Development of a bicistronic vector for multimodality imaging of estrogen receptor activity in a breast cancer model: preliminary application
Development of a bicistronic vector for multimodality imaging of estrogen receptor activity in a breast cancer model: preliminary application
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DOI：
10.1007/s00259-007-0578-z
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发表时间：
2008-02-01
影响因子：
9.1
通讯作者：
Lucignani, Giovanni
中科院分区：
文献类型：
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作者：
Ottobrini, Luisa;Ciana, Paolo;Lucignani, Giovanni
目的 本研究的目的是通过使用正电子发射断层扫描 (PET) 和生物发光成像 (BLI) 开发报告基因表达同步成像的细胞模型，以评估乳腺癌模型体内雌激素受体活性。方法选择了两种报告基因：用于 PET 成像的多巴胺能 D2 受体 (D(2)R80A) 的突变形式，以及用于 BLI 的萤火虫荧光素酶。两个报告基因之间 IRES 序列的存在确保了由包含雌激素反应元件 (ERE) 的相同调控序列驱动的协调表达。为了防止染色质对报告基因表达的影响，该构建体的两侧是绝缘子序列（基质附着区域，MAR）。结果体外研究表明该载体能够有效协调两个基因的表达。此外，植入受体动物体内的稳定转染细胞保持了表达报告基因并对全身治疗做出反应的能力，从而允许通过 PET 和 BLI 成像对 ER 活性进行体内研究。长期治疗后的体外表达分析显示，两种报告蛋白在监测雌激素依赖性转录方面的不同行为，概述了多报告系统的重要性。通过该模型，PET和BLI可用于通过使用双顺反子构建体同时评估雌激素及其类似物诱导的基因表达。结论快速、灵敏、连续的BLI与断层扫描和定量PET成像的组合特征将允许将该策略用于分子过程的体内评估以及药效学研究。
Purpose The aim of this study was to develop a cellular model for the concurrent imaging of reporter genes expression by using positron emission tomography (PET) and bioluminescence imaging (BLI) for the assessment of estrogen receptor activity in vivo in a breast cancer model.Methods Two reporters were chosen: a mutated form of the dopaminergic D2 receptor (D(2)R80A) for PET imaging, and the Firefly Luciferase for BLI. The presence of an IRES sequence between the two reporters ensured the coordinated expression driven by the same regulatory sequence containing an estrogen responsive element (ERE). To prevent chromatin effects on reporter expression, the construct was flanked by insulator sequences (Matrix Attachment Region, MAR).Results In vitro studies showed that the vector was efficient in coordinating the expression of the two genes. Moreover, stably transfected cells implanted in recipient animals maintained their capacity to express the reporters and react to systemic treatments permitting the in vivo study of ERs activity by PET and BLI imaging. In vitro expression analysis after long-term treatments showed different behaviour of the two reporter proteins in monitoring estrogen-dependent transcription outlining the importance of multi-reporter systems. With this model, PET and BLI can be applied to the concurrent evaluation of gene expression induced by estrogen and its analogues by using a bicistronic construct.Conclusion The combined features of rapid, sensitive, sequential BLI and tomographic and quantitative PET imaging will allow the use of this strategy for the in vivo evaluation of molecular processes also for pharmacodynamic studies.