Lentivirus-mediated Runx2 gene transfection into MC3T3-E1 cells

doi:10.3969/j.issn.2095-4344.0951

Abstract

Abstract:

BACKGROUND: Heterozygous mutation, gene insertion and gene deletion of Runt-related transcription factor 2 (Runx2) have been found to be important causes of cleidocranial dysostosis. However, there is a lack of studies on the changes of osteoblasts after Runx2 silencing and how to perform gene silencing.
OBJECTIVE: To construct the lentiviral vector targeting Runx2 gene in mouse osteoblast-like cells (MC3T3-E1), so as to provide experimental basis for the research of cleidocranial dysostosis.
METHODS: According to the preliminary experimental results, the lentiviral vector was infected by MOI=40 in the ENi.S medium containing 5 mg/L Polybrene for 10 hours, and divided into four groups according to the viral number: group NC: pFU-GW-016PSC53349-1; group KD1: LVpFU-GW-016PSC60107-1; group KD2: LVpFU-GW-016PSC60108-1 and group KD3: LVpFU-GW-016PSC60109-1, then replaced with conventional medium at 16 hours after infection. The target gene expression was detected by Celigo® Image Cytometer at 72 hours after transfection. The two step method of real-time PCR was utilized to detect the silencing effect of Runx2 gene.
RESULTS AND CONCLUSION: At 72 hours after transfection, there was no green fluorescence in the group NC; a small amount of fluorescent expression in the group KD1; enhanced fluorescence expression in the group KD2 and obvious green fluorescence in the group KD3. Real-time PCR curves showed the better silencing effect of Runx2 gene in the group KD3. In summary, we successfully constructed the lentiviral vector targeting Runx2 gene into MC3T3-E1 cells, screened the virus inhibiting Runx2 gene, and clarified the action time and related measurement.

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: Genes, Osteoblasts, Lentivirus, Transfection, Tissue Engineering

CLC Number:

R318

Qin Han, Gong Yong-qing, Xu Hong-zhi. Lentivirus-mediated Runx2 gene transfection into MC3T3-E1 cells[J]. Chinese Journal of Tissue Engineering Research, 2018, 22(28): 4429-4433.

Figures/Tables 3

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