NHGRI/DIR Cytogenetics and Microscopy Core

NHGRI/DIR 细胞遗传学和显微镜核心

• 批准号: 8177745
• 负责人: Leslie Biesecker
• 金额: $ 109.07万
• 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/8177745
• 关键词: 18p; Abnormal Karyotype; Acute Myelocytic Leukemia; Animals; Area; Atopic Dermatitis; Base Sequence; Biological; Biological Assay; Bone Development; Bone Morphogenetic Proteins; Candidate Disease Gene; Cell Culture Techniques; Cell Nucleus; Cell-Cell Adhesion; Cells; Cellular Morphology; Chest; Chromatin Structure; Chromosomal Duplication; Chromosome Breakage; Chromosome abnormality; Chromosomes; Clear Cell; Cobalamin; Collection; Complementary DNA; Complex; Computer Workstations; Congenital Abnormality; Cytogenetic Analysis; Cytogenetics; Cytoplasm; Cytoplasmic Organelle; DNA; DNA Damage; DNA mapping; Data; Data Set; Defect; Development; Disease; Disease Progression; Drosophila genus; Embryo; Endometrial; Endometrial Carcinoma; Endometrial Neoplasms; Fiber; Fluorescence; Fluorescence Resonance Energy Transfer; Fluorescent in Situ Hybridization; Four-dimensional; G-Banding; Ganglia; Gaucher Disease; Gene Deletion; Gene Dosage; Genes; Genomic Instability; Goals; Green Fluorescent Proteins; Hair Cells; Hematopoiesis; Hereditary Disease; Holoprosencephaly; Homologous Gene; Hour; Human; Human Genetics; Image; Imagery; In Situ; Institutes; Karyotype; Karyotype determination procedure; Label; Laboratories; Laser Scanning Confocal Microscopy; Life; Malignant Neoplasms; Maps; Membrane Proteins; Metaphase; Methodology; Metric; Microscope; Microscopy; Microtubules; Mission; Mitosis; Mitotic; Monitor; Motor Neurons; Movement; Mus; Mutation; Myxoid cyst; National Heart, Lung, and Blood Institute; National Human Genome Research Institute; National Institute of Neurological Disorders and Stroke; Nerve Degeneration; Nuclear; Nucleic acid sequencing; PKD2 protein; PTK2 gene; Parkinsonian Disorders; Pathway interactions; Patients; Pharmaceutical Preparations; Phenotype; Photons; Physiology; Point Mutation; Polydactyly; Preparation; Principal Investigator; Procedures; Process; Progeria; Protein Dynamics; Proteins; Pseudoxanthoma Elasticum; Recovery; Research Personnel; Resolution; Resources; Role; Sampling; Screening procedure; Series; Serous; Services; Signal Transduction; Sister Chromatid Exchange; Skin; Specimen; Spectral Karyotyping; Stimulus; Stretching; Syndrome; System; Technology; Thick; Three-Dimensional Imaging; Time; Tissues; Transgenes; Transgenic Mice; United States National Institutes of Health; Wiskott-Aldrich Syndrome; Yang; Zebrafish; assay development; base; cancer cell; charge coupled device camera; fluorescence microscope; follow-up; gastrulation; investigator training; leukemia; melanoma; methylmalonic aciduria; mouse model; multi-photon; nerve supply; protein expression; research study; response; smoothened signaling pathway; telomere; two-dimensional

