Construction of mouse chemerin gene knock-down cell lines mediated by adenovirus vector

doi:10.3969/j.issn.2095-4344.0326

Abstract

Abstract:

BACKGROUND: Level of chemerin is elevated in atherosclerosis, indicating that chemerin may participate in the pathological process of atherosclerosis.
OBJECTIVE: To construct and effectively silence the expression of chemerin gene by adenovirus-mediated RNA interference technique, so as to lay an experimental foundation for the study on the mechanism of atherosclerosis.
METHODS: Four chemerin shRNA sequences were designed by pCD316-ZsGreen-shRNA adenovirus vector for RNAi targeting the coding region of mouse chemerin mRNA, and another control vector containing a nonsense sequence was constructed. The best pCD316-shRNA was picked up to package recombinant adenovirus in 293T cells, which was infected in cultured mouse melanoma B16F10 cells. The chemerin mRNA and protein levels in B16F10 cells were detected by qPCR and western blot assay.
RESULTS AND CONCLUSION: The adenovirus vectors chemerin shRNA was successfully constructed, which was confirmed by PCR and DNA sequencing. The titer of adenovirus reached 2×1010 PFU/mL in the packaging cells, and pDC-shRNA4 showed the most significant interfering effects on chemerin gene (P < 0.001). QPCR and western blot assay showed that the expression levels of chemerin mRNA and protein in B16F10 cells were significantly inhibited, suggesting that chemerin-shRNA4 showed the interfering effects on chemerin gene. We finally confirmed that the expression of chemerin gene can be effectively silenced by adenovirus-mediated siRNA in mouse B16F10.

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

Key words: Chemotactic Factors, Adenoviridae, RNA interference, Atherosclerosis, Plasmids, Tissue Engineering

CLC Number:

R318

Chen Ting-ting, Cao Yuan-zhi, Xiong Wei, Dong Shao-hong. Construction of mouse chemerin gene knock-down cell lines mediated by adenovirus vector[J]. Chinese Journal of Tissue Engineering Research, 2018, 22(24): 3875-3879.

Figures/Tables 2

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