Construction of a recombinant adenovirus vector expressing shRNA targeting interleukin-1beta gene in rats

doi:10.3969/j.issn.2095-4344.2015.18.024

Abstract

Abstract:

BACKGROUND: Specific down-regulation of interleukin 1 beta (IL-1β) may alleviate the pain behaviors effectively after peripheral nervous injury. Compared with small interference RNA (siRNA), short hairpin RNA (shRNA) could inhibit the expression of target gene more stably and efficiently. However, simple shRNA could not enter target cells to down-regulate target gene efficiently. Adenovirus vectors have wide host range, high infection efficiency and stable expression in host cells.
OBJECTIVE: To construct recombinant adenovirus vector expressing shRNA targeting IL-1β gene and detect its effect on the expression of target gene.
METHODS: Three siRNAs were designed on the basis of the nucleotide sequence of IL-1β obtained from NCBI
and then three shRNAs (shRNA1, shRNA2 and shRNA3) were synthesized. The annealed shRNA product and adenovirus vector pHBAd/U6/GFP digested by BamH I and EcoR I were connected to construct the recombinant adenovirus vector shuttle plasmid expressing shRNA targeting IL-1β. After sequencing, HEK 293 cells were co-transfected by the shuttle plasmid and skeleton vector, and three recombinant adenovirus vector expressing shRNA targeting IL-1β (rAd/shRNA1, rAd/shRNA2 and rAd/shRNA3) were packaged and amplified. Rats H9C2 cells were infected by recombinant adenovirus vector expressing shRNA targeting IL-1β and fluorescence microscope was used to observe the infection efficiency. The effect of recombinant adenovirus vector expressing shRNA targeting IL-1β on the expression of target gene was detected by western blot assay.
RESULTS AND CONCLUSION: The sequencing results showed that the sequences of three shRNAs adenovirus vector shuttle plasmid were consistent with the sequences of three designed shRNAs. rAd/shRNA1, rAd/shRNA2 and rAd/shRNA3 were constructed successfully. rAd/shRNA1, rAd/shRNA2 and rAd/shRNA3 could down-regulate the expression of IL-1β in rat H9C2 cells and the down-regulation effect of rAd/shRNA2 was the most significant.

中国组织工程研究杂志出版内容重点：肾移植；肝移植；移植；心脏移植；组织移植；皮肤移植；皮瓣移植；血管移植；器官移植；组织工程

全文链接：

Key words: Pain, Interleukin-1beta, RNA Interference

CLC Number:

R318

Zhao Xiao-long, Chen Jian-ping, Zhang Yu, Li Hang, Liu Yan-fang, Gao Wen-yan, Han Lei, Deng Ya-nan . Construction of a recombinant adenovirus vector expressing shRNA targeting interleukin-1beta gene in rats[J]. Chinese Journal of Tissue Engineering Research, 2015, 19(18): 2923-2927.

Figures/Tables 2

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