Summary: In total, 1336 cytogenetic experiments were performed in 2010 from January to July. Microscopy services included training investigators and institute trainees in how to use Confocal Laser Scanning Microscopy in studies that included Fluorescence Recovery After Photo-bleaching (FRAP), Fluorescence Resonance Energy Transfer (FRET), Photo-activation of Green Fluorescent Protein (PA-GFP), nuclear/organelle/cytoplasmic colocalization studies, Two-Dimensional (2D), Three-Dimensional (3D) and Four-Dimensional (4D) cell morphology and volumetric studies, response to stimuli (drug), quantitative analysis (fluorescence, area, counts, etc), and live-cell and deep-tissue imaging (with multi-photon microscopy). Microscopy usage described from January to July by the metric of hours logged by Principal Investigators or their trainees. For 2010 this usage to date involved 1380 Confocal hours of PI and trainee usage, 1121 long-term live-cell hours, 674 epi-fluorescent hours and 186 post-processing hours on the Core's computer workstation. The Core maintains two Confocal systems (UV and NLO), one long-term live-cell system, two epi-fluorescence microscopes all fitted with CCD cameras and two computer workstations. The Core collaborated with the following projects in the past year: The laboratory of Dr. Bell (GTB) is performing studies on endometrial (uterine corpus) cancer. The Core performed FISH and SKY analyses on endometrial cancer cells to determine chromosomal abnormalities, with the overall goal of assisting the investigator in defining the molecular genetic abnormalities that give rise to serous and clear cell endometrial tumors. The laboratory of Dr. Collins (GTB) is studying Hutchinson-Gilford progeria syndrome (HGPS), a rare genetic disorder caused by a de novo point mutation in the LMNA gene, which encodes progerin. The Core is assisting in functional characterization of progerin during mitosis and analysis of the potential mitotic defects caused by progerin accumulation using classic cytogenetics techniques and FISH with telomeric probes. Also, this lab has developed a qPCR assay for identifying LMNA transgenic mice carrying two putative LMNA copies but, they rely heavily on the accuracy of the FISH analysis performed by the Core to confirm the existence of both copies of the transgene in selecting their mice for follow up analysis. The laboratory of Dr. Green (GTB) is studying ABCC6 gene deletions in samples from patients with pseudoxanthoma elasticum by performing FISH analyses using fosmid clones. The laboratory of Dr. Muenke have potential candidate gene, Twisted Gastrulation Homolog 1 (TWSG1), was previously suggested as a contributor to the complex genetics of human (Holoprosencephaly) HPE based on (1)cytogenetic studies of patients with 18p deletions, (2) animal studies of TWSG1 deficient mice, and (3) the relationship of TWSG1 to bone morphogenetic protein (BMP) signaling, which modulates the primary pathway implicated in HPE, Sonic Hedgehog (SHH) signaling. Our core performed FISH analyses using BAC clones to do a fine mapping of 18p for a subset of patients with partial 18p deletions The laboratory of Dr. Myung (GMBB) has identified a human protein, ELG1, which responds to multiple DNA damaging agents and localizes on chromosomes at the place of DNA breakage. The Core assisted with spectral karyotyping experiments in metaphase chromosomes and studies of genomic instabilities by chromosome breakage analyses, sister chromatid exchange as well as FISH with telomeric probes. Also, the Core performed experiments analyzing telomere dynamics, protein movement in cells and localization of damage DNA