NHGRI/DIR Cytogenetics and Microscopy Core

NHGRI/DIR 细胞遗传学和显微镜核心

• 批准号: 7734916
• 负责人: Leslie Biesecker
• 金额: $ 86.83万
• 依托单位: NATIONAL HUMAN GENOME RESEARCH INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7734916
• 关键词: Acute Myelocytic Leukemia; Adult; Alleles; Allergens; Amyotrophic Lateral Sclerosis; Area; Asthma; Autosomal Dominant Polycystic Kidney; Bacterial Artificial Chromosomes; Beta Cell; Binding; Biological Assay; Carbon Dioxide; Cardiovascular system; Cell Cycle; Cell Cycle Progression; Cell Line; Cell Nucleus; Cell Polarity; Cells; Cellular Morphology; Chimeric Proteins; Chinese People; Chromosomal Duplication; Chromosomal Loss; Chromosome Breakage; Chromosome Mapping; Chromosome Painting; Chromosome abnormality; Chromosomes; Clear Cell; Clinical; Coloboma; Color; Complex; Computer Workstations; Computer software; Confocal Microscopy; Connective Tissue Diseases; Controlled Environment; Cosmids; Count; Cyclin-Dependent Kinases; Cystic Fibrosis; Cytogenetics; Cytoplasm; Cytoplasmic Organelle; DNA; DNA Damage; DNA Probes; DNA Sequence Rearrangement; Deer; Depth; Development; Diabetes Mellitus; Diagnosis; Diagnostic; Diffusion; Disease; Disease Progression; Drosophila genus; Educational process of instructing; Embryo; Endometrial Neoplasms; Energy Transfer; Evaluation; Evolution; Eye; Failure; Family; Fiber; Fluorescence; Fluorescent in Situ Hybridization; G-Banding; Gaucher Disease; Gene Deletion; Gene Rearrangement; Generations; Genes; Genetic; Genome; Genomics; Genotype; Goals; Green Fluorescent Proteins; Hematopoiesis; Hereditary Disease; Histocompatibility Testing; Hour; Human; Humidity; Image; Image Analysis; Imagery; Imaging Techniques; Immunofluorescence Immunologic; Institutes; Interphase; Investigation; Karyotype; Karyotype determination procedure; Lamin Type A; Laser Scanning Confocal Microscopy; Lead; Length; Life; Localized; Malignant Neoplasms; Maps; Measurement; Measures; Meiosis; Membrane; Messenger RNA; Metaphase; Microcephaly; Microscope; Microscopy; Mission; Mitochondria; Mitosis; Molecular; Monitor; Morphology; Mucins; Mucous body substance; Muntiacus; Mus; Mutation; Nerve Degeneration; Neuraxis; Neurodegenerative Disorders; Neurofibromatosis 1; Neurons; Non-Insulin-Dependent Diabetes Mellitus; Nuclear; Nuclear Protein; Nuclear Proteins; Optics; Organelles; Osteoporosis; Outcome; PKD2 protein; Parkinson Disease; Parkinson' s Dementia; Parkinsonian Disorders; Pathway interactions; Patients; Pharmaceutical Preparations; Play; Polycystic Kidney Diseases; Polymerase Chain Reaction; Post-Transcriptional Regulation; Preparation; Production; Progeria; Property; Proprotein Convertase 2; Protein Tyrosine Kinase; Proteins; Protocols documentation; Pseudoxanthoma Elasticum; Publications; Recovery; Reporting; Research Personnel; Resources; Retina; Role; Screening procedure; Serous; Services; Signal Transduction Pathway; Skin; Solutions; Source; Spectral Karyotyping; Spinal; Spleen; Staining method; Stains; Stimulus; Support of Research; Syndrome; System; Techniques; Technology; Temperature; Testing; Tissues; Touch sensation; Training; Transfer RNA; Transgenes; Transgenic Mice; Translocation Breakpoint; Triage; Tubulin; Tumor Cell Line; Vascular Endothelial Growth Factors; X Chromosome; Yeasts; Zebrafish; base; calcification; cell type; cellular imaging; charge coupled device camera; comparative; comparative genomic hybridization; data management; day; deafness; embryonic stem cell; fluorescence microscope; glucosylceramidase; human disease; image processing; insight; interest; intracellular protein transport; investigator training; knock-down; knockout gene; leukemia; malformation; malignant breast neoplasm; melanoma; member; methylmalonic aciduria; mouse model; mutant; normal aging; novel; positional cloning; protein misfolding; protein transport; research study; response; synuclein; telomere; tool; trafficking; tumor; tumorigenesis; two-photon

