Studies on the Regulation of MBD1-induced Expression by Dox in the Tet-On System

doi:10.13560/j.cnki.biotech.bull.1985.2024-0015

Abstract

Abstract:

【Objective】This work aims to construct the Tet-On inducible expression plasmid of methyl-binding protein 1（MBD1）and 293T cells, to explore the effect of doxycycline（Dox）on the expression of MBD1, and to reflect the regulatory mechanism of MBD1 by analyzing the CpG methylation level of the plasmid promoter in the cells.【Method】The MBD1 expression sequence of 293T was amplified using MBD1 primers and ligated into the polyclonal site of Tet-On inducible expression plasmid. The successfully constructed plasmid was transfected into MBD1 KO 293T by electro-transfection, and the obtained MBD1-restored 293T was verified using fluorescence microscope and Western blot（MBD1 RE）. MBD1 expression was induced using different concentrations of Dox, and the expression of MBD1 in monoclonal cells was detected by Western blot. MBD1 expression was induced using the same concentration of Dox, and the cells were collected at 12, 24, 48, 96, 144 and 192 h. The expression of MBD1 in the cells was detected by Western blot; and MBD1 expression in the cells was detected by bisulfite sequencing. The expression of MBD1 in the cells was detected by Western blot, and the CpG methylation of inducible plasmid promoter was detected by bisulfite sequencing after Dox induction.【Result】Tet-On inducible MBD1 plasmid was successfully constructed; MBD1 protein was re-expressed in the transfected cells after Dox induction compared with uninduced cells. The expression of MBD1 in the cells increased gradually with the increase of Dox concentration; the expression of MBD1 in the cells increased gradually from 12 to 96 h after induction with the same concentration of Dox, but the expression decreased after 144 h. The expression of CpG methylation of plasmid promoter was detected by bisulfite sequencing method; the CpG methylation of plasmid promoter was detected by bisulfite sequencing method. CpG methylation of the plasmid promoter showed that the methylation level of the plasmid promoter gradually increased at 144 and 192 h compared with that at 96 h. The methylation level of the plasmid promoter decreased and the expression of MBD1 increased after the addition of the methyltransferase inhibitor, decitabine, at 144 h. The expression of MBD1 in the cells also decreased by the addition of the methyltransferase inhibitor, decitabine, at 144 h after the induction.【Conclusion】Tet-On induced the re-expression of MBD1 protein in the cells and the expression was positively correlated with the Dox concentration, but the elevated level of intracellular plasmid CpG methylation after prolonged induction affected the expression of MBD1 in the cells.

Key words: Tet-On, MBD1, MBD1 ΔCXXC3, methylation, HEK293T

LU Xi, YUAN Yue, LI Dan, ZHANG Peng. Studies on the Regulation of MBD1-induced Expression by Dox in the Tet-On System[J]. Biotechnology Bulletin, 2024, 40(8): 47-52.

Figures/Tables 3

Fig. 1 Construction of Tet-On MBD1ΔCXXC3 plasmid A: The electrophoresis of MBD1 expression sequence amplified from cDNA obtained by RNA reverse transcription in HEK293T; B: the schematic diagram of Tet-On MBD1ΔCXXC3 plasmid construction; C: the electrophoresis diagram of Tet-On MBD1ΔCXXC3 plasmid enzyme digestion, in which M is the 1 000 bp DNA Ladder, and 1 is the restriction nuclease endonuclease BamH I added to the experimental group, and 2 is the negative control group without restriction nucleic acid endonuclease; D: the red fluorescence expression of cells after electrotransformation by Tet-On MBD1ΔCXXC3 plasmid, where the scale bar is 50 μm; E: the MBD1 expression of cells before and after induction by Dox

Fig. 2 Effects of Dox on MBD1 expression in Tet-On MBD1 cells A: Expression of MBD1 when induced by different Dox concentrations. B: Expression of MBD1 at different induction times, *P < 0.05. The same below

Fig. 3 Effects of Dox induction time on the promoter methylation of Tet-On MBD1ΔCXXC3 plasmid A: Methylation of 20 CpG sites in the promoter region of Tet-On MBD1ΔCXXC3 plasmid under different time of induction; B: expression of MBD1 in the cells after the addition of Decitabine after 96 h of induction by Dox, where 144+ is after 96 h of induction by DOX followed by the addition of Decitabine, and 144- is after 96 h of induction by DOX after DOX induction for 96 h without Decitabine

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