Construction of circular RNA mmu_circ_Rab11a overexpression vector to transfect 293T cells

doi:10.3969/j.issn.2095-4344.1822

Abstract

Abstract:

BACKGROUND: Rab11a plays an important role in the cell autophagy and proliferation, and circular RNA is involved in the regulation of biological processes by regulating the expression of parental genes. Therefore, whether mmu_circ_Rab11a also plays an important role in the process of cell autophagy and proliferation deserves further study.
OBJECTIVE: To investigate the feasibility of circular RNA (mmu_circ_Rab11a) plasmid vector construction and transfection of 293T cells.
METHODS: We constructed PLC5+mmu_circ_Rab11a overexpressed plasmid vectors, verified the recombinant plasmid by PCR and gene sequencing. mmu_circ_Rab11a overexpression vector was transiently transfected into 293T cells using Lipofectamine 3000 transfection reagent. Expression of mmu_circ_Rab11a gene in control, pLC5-ciR and mmu_circ_Rab11a overexpression groups was detected by RT-PCR, and PCR products were verified by Sanger sequencing. mmu_circ_Rab11a overexpression plasmid vector was transfected into MC3T3 cells and cell counting kit-8 was used to detect cell proliferation ability.
RESULTS AND CONCLUSION: mmu_circ_Rab11a overexpression vector was successfully constructed, which could efficiently promote mmu_circ_Rab11a transcribed into 293T cells. Over-expression of mmu_circ_Rab11a could enhance the proliferation of MC3T3 cells. Our findings indicate that gene engineering technology can be used to construct mmu_circ_Rab11a overexpression vector, which is then transfected into 293T cells by liposomal transfection method to make efficient transcription of mmu_circ_Rab11a, laying an experimental foundation for further investigation on the biological function of mmu_circ_Rab11a. The proliferation of MC3T3 cells could be affected by regulating the expression of mmu_circ_Rab11a.

Key words: circular RNA, mmu_circ_Rab11a, vector construction, 293T cells, liposome transfection, overexpression vector, MC3T3 cells, cell proliferation

CLC Number:

Yuan Peiyan, Chen Lei, Xu Shuaimei, Tong Fangli, Xu Pingping. Construction of circular RNA mmu_circ_Rab11a overexpression vector to transfect 293T cells[J]. Chinese Journal of Tissue Engineering Research, 2019, 23(33): 5372-5377.

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