Cytogenetics services include support to gene mapping, positional cloning, cytogenetic diagnostics, tumor cytogenetics, comparative evolution studies, transgenic mouse analysis, karyotyping, metaphase & interphase Fluorescent In Situ Hybridization (FISH), chromosome painting, fiber-FISH, Spectral Karyotyping (SKY), and chromosome Comparative Genomic Hybridization techniques. These services provide a spectrum of critically important techniques that are required to support the research efforts of many investigators within the institute. The culturing of cell lines, embryos, tumors, spleen and other sources of tissue are also provided. This section maintains two upright fluorescent scopes (FISH and SKY), cameras, spectracube and image analysis software. In total 1383 cytogenetic experiments were performed last year. Microscopy services include training investigators on how to properly use confocal laser scanning microscopy in studies which include Fluorescence Recovery After Photo-bleaching (FRAP), Fluorescent Resonant Energy Transfer (FRET), Photo-activation of Green Fluorescent Protein (PA-GFP), nuclear/organelle/cytoplasmic co-localization studies, 2-dimentional (2D), 3-dimentional (3D) & 4-dimentional (4D) cell morphology and volumetric studies, response to stimuli (drug), quantitative analysis (fluorescence, area, counts, etc), live cell and deep tissue imaging (with two-photon microscopy). The long-term live cell system gives investigators the ability to study live cells from hours to days with a temperature, humidity and CO2 controlled environment. This section maintains two confocal systems (UV and NLO), one long-term live-cell system, three epi-fluorescent microscopes all fitted with CCD cameras and two computer workstations. Microscopy usage for the past year; 1792 confocal hours, 4217 long-term live-cell hours, 1265 epi-fluorescent hours and 167 (only 4 months) workstation hours. The Core contributes in the following projects utilizing molecular cytogentic tools: Determination of chromosomal abnormalities using SKY, prior to sequence serous and clear cell endometrial tumors genomes; Analysis of chromosomal amplifications of specific locus in Melanoma disease progression; Evaluation of whole-gene deletion in Neurofibromatosis type 1 patients-cell lines and tumors touch-preps; Mapping of translocation breakpoints in a Hapomelanosis patient between chromosomes X and 16; Identification of whole-gene deletions for ATP-binding cassette, sub-family C member 6 that are the result of many segmental duplications known to lead to genomic rearrangements in Pseudoxanthoma Elasticum a rare autosomal recessive connective tissue disorder involving calcification of tissues in the retina, cardiovascular system, and flexural areas of the skin; Visualization of chromosome breakage in response to DNA damage caused by ELG1 protein; Analysis of chromosomes alterations by SKY in human disease mouse model of microcephaly; Studying the region of the insertion of