IN VIVO VISUALIZATION AND QUANTIFICATION OF EPIGENETIC ACTIVITIES USING MAGNETIC

使用磁性对表观遗传活动进行体内可视化和量化

• 批准号: 8145600
• 负责人: ERIC T. AHRENS
• 金额: $ 30.26万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-17 至 2013-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8145600
• 关键词: Adult; Anatomy; Animal Experimentation; Bacteria; Biological; Breeding; Cells; Characteristics; Chemical Shift Imaging; Chemicals; Chimera organism; Clinic; Clinical; Code; Color; Communities; Computer software; Confocal Microscopy; Contrast Media; Cytosine deaminase; DNA Methyltransferase; DNA Modification Methylases; Databases; Diagnostic; Diet; DsRed; ES Cell Line; Embryo; Embryonic Development; Environment; Enzymes; Epigenetic Process; Event; Ferritin; Flucytosine; Fluorine; Fluorine Compounds; Fluorouracil; Frequencies; Gene Expression; Gene Transfer; Gene-Modified; Genes; Genetic; Genome; Goals; Gray unit of radiation dose; Health; Image; Imagery; Imaging Techniques; Imaging technology; Iron; Knock-in Mouse; Life; Light; Location; Longitudinal Studies; Magnetic Resonance Imaging; Magnetic Resonance Spectroscopy; Magnetism; Maps; Measurement; Measures; Metalloproteins; Microscope; Microscopy; Modeling; Molecular; Monitor; Mus; Nature; Nutrient; Optics; Organ; Organism; Outcome; Output; Patients; Population; Reporter; Research; Research Personnel; Resolution; Route; Spatial Distribution; Staging; Technology; Teratoma; Testing; Tetanus Helper Peptide; Three-Dimensional Image; Time; Tissues; Tube; United States National Institutes of Health; Whole Organism; Yeasts; base; environmental chemical; flexibility; imprint; in utero; in vivo; indexing; innovation; insight; interest; magnetic field; mouse model; novel strategies; promoter; ratiometric; soft tissue; three dimensional structure

DESCRIPTION (provided by applicant): MRI is non-invasive and allows longitudinal studies over the course of days and weeks to view into optically opaque subjects, and provides contrast among soft tissues at relatively high spatial resolution. In vivo imaging of gene expression using MRI has become a popular non-invasive imaging technology for biologists through the use of cellular metalloproteins, ex. ferritin, as contrast agents to monitor transferred gene expression under the control of interested promoter, such as DNA methyltransferase 1 (DNMT1). Metalloproteins load iron and become paramagnetic, thus enhancing the magnetic contrast in gene-transferred cells. This approach enables us to use high-resolution MRI to localize and image gene expression in a tissue or whole organism in three-dimensional (3D) format. Another novel approach proposed in this application is using fluorine-19 magnetic resonance spectroscopy/imaging (19F MRS/ MRI) as an in vivo "confocal microscope" to measure DNMT1 gene activity based on the principle of fluorine chemical shift imaging. A yeast cytosine deaminase (CD) will be used as a reporter to convert 5-fluorocytosine (5-FC) to 5-fluorouracil (5-FU). Both 5-FC and 5-FU will be measured by 19F MRS/ MRI in vivo as a 5-FC/5-FU ratiometric index to indicate DNMT1 gene activity in a living organism and then superimposed to 3D anatomic MR images to illustrate the spatial distribution of DNMT1 gene activity. We will use both ferritin and CD/fluorine as MRI reporters along with optical markers (GFP/RFP) to visualize and quantify DNMT1 gene expression in mouse models. The outcomes of this application would produce an anatomic 3D image of DNMT1 gene activity in a living mouse throughout its lifetime from embryo to adult. The mouse model established by the synthetic genetic circuit proposed in this application is flexible and can be adapted to the study of other epigenetic activity. This mouse model, e.g. DNMT1 in this application, can serve as a living test tube for the treatments of different nutrients, diet, chemicals and environmental challenges to study the genome-environment interaction at cellular level or whole organism. The epigenetic effects resulted from these treatments can be quantified and visualized in vivo by MRI. The measurement of biological events by 19F MRS is precise and sensitive. Using both ferritin and CD/19F MRI/MRS can digitize the measured promoter activity in different tissues and organs in the same organism before and after experimental treatments; thereby, a database of the 3D anatomic MR images with gene activity digitized and pseudo-colored for computing and visualization can provide invaluable insights into the temporal and spatial nature of epigenetic change.

描述(由申请人提供)： MRI是非侵入性的，允许在几天和几周的过程中进行纵向研究，以观察光学不透明的对象，并以相对较高的空间分辨率提供软组织之间的对比。利用核磁共振进行基因表达的活体成像已经成为生物学家通过使用细胞金属蛋白等非侵入性成像技术的流行技术。铁蛋白作为造影剂，在感兴趣的启动子，如DNA甲基转移酶1(DNMT1)的控制下，监测转移基因的表达。金属蛋白负载铁并变得顺磁性，从而增强了转基因细胞的磁性对比度。这种方法使我们能够使用高分辨率MRI来定位和成像三维(3D)格式的组织或整个生物体中的基因表达。本申请中提出的另一种新方法是利用氟-19磁共振波谱/成像(19F MRS/MRI)作为体内的共焦显微镜，基于氟化学位移成像的原理来测量DNMT1基因的活性。利用酵母胞嘧啶脱氨酶(CD)催化5-氟胞嘧啶(5-FC)转化为5-氟尿嘧啶(5-FU)。用~(19)F MRS/MRI在活体内测量5-FC和5-FU，作为5-FC/5-FU比值指数来指示活体中DNMT1基因的活性，然后将其叠加到3D解剖MR图像上，以显示DNMT1基因活性的空间分布。我们将使用铁蛋白和CD/氟作为MRI报告，并结合光学标记(GFP/RFP)来可视化和量化DNMT1基因在小鼠模型中的表达。这一应用的结果将产生从胚胎到成年的整个生命周期中活着的小鼠DNMT1基因活动的解剖3D图像。本应用中提出的合成遗传电路建立的小鼠模型具有灵活性，可用于其他表观遗传活动的研究。这个小鼠模型，例如本应用中的DNMT1，可以作为一个活的试管，用于不同营养、饮食、化学物质和环境挑战的处理，以在细胞水平或整个生物体水平上研究基因组与环境的相互作用。这些治疗所产生的表观遗传效应可以通过核磁共振在体内进行量化和可视化。用~(19)F-MRS对生物事件的测量是精确和灵敏的。同时使用铁蛋白和CD/19F MRI/MRS可以将实验治疗前后同一生物体不同组织和器官中测量的启动子活性数字化；因此，具有基因活性的三维解剖MR图像数据库可以为计算和可视化提供宝贵的深入了解表观遗传变化的时间和空间性质。