Developing a CEST Reporter Gene

开发 CEST 报告基因

• 批准号: 7140295
• 负责人: Jeff W. Bulte
• 金额: $ 34.08万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-09-15 至 2009-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7140295
• 关键词: NIH Roadmap Initiative tag; NOD mouse; SCID mouse; bioimaging /biomedical imaging; cell differentiation; cell migration; cell proliferation; contrast media; cytotoxicity; gene expression; glioma; magnetic resonance imaging; molecular /cellular imaging; molecular cloning; molecular probes; nanotechnology; nerve stem cell; reporter genes; technology /technique development; transfection

DESCRIPTION (provided by applicant): Non-invasive imaging of cell migration, trafficking, and homing is an emerging new field that can provide us with a deeper insight into the dynamics of cell-tissue interactions, as well as provide guidance to the development of novel cell therapies using stem cells and progenitors. Compared to other imaging modalities (i.e., PET, SPECT, and bioluminescent imaging), MR imaging has the highest spatial resolution and can provide both anatomical and functional information, but it suffers from a severely high signal-to noise threshold for the detection of cells using suitable labels/tracers. To a certain degree, this limitation has been resolved using intracellular endosomal tagging with super-paramagnetic nanoparticles. However, the magnetic susceptibility-based T2 (*) contrast induced this way has significant drawbacks, including the creation of hypointense "black holes" (obscuring tissue morphology), difficult differentiation between live and dead cells, the presence of hypointense imaging artifacts, uncertainty about long-term metal toxicity, and, most important, dilution of label following cell proliferation. We are proposing a new approach for the MR detection of labeled cells using a CEST (Chemical Exchange Saturation Transfer) reporter gene. The method is based on the expression of amide-enriched artificial proteins, i.e., lysine-rich protein (LRP) and argenine-rich protein (ARP) that can be detected by CEST imaging in the nanomolar range. The advantages of using these molecular probes are: 1) the gene product can be visualized directly without the need of a substrate (no tissue penetration needed); 2) detection sensitivity is not limited by cell proliferation; 3) only live cells should provide CEST contrast; 4) the contrast can be "switched-on" and "switched-off" repeatedly; and 5) double- or triple-cell labeling strategies may be pursued. We have initial data showing that a CEST reporter gene can be cloned, expressed in transfected cells, and specifically detected by MR CEST imaging in phantoms, without affecting cell viability or proliferation. We hypothesize that this detection is also possible in vivo. To achieve this goal, our aim is to synthesize novel, more efficient CEST reporter genes, and to detect double-labeled LRP/ARP transfected glioma cells and neural stem cells individually in live animals.

描述（由申请人提供）： 细胞迁移、运输和归巢的非侵入性成像是一个新兴的新领域，它可以让我们更深入地了解细胞与组织相互作用的动态，并为利用干细胞和细胞因子开发新型细胞疗法提供指导。祖先。与其他成像方式（即 PET、SPECT 和生物发光成像）相比，MR 成像具有最高的空间分辨率，可以提供解剖和功能信息，但其细胞检测的信噪比阈值非常高使用合适的标签/示踪剂。在某种程度上，使用超顺磁性纳米颗粒的细胞内内体标记已经解决了这一限制。然而，以这种方式诱导的基于磁化率的 T2 (*) 对比具有显着的缺点，包括产生低信号“黑洞”（模糊组织形态）、难以区分活细胞和死细胞、存在低信号伪影、不确定性关于长期金属毒性，最重要的是，细胞增殖后标记的稀释。 我们提出了一种使用 CEST（化学交换饱和转移）报告基因对标记细胞进行 MR 检测的新方法。该方法基于富含酰胺的人工蛋白的表达，即富含赖氨酸的蛋白（LRP）和富含精氨酸的蛋白（ARP），可以通过 CEST 成像在纳摩尔范围内检测到。使用这些分子探针的优点是：1）无需底物（无需组织穿透）即可直接观察基因产物； 2）检测灵敏度不受细胞增殖的限制； 3）只有活细胞才能提供CEST对比； 4）对比度可反复“开启”和“关闭”； 5) 可以采用双细胞或三细胞标记策略。我们的初步数据表明，CEST 报告基因可以在转染细胞中克隆、表达，并通过体模中的 MR CEST 成像进行特异性检测，而不影响细胞活力或增殖。我们假设这种检测在体内也是可能的。为了实现这一目标，我们的目标是合成新颖、更高效的CEST报告基因，并在活体动物中单独检测双标记LRP/ARP转染的神经胶质瘤细胞和神经干细胞。