Lentiviral-mediated shRNA silencing of Notch-1 inhibits expression of Notch signaling pathway in rat bone marrow mesenchymal stem cells

doi:10.3969/j.issn.2095-4344.1704

Abstract

Abstract:

BACKGROUND: Number studies have insight into the mechanism of Notch signaling pathway in the differentiation of bone marrow mesenchymal stem cells into cardiomyocytes, but the mechanism of silencing Notch signaling pathway in the engineered myocardial-like tissue vascular network is less elucidated.
OBJECTIVE: To investigate the role of Notch signaling pathway in bone marrow mesenchymal stem cells in the construction of engineered myocardial-like tissue vascular network.
METHODS: The lentivirus expression vector was constructed by pLV[shRNA]-EGFP: T2A: Puro-U6] {rNotch1 [shRNA]_19 nt}(PLV-Notch) and pLV[shRNA]-EGFP: T2A: Puro-U6>Scramble_shRNA(PLV-Scramble) and packaged with the lentivirus vector. The mixture was co-transfected into HEK-293 T cells. The lentivirus suspension was collected and the titer of recombinant virus was detected by quantitative PCR. The constructed PLV-Notch and PLV-Scramble were infected with bone marrow mesenchymal stem cells respectively. The expression of enhanced green fluorescent protein gene and target gene rNotch1 in the transfected cells was identified by fluorescence microscopy, RT-qPCR and western blot.
RESULTS AND CONCLUSION: The titer of lentivirus vector was more than 1×10⁸ TU/mL. The mature cell lines were screened. The fluorescence rate of the two transfection groups was 80%, and the expression of rNotch1 gene in the interference group was 75.2% of that in the scrambled group. Therefore, lentiviral-mediated rNotch1 gene transfection of bone marrow mesenchymal stem cells can be successfully constructed and silence the expression of Notch signal pathway.

Key words: bone marrow mesenchymal stem cells, Notch gene, lentiviral vector, RNA interference, Notch signaling pathway, Natural Science Foundation of Shanxi Province

CLC Number:

Yan Jinyu, Xing Wanhong. Lentiviral-mediated shRNA silencing of Notch-1 inhibits expression of Notch signaling pathway in rat bone marrow mesenchymal stem cells[J]. Chinese Journal of Tissue Engineering Research, 2019, 23(17): 2659-2664.

Figures/Tables 2

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