Spatial, High-Accuracy, Multiplexed Mapping of Genes in Human Tissue Sections

人体组织切片中基因的空间、高精度、多重定位

• 批准号: 8300976
• 负责人: ELISABETH SMELA
• 金额: $ 12.8万
• 依托单位: UNIV OF MARYLAND, COLLEGE PARK
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-08-05 至 2015-12-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8300976
• 关键词: Abnormal Cell; Affect; Amaze; Animal Model; Antibodies; Area; Atypia; Bacteria; Baltimore; Base Sequence; Benign; Binding; Breast; Cancer Patient; Cell Culture Techniques; Cells; Chromosome Mapping; Clinical; Complex; Consensus; Cyclin-Dependent Kinase Inhibitor 2A; DNA; DNA Methylation; DNA Sequence; DNA Sequence Alteration; DNA amplification; DNA lesion; DNA mapping; Data; Detection; Disease; Dissection; Ductal Carcinoma; Environment; Epigenetic Process; Epithelial; Eukaryota; Freezing; Future; GSTP1 gene; Gene Deletion; Gene Expression; Gene Expression Profile; Gene Silencing; Genes; Genetic; Genetic Variation; Genome; Goals; Hematoxylin and Eosin Staining Method; Histology; Human; Human Genome; Hypermethylation; Imagery; In Situ; Individual; Intraductal Hyperplasia; Intraepithelial Neoplasia; Knowledge; Laboratories; Lasers; Left; Lesion; Letters; Lobule; Location; Malignant Neoplasms; Maps; Maryland; Measures; Membrane; Messenger RNA; Methods; Methylation; Miniaturization; Molecular; Mutation; Noninfiltrating Intraductal Carcinoma; Nucleotides; Organism; Papilloma; Paraffin; Pathology; Patients; Premalignant; Promoter Regions; Prostate; Prostate carcinoma; Proteins; Publications; Publishing; Reproducibility; Research; Resected; Resolution; Reverse Transcriptase Polymerase Chain Reaction; Sampling; Shapes; Site; Spatial Distribution; Spottings; Staging; Staining method; Stains; System; Tail; Techniques; Technology; Testing; Time; Tissue Sample; Tissues; Tube; Universities; Variant; Vial device; Virus; Work; anticancer research; base; cancer genetics; cancer therapy; carcinogenesis; cost; genetic analysis; genome sequencing; human tissue; infiltrating duct carcinoma; interest; laser capture microdissection; malignant breast neoplasm; medical schools; meetings; millimeter; miniaturize; neoplastic cell; new technology; novel strategies; promoter; technology validation; theories; tissue fixing; tumor

DESCRIPTION (provided by applicant): The aim of the proposed research is to demonstrate a technology to visualize the spatial distribution of genes and gene methylation in tissue sections. Our novel approach combines the key advantages of existing techniques: the amplification of in-situ PCR, the per-few-cells resolution of laser capture microdissection, and the multiplexing of FISH. We have already demonstrated that our technology can extract, amplify, and detect DNA in a spatially resolved manner using a substrate with mini-vials (so to a resolution of 1.6 millimeter per spot). Our aims now are to Aim 1 (achieve DNA methylation): Map GSTP1 promoter methylation over whole tissue sections from prostates with cancer. Aim 2 (achieve high spatial resolution): Miniaturize the technology to achieve a resolution of 100 ¿m. Aim 3 (multiplexing): Map multiple genetic alterations at once. We will demonstrate 3 but hundreds at once are possible in principle. It is also possible to spatially map gene expression (mRNA) by reverse-transcriptase PCR, and to quantify the amount of mRNA (by quantitative real-time PCR). Though we have initial results for both these directions and the path to them is open, we must meet aims 1 to 3 before we can move on to these more challenging areas. Consequently, multiplexed mRNA mapping and quantification will be done in future work. We will demonstrate a technology to spatially map genetic changes in tissue sections. Our method will allow the mapping of gene deletions, mutations, insertions, and methylations (silencing) in tumors, neighboring abnormal cells, and surrounding healthy tissue. The technology will be validated on frozen and paraffin fixed tissues supplied to us by Dr. Emmert- Buck at NCI and Dr. Olga Ioffe at UMB Medical School.

描述（由适用提供）：拟议的研究的目的是证明一种技术，可视化基因和基因甲基化的空间分布。我们的新方法结合了现有技术的关键优势：原位PCR的扩增，激光捕获微解析的每触点分辨率和鱼类的多路复用。我们已经证明，我们的技术可以使用带有迷你瓶的底物以空间分辨的方式提取，放大和检测DNA（因此每点的分辨率为1.6毫米）。我们的目的是AIM 1（实现DNA甲基化）：从癌症前列腺的整个组织切片上MAP GSTP1启动子甲基化。 AIM 2（实现高空间分辨率）：小型化技术以达到100€的分辨率。 AIM 3（多路复用）：立即绘制多个遗传改变。我们将在原则上证明3个但数百个。也可以通过反向转录酶PCR在空间上绘制基因表达（mRNA），并量化mRNA的量（通过定量实时PCR）。尽管我们对这些方向和通往它们的途径都有最初的结果是开放的，但我们必须达到目标1到3，才能继续前进这些更多挑战领域。因此，将在以后的工作中进行多路复用的mRNA映射和定量。我们将展示一项技术，可以在组织切片中绘制遗传变化。我们的方法将允许在肿瘤，邻近异常细胞和周围健康组织中映射基因缺失，突变，插入和甲基化（沉默）。该技术将在NCI的Emmert-Buck博士和UMB医学院的Olga Ioffe博士提供给我们的冷冻和石蜡固定组织上进行验证。

项目成果

Dynamic inversion enables external magnets to concentrate ferromagnetic rods to a central target.

• DOI: 10.1021/nl503654t
• 发表时间: 2015-01-14
• 期刊: Nano letters
• 影响因子: 10.8
• 作者: Nacev A;Weinberg IN;Stepanov PY;Kupfer S;Mair LO;Urdaneta MG;Shimoji M;Fricke ST;Shapiro B
• 通讯作者: Shapiro B

Movement of magnetic nanoparticles in brain tissue: mechanisms and impact on normal neuronal function.

• DOI: 10.1016/j.nano.2015.06.003
• 发表时间: 2015-10
• 期刊: Nanomedicine : nanotechnology, biology, and medicine
• 作者: Ramaswamy B;Kulkarni SD;Villar PS;Smith RS;Eberly C;Araneda RC;Depireux DA;Shapiro B
• 通讯作者: Shapiro B