Effects of epigenetic modulation on reporter gene expression: implications for stem cell imaging
Effects of epigenetic modulation on reporter gene expression: implications for stem cell imaging
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DOI：
10.1096/fj.05-4551fje
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发表时间：
2005-10-01
期刊：
影响因子：
4.8
通讯作者：
Wu, JC
中科院分区：
文献类型：
--
作者：
Krishnan, M;Park, JM;Wu, JC
跟踪活体干细胞移植后的定位、存活、分化和增殖对于理解干细胞生物学和生理学是必不可少的。在这项研究中，我们研究了报告基因表达的长期稳定性在胚胎大鼠心肌细胞系和逆转报告基因沉默的表观遗传调制的作用。用携带巨细胞病毒启动子驱动萤火虫荧光素酶报告基因（CMV-Fluc）的质粒稳定转染细胞，并重复传代3-8个月。在最高表达克隆中，萤火虫荧光素酶活性从第1代（843 +/-28）至第20代（250 +/-10）至第40代（44 +/-3）至第60代（3 +/-1 RLU/μ g; P < 0.05 vs.第1代）逐渐降低。5-氮杂胞苷（DNA甲基转移酶抑制剂）处理后萤火虫荧光素酶活性最大程度地恢复，与组蛋白脱乙酰酶抑制剂和视黄酸（转录激活剂）相比，P < 0.05。增加5-氮杂胞苷处理的剂量导致萤火虫荧光素酶mRNA（RT-PCR）和蛋白质（Western印迹）的水平更高，并且CMV启动子（DNA核苷酸序列）中的甲基化水平相反地更低。这些体外结果被扩展到活体动物细胞移植的体内生物发光成像（BLI）。用5-氮胞苷处理的细胞监测2 wk，而未处理的细胞监测1 wk（P < 0.05）。这些发现对基于报告基因的干细胞移植成像具有重要意义。
Tracking stem cell localization, survival, differentiation, and proliferation after transplantation in living subjects is essential for understanding stem cell biology and physiology. In this study, we investigated the long-term stability of reporter gene expression in an embryonic rat cardiomyoblast cell line and the role of epigenetic modulation on reversing reporter gene silencing. Cells were stably transfected with plasmids carrying cytomegalovirus promoter driving firefly luciferase reporter gene (CMV-Fluc) and passaged repeatedly for 3-8 months. Within the highest expressor clone, the firefly luciferase activity decreased progressively from passage 1 ( 843 +/- 28) to passage 20 ( 250 +/- 10) to passage 40 ( 44 +/- 3) to passage 60 ( 3 +/- 1 RLU/mu g; P < 0.05 vs. passage 1). Firefly luciferase activity was maximally rescued by treatment with 5-azacytidine ( DNA methyltransferase inhibitor) compared with trichostatin A ( histone deacetylase inhibitor) and retinoic acid ( transcriptional activator; P < 0.05). Increasing dosages of 5-azacytidine treatment led to higher levels of firefly luciferase mRNA ( RT-PCR) and protein ( Western blots) and inversely lower levels of methylation in the CMV promoter ( DNA nucleotide sequence). These in vitro results were extended to in vivo bioluminescence imaging (BLI) of cell transplant in living animals. Cells treated with 5-azacytidine were monitored for 2 wk compared with 1 wk for untreated cells ( P < 0.05). These findings should have important implications for reporter gene-based imaging of stem cell transplantation.