Differences in the construction of thioredoxin-interacting protein overexpression model in INS-1 cells by two adenovirus infection methods

doi:10.3969/j.issn.2095-4344.0839

Abstract

Abstract:

BACKGROUND: There are two methods for constructing the protein overexpression model in cells by adenovirus infection. In Method I, 3 mL of 1640 medium was supplemented 4 hours after cell infection by adenovirus, and 24 hours later, culture medium was refreshed with complete medium for further 48 hour culture, In Method II, 4 hours after cell infection by adenovirus, PBS was used to wash 1640 culture medium containing adenovirus, and then 4 mL of complete culture was added for further culture till 48 hours. The main difference between the two methods is that the adenovirus in Method I has a longer action time and a better infection effect, but the damage of the adenovirus itself to cells is more severe, and it disturbs the stability of the experimental model. The effect of adenovirus in Method II is short lasting, and the toxicity of adenovirus to cells is lower, but the efficiency of adenovirus infection is weaker, and the expression of target protein is weaker.
OBJECTIVE: To find a more stable and efficient way to establish the thioredoxin-interacting protein (TXNIP) overexpression model by adenovirus infection in INS-1 islet β-cells.
METHODS: The adenovirus vector (Ad-GFP) with green fluorescent protein (GFP) and TXNIP overexpressing adenovirus vector (Ad-TXNIP-GFP) with green fluorescent protein (GFP) were constructed. Three groups (normal, Ad-GFP, Ad-TXNIP-GFP) were transfected with methods I and II, respectively.
RESULTS AND CONCLUSION: The GFP fluorescence intensity and the GFP positive percent in Method II were greater than those in the Method I. The cell morphology and survival rate in Method II were better than those in Method I. The expression of TXNIP at mRNA and protein levels in Method II was significantly higher than that in Method I. These findings show that the method II is more conducive to INS-1 cells in the construction of TXNIP overexpression model.

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: Adenoviridae Infections, Gene Expression Regulation, Genes, Regulator, Insulin-Secreting Cells, Tissue Engineering

CLC Number:

R318

Cao Zhu-jie, Feng Yan-jin, Li Dan, Wang Jin, Jiao Xiang-ying. Differences in the construction of thioredoxin-interacting protein overexpression model in INS-1 cells by two adenovirus infection methods[J]. Chinese Journal of Tissue Engineering Research, 2018, 22(28): 4487-4492.

Figures/Tables 2

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