摘要
目的构建针对小鼠Jmjd3基因的RNAi慢病毒载体,感染小鼠骨髓间充质干细胞(mouse bone marrow mesenchymal stem cells,mBM-MSCs)后观察其对Jmjd3基因表达的影响及对组蛋白H3K27me3的去甲基化作用。方法根据Jmjd3mRNA序列设计并合成3对shRNA寡核苷酸片段,退火形成双链并连接入PLL3.7慢病毒干扰载体,双酶切和DNA测序鉴定重组克隆,重组质粒与其他3种辅助包装质粒(pRsv-REV,pMDlg-pRRE,pMD2G)共转染293T细胞生产病毒液,病毒液感染mBM-MSCs,荧光定量PCR和Western blot检测目的基因Jmjd3的表达,筛选出最有效的干扰序列。Western blot检测组蛋白H3K27me3的表达量,分析Jmjd3的生物学功能。结果双酶切和测序结果证实Jmjd3基因shRNA序列正确插入慢病毒载体。荧光定量PCR和Western blot结果显示,感染后Jmjd3表达显著下调,与空载体对照组相比,Jmjd3-shRNA1组、Jmjd3-shRNA2组和Jmjd3-shRNA3组分别下调88.9%(P<0.001)、67.9%(P<0.001)和72.4%(P<0.001),Jmjd3-shRNA1组为最佳干扰组。Western blot结果显示干扰Jmjd3之后,H3K27me3的表达较空载体对照组升高。结论成功构建了针对小鼠Jmjd3基因的慢病毒干扰载体,并能有效抑制mBM-MSCs中Jmjd3基因的表达,Jmjd3具有组蛋白H3K27me3的去甲基化作用。
Objective To construct a specific lentiviral RNAi expression vector targeting mouse Jmjd3 gene and study the effect of Jmjd3-RNAi vector on expression of Jmjd3 in mouse bone marrow mesenchymal stem cells （mBM-MSCs） and its effect on H3K27me3 demethylation.Methods Three pairs of shRNA oligonucleotides were designed and synthesized based on the sequence of Jmjd3 mRNA.These oligonucleotides were annealed and cloned into PLL3.7 vector.The recombinant vectors were identified by double restriction enzyme digestion and direct sequencing.Then the recombinant PLL3.7 plasmids were co-transfected with three packaging plasmids （pRsv-REV,pMDlg-pRRE,pMD2G）into 293T cells.The lentivirus was used to infect the mBM-MSCs.Expression of Jmjd3 was detected by Real-Time PCR and Western blot.The level of H3K27me3 was examined by Western blot.Results All of the recombined vectors were found to contain the correct shRNA sequences,as confirmed by enzymatic digestion and sequencing.Results of Real-Time PCR and Western blot showed that the abundances of Jmjd3 mRNA and protein were significantly decreased.Compared to the negative control group,the group of Jmjd3-shRNA1,Jmjd3-shRNA2 and Jmjd3-shRNA3 can suppress the expression of Jmjd3 by 88.9% （P 〈0.001）,67.9% （P 〈 0.001） and 72.4% （P 〈 0.001） respectively.Western blot showed that the level of H3K27me3 was significantly increased.Conclusion Specific lentiviral RNAi vector targeting mouse Jmjd3 gene was successfully constructed and they can efficiently down-regulate the expression of Jmjd3 in mBM-MSCs.Jmjd3 is a H3K27me3 demethylase.
出处
《医学研究杂志》
2013年第9期63-67,共5页
Journal of Medical Research
基金
浙江省自然科学基金资助项目(Y2090337)
浙江省市共建重点学科(GJSX-010-003)