a transgene (NSE-VEGF) in a mouse model for coloboma, a potentially blinding congenital eye malformation caused by the failure of the optic fissure to close during development; Screening the integration by FISH to identify putative two copies Lamin A transgenic mice; Screening by FISH of correct integration of modified Bacterial Artificial Chromosome DNA into the mouse embryonic stem cells for the generation of gene knockouts of different tyrosine kinases to study their role in immuno-deficiencies; Quantification of telomere lengths in normal and progeria cells from Hutchinson-Guilford Progeria Syndrome (HGPS) patient-cell lines; Comparative analysis on Indian and Chinese muntjac deer chromosomes, two species that are closely related but their genomes have drastically different organizations. The Core participates in the following projects utilizing confocal microscopy and image processing techniques: Immuno-flourescence studies include: Characterization of Neurofibromatosis type 1 (NF1); GFP-tagged protein foci in yeast; mRNA triage post-transcriptional regulation of messages in the beta cell in Type 2 Diabetes; Studying the relevance of a novel binding partner for the Cystic Fibrosis Trans-membrane Conductance Regulator protein (CFTR) and to elucidate its role in CFTR trafficking; Yes-associated protein 65 (Yap65) is a transcriptional co-activator important in cancer and development using confocal microscopy to monitor endogenous Yap translocation from the nucleus to the cytoplasm and vice-versa in response to stimuli as well as during the cell cycle in various cell types; Neuron-specific cyclin-dependent kinase Cdk5 is strongly implicated in the progression of many mammalian neurodegenerative diseases, including Alzheimers, Parkinson and Amyotrophic lateral sclerosis (ALS). We found that Drosophila lacking Cdk5 activator protein, p35, displays an adult-onset neurodegenerative central nervous system (CNS) syndrome. The goal of this project is to study how Cdk5/p35 causes neurodegeneration; The study of acute myeloid leukemia protein, Cbfb-MYH11 by confocal microscopy; examine the morphology of mitochondria from patients and mice with methylmalonic acidemia (MMA); Determine the sub-cellular localization of GFP-tagged TRMT12 fusion protein. Previously, we reported that the tRNA methyltransferase12 (TRMT12) gene is over-expressed in breast cancer and we are interested in studying the role of TRMT12 in tumorigenesis. Co-localization studies include: Evaluation of mechanisms in a mouse model of microcephaly; Signal transduction pathways utilized by the Wnt family of secreted proteins, dysregulation of this pathway has been strongly implicated in cancer and osteoporosis. The planar cell polarity pathway has been associated with spinal bifida, deafness, polycystic kidney disease and diabetes; How human ELG1 protein responds to multiple DNA damages and localizes where there is DNA damage using confocal microscopy and live cell imaging techniques; Dhx8 was identified from a zebrafish screen looking for genes involved in normal hematopoiesis and leukemia. To determine if dhx8 co-localizes with DNA or tubulin during mitosis and to observe the effect of knock-down on cell cycle progression by using confocal microscopy; Mutations in glucocerebrosidase gene(GBA) are seen in Gaucher disease, Parkinson disease and dementias with parkinsonism. Using several organelle markers and immunofluorescence assays for a-synuclein