Tet-On系统中多西环素对MBD1诱导表达的调控研究

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

摘要/Abstract

摘要：

【目的】构建甲基结合蛋白1（MBD1）的Tet-On可诱导表达质粒和293T细胞，探讨多西环素（Dox）对MBD1表达的影响，并通过分析细胞中质粒启动子CpG甲基化水平反映MBD1调控机制。【方法】使用MBD1引物扩增293T的MBD1表达序列，将其连接于Tet-On诱导表达质粒的多克隆位点；将构建成功的质粒用电转染的方法转入MBD1 KO 293T，使用荧光显微镜及Western blot验证获得的MBD1 恢复的293T（MBD1 RE 293T）；使用不同浓度Dox诱导MBD1表达，利用Western blot检测单克隆细胞中MBD1的表达情况；使用同一浓度Dox诱导MBD1表达，在12、24、48、96、144、192 h收集细胞，利用Western blot检测细胞中MBD1的表达情况；使用亚硫酸氢盐测序方法检测Dox诱导后细胞内可诱导质粒启动子CpG甲基化情况。【结果】成功构建Tet-On诱导表达MBD1质粒；与未诱导细胞相比，Dox诱导后转染细胞MBD1蛋白重新表达；随着Dox浓度的升高细胞中MBD1的表达逐渐增多；相同浓度Dox诱导后，从12-96 h细胞内MBD1的表达逐渐升高，但144 h 后MBD1表达下降；细胞内质粒启动子CpG甲基化检测显示，与96 h相比，144和192 h质粒启动子甲基化水平逐渐升高；在诱导144 h加入甲基转移酶抑制剂地西他滨（decitabine）后，质粒启动子甲基化水平降低，MBD1表达升高。【结论】Tet-On可诱导细胞中重新表达MBD1蛋白，并且表达量与Dox浓度正相关，但长时间诱导后细胞内质粒CpG甲基化水平升高，影响细胞中 MBD1的表达。

关键词: Tet-On, MBD1, MBD1 ΔCXXC3, 甲基化, HEK293T

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

卢茜, 袁月, 李丹, 张鹏. Tet-On系统中多西环素对MBD1诱导表达的调控研究[J]. 生物技术通报, 2024, 40(8): 47-52.

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.

图/表 3

图1 Tet-On可诱导MBD1质粒及细胞 A：HEK293T中RNA逆转录获得的cDNA扩增出的MBD1表达序列电泳；B：Tet-On MBD1ΔCXXC3质粒构建示意图；C：Tet-On MBD1ΔCXXC3质粒酶切电泳图，其中M为1 000 bp DNA Ladder，1：加入限制性核酸内切酶BamH I的实验组，2：不加入限制性核酸内切酶的阴性对照组；D：细胞经Tet-On MBD1ΔCXXC3质粒电转后红色荧光表达情况，其中标尺为50 μm；E：细胞在Dox诱导前后MBD1表达情况

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

图2 Dox对Tet-On MBD1细胞中MBD1表达的影响 A：不同Dox浓度诱导时MBD1的表达情况；B：不同诱导时间MBD1的表达情况，*P<0.05。下同

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

图3 Dox诱导时间对Tet-On MBD1ΔCXXC3质粒启动子甲基化的影响 A：不同时间诱导下Tet-On MBD1ΔCXXC3质粒启动子区域20个CpG位点的甲基化情况；B：Dox诱导96 h后加入Decitabine后细胞中MBD1表达量，其中144+为在DOX诱导96 h后，再加入Decitabine；144-为在DOX诱导96 h后，不加入Decitabine

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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