen

Wu Li-hua, Yang Ru-de   

  1. College of Bioscience and Bioengineering, South China University of Technology, Guangzhou  510640, Guangdong Province, China
  • Online:2010-03-12 Published:2010-03-12
  • Contact: Yang Ru-de, Professor, College of Bioscience and Bioengineering, South China University of Technology, Guangzhou 510640, Guangdong Province, China rdyang@163.com
  • About author:Wu Li-hua, Studying for master’s degree, College of Bioscience and Bioengineering, South China University of Technology, Guangzhou 510640, Guangdong Province, China andyxian@21cn.com

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Abstract:

BACKGROUND: In mammalian cells, introduction of double-stranded small interfering RNA (19-25 bp) can cleave and destroy the cognate RNA, which can result in suppression of gene expression.
OBJECTIVE: To construct siRNA expression plasmid for interference angiotensinogen (AGT), thereby, to resist AGT expression in adipose cells.
METHODS: The mRNA sequence of AGT gene was searched from NCBI (NM000029). Utilize of GenScript siRNA technology, AGT-siRNA oligonucletides were chemically synthesized and inserted into pRNAT-U6.1/Neo vector after annealing, then transformed into TOP10. The recombinant plasmid was identified by restriction endonuclease and DNA sequencing.
RESULTS AND CONCLUSION: The recombinant plasmid psiRNAT-U6.1/Neo-AGT was obtained by connecting 19 bp segment containing AGT-mRNA sequence to pRNAT-U6.1/Neo. After EcoR Ⅰ and Hind Ⅲ digestion, 351 bp segment was obtained from empty vector, and 397 bp fragment band was obtained form recombinant plasmid, which was coincidence to the expectation. DNA sequencing showed Targeting siRNA oligonucleotides were correctly inserted into the eukaryotic expression vector pRNAT-U6.1/Neo without base mutation. The interference vector psiRNAT-U6.1/Neo-AGT was successfully constructed.

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