aggregation to show that GBA mutations lead to misfolding of protein and improper trafficking; Understanding the mechanisms behind the development of Autosomal Dominant Polycystic Kidney Disease by using confocal microscopy techniques to assess the role of Polycystin-2 (PC-2) binding partners on localization of PC-2 and cell-cell contacts and for co-localization with PC2. Fluorescent intensity measurements studies include: Assessing the degree of cytoplasmic sequestration of the primarily nuclear protein Runx1 through its interaction with the acute myelogenous leukemia protein, inversion 16 and its deletions mutants; Quantifying mucus production based on a modified mucin stain in asthma related-allergen challenged mice; To understand the cellular mechanisms of Hutchinson-Guilford Progeria Syndrome (HGPS) and normal aging by quantitatively measuring the telomere lengths in normal and progeria cells; Fluorescence Recovery After Photo-bleaching (FRAP) studies include: Analysis of GFP-progeria cells in mitosis; Quantifying the diffusion properties of the IGF2BP2 protein.

细胞遗传学服务包括支持基因作图、定位克隆、细胞遗传学诊断、肿瘤细胞遗传学、比较进化研究、转基因小鼠分析、核型分析、中期和间期荧光原位杂交 (FISH)、染色体涂色、纤维 FISH、光谱核型分析 (SKY)和染色体比较基因组杂交技术。这些服务提供了支持研究所内许多研究人员的研究工作所需的一系列至关重要的技术。还提供细胞系、胚胎、肿瘤、脾脏和其他组织来源的培养。该部门拥有两台直立荧光显微镜（FISH 和 SKY）、相机、spectrumcube 和图像分析软件。去年总共进行了 1383 次细胞遗传学实验。 显微镜服务包括培训研究人员如何在研究中正确使用共焦激光扫描显微镜，包括光漂白后的荧光恢复 (FRAP)、荧光共振能量转移 (FRET)、绿色荧光蛋白的光激活 (PA-GFP)、核/细胞器/细胞质共定位研究，2 维 (2D)、3 维 (3D) 和 4 维 (4D) 细胞形态和体积研究、对刺激（药物）的反应、定量分析（荧光、面积、计数等）、活细胞和深层组织成像（使用双光子显微镜）。 长期活细胞系统使研究人员能够在温度、湿度和二氧化碳受控的环境下研究活细胞数小时至数天。 该部门拥有两套共焦系统（UV 和 NLO）、一套长期活细胞系统、三台落射荧光显微镜（均配有 CCD 摄像头）和两台计算机工作站。 过去一年显微镜的使用情况； 1792 共聚焦小时、4217 长期活细胞小时、1265 落射荧光小时和 167（仅 4 个月）工作站小时。 核心利用分子细胞遗传学工具为以下项目做出贡献： 在对浆液性和透明细胞子宫内膜肿瘤基因组进行测序之前，使用 SKY 确定染色体异常； 黑色素瘤疾病进展中特定位点的染色体扩增分析； 1 型神经纤维瘤病患者细胞系和肿瘤接触准备中全基因缺失的评估； Hapomelanosis 患者 X 染色体和 16 号染色体之间的易位断点图谱； 鉴定 ATP 结合盒（亚家族 C 成员 6）的全基因缺失，这是许多片段重复的结果，已知会导致弹性假黄瘤的基因组重排，这是一种罕见的常染色体隐性结缔组织疾病，涉及视网膜组织钙化，心血管系统和皮肤弯曲区域； ELG1 蛋白引起的 DNA 损伤导致染色体断裂的可视化； SKY在人类小头畸形小鼠模型中的染色体改变分析 研究在小鼠缺损模型中插入转基因（NSE-VEGF）的区域，缺损是一种可能致盲的先天性眼部畸形，是由于视裂在发育过程中未能闭合而引起的； 通过 FISH 筛选整合，鉴定假定的两个拷贝 Lamin A 转基因小鼠； 通过 FISH 筛选修饰的细菌人工染色体 DNA 是否正确整合到小鼠胚胎干细胞中，以产生不同酪氨酸激酶的基因敲除，以研究它们在免疫缺陷中的作用； 对来自哈钦森-吉尔福德早衰综合症 (HGPS) 患者细胞系的正常细胞和早衰细胞的端粒长度进行定量； 印度和中国麂鹿染色体的比较分析，这两个物种密切相关，但它们的基因组组织却截然不同。 该核心利用共焦显微镜和图像处理技术参与了以下项目： 免疫荧光研究包括： 1 型神经纤维瘤病 (NF1) 的特征； 酵母中 GFP 标记的蛋白焦点； 2 型糖尿病 β 细胞中信息的 mRNA 分类转录后调节； 研究囊性纤维化跨膜电导调节蛋白（CFTR）的新型结合伴侣的相关性，并阐明其在 CFTR 运输中的作用； Yes 相关蛋白 65 (Yap65) 是一种在癌症和发育中重要的转录共激活因子，使用共聚焦显微镜监测内源性 Yap 从细胞核到细胞质的易位，反之亦然，以响应刺激以及在各种细胞周期中细胞类型； 神经元特异性细胞周期蛋白依赖性激酶 Cdk5 与许多哺乳动物神经退行性疾病的进展密切相关，包括阿尔茨海默病、帕金森病和肌萎缩侧索硬化症 (ALS)。我们发现，缺乏 Cdk5 激活蛋白 p35 的果蝇表现出成人发病的神经退行性中枢神经系统 (CNS) 综合征。该项目的目标是研究Cdk5/p35如何引起神经变性； 急性髓系白血病蛋白Cbfb-MYH11的共聚焦显微镜研究检查甲基丙二酸血症（MMA）患者和小鼠的线粒体形态； 确定 GFP 标记的 TRMT12 融合蛋白的亚细胞定位。 此前，我们报道了tRNA甲基转移酶12（TRMT12）基因在乳腺癌中过度表达，我们有兴趣研究TRMT12在肿瘤发生中的作用。 共定位研究包括： 小头畸形小鼠模型的机制评估； Wnt 分泌蛋白家族利用信号转导途径，该途径的失调与癌症和骨质疏松症密切相关。平面细胞极性通路与脊柱裂、耳聋、多囊肾病和糖尿病有关； 人类 ELG1 蛋白如何响应多重 DNA 损伤并使用共焦显微镜和活细胞成像技术定位 DNA 损伤的位置； Dhx8 是从斑马鱼筛选中鉴定出来的，该筛选寻找与正常造血和白血病有关的基因。确定 dhx8 在有丝分裂过程中是否与 DNA 或微管蛋白共定位，并使用共聚焦显微镜观察敲除对细胞周期进展的影响； 葡萄糖脑苷脂酶基因（GBA）突变见于戈谢病、帕金森病和帕金森病痴呆。使用多种细胞器标记和免疫荧光测定法进行α-突触核蛋白聚集，表明 GBA 突变导致蛋白质错误折叠和不当运输； 通过使用共聚焦显微镜技术评估多囊蛋白-2 (PC-2) 结合伴侣对 PC-2 和细胞间接触的定位以及与 PC2 共定位的作用，了解常染色体显性多囊肾病的发展机制。 荧光强度测量研究包括： 通过与急性髓性白血病蛋白、倒位 16 及其缺失突变体的相互作用，评估主要核蛋白 Runx1 的细胞质隔离程度； 基于改良的粘蛋白染色来量化哮喘相关过敏原攻击小鼠的粘液产生； 通过定量测量正常细胞和早衰细胞的端粒长度，了解哈钦森-吉尔福德早衰综合症（HGPS）和正常衰老的细胞机制； 光漂白后的荧光恢复 (FRAP) 研究包括： GFP-早衰细胞有丝分裂的分析； 量化 IGF2BP2 蛋白的扩散特性。