studies. The laboratory of Dr. Pavan (GDRB) is analyzing chromosomal amplifications of murine loci involved in melanoma disease progression. We assisted with the characterization of the effect of Sox10 mutations by establishing the sub-cellular localization and pixel quantification in post-processing analyses from in situ images of the Sox10 gene product... Karyotype analyses were done for a Rps7 mutation. The laboratory of Dr. Yang group (GDRB) is studying hair cell orientation in the cochlear region. We assisted them by providing FRET of membrane proteins, interactions of the Wnt and BMP pathways, and Hedgehog signaling in bone development. In addition to these major projects, the Core has performed or assisted in many other projects with NHGRI and some other NIH investigators. These include: polydactyly mapping (Biesecker, GDRB), NF1 deletion mapping (Stewart GDRB), 2D and Time lapse imaging in Wiskott-Aldrich Syndrome in a mouse model (Candiotti, GMBB), FISH, 2D imaging and post processing in the cooperation of mutations between leukemia phenotypes in a mouse model, mapping markers in Zebra- fish chromosomes and screening genes involved in normal hematopoiesis and leukemia and studying acute myeloid leukemia protein, Cbfb-MYH11 (Liu, GMBB), development of an assay to identify bacterial strains in mouse skin sections and a mouse model of atopic dermatitis, and imaging protein expression in embryonic mouse epidermal sections (Segre, GMBB), 2D and Time lapse in methylmalonic acidemia, cobalamin disorders (Venditti, GMBB), FISH mapping in the Progerin mouse model (Nabel, GTB), co-localization studies on the influence of GBA mutations in the development of parkinsonism and gene dosage analysis in patient with Gaucher disease (Sidransky, MGB), 2D imaging, co-localization and Time Lapse in monitoring endogenous (Yap65) translocation from the nucleus to the cytoplasm and colocalization of polycystin-2 and interacting proteins, the effect of microtubules on Polycystin-2 localization and the role of FAK on cell-cell adhesion (Milgram, adjunct NHLBI) and 3D imaging in motor neuron innervations of the Drosophila thoracic ganglion and absence of the Cdk5 activator, p35, causes that neurodegeneration in Drosophila (Giniger Adjunct NINDS).

摘要：总共1336个细胞遗传学实验于2010年1月至7月进行。显微镜服务包括培训研究者和研究人员在如何使用共聚激光扫描显微镜的研究中进行的研究，其中包括荧光恢复后荧光恢复（FRAP），荧光共振能量转移（FRET），绿色荧光蛋白（PA-GFP）的光激活，核/细胞器/细胞质量研究（两位），两位 - 两位（两位），两位（两位）（两位），四维（4D）细胞的形态和体积研究，对刺激（药物）的反应，定量分析（荧光，面积，计数等）以及活细胞和深部组织成像（具有多光子显微镜）。 首席调查员或其受训者记录的时间的指标从1月至7月描述了显微镜使用情况。在2010年，迄今为止，这种使用涉及1380小时的PI和受训者使用情况，1121个长期的活细胞时间，674个Epi-fluorescent小时和核心计算机工作站的186个后处理时间。核心维护两个共聚焦系统（UV和NLO），一个长期的活细胞系统，两个Epi荧光显微镜都配备了CCD摄像头和两个计算机工作站。 核心与过去一年的以下项目合作： 贝尔博士（GTB）的实验室正在进行有关子宫内膜（子宫菌群）癌症的研究。核心对子宫内膜癌细胞进行了鱼类和天空分析，以确定染色体异常，其总体目标是协助研究者定义分子遗传异常，从而引起浆液性和清晰的子宫内膜肿瘤。 Collins博士（GTB）的实验室正在研究Hutchinson-Gilford Forperia综合征（HGP），这是一种由LMNA基因中的从头点突变引起的罕见遗传疾病，它编码了孕激素。核心是在有丝分裂过程中有助于对肌蛋白的功能表征，并分析使用经典的细胞遗传学技术和带有端粒探针的FISH引起的孕激素积累引起的潜在有丝分裂缺陷。 此外，该实验室已经开发了一种QPCR分析，用于鉴定带有两种推定LMNA副本的LMNA转基因小鼠，但是，它们在很大程度上依赖于核心执行的鱼类分析的准确性来确认在选择其小鼠以进行后续分析中的转基因的两种副本。 Green博士（GTB）的实验室正在研究使用FOSMID克隆进行鱼类分析的弹性瘤患者的ABCC6基因缺失。 Muenke博士的实验室具有潜在的候选基因，即抗胃同源物1（TWSG1），以前被认为是基于（1）18p缺失患者的细胞遗传学研究的人（Holoprosencephaly）HPE的复杂遗传学的贡献者信号传导调节与HPE，声音刺猬（SHH）信号传导有关的主要途径。我们的核心使用BAC克隆进行了鱼类分析，以对部分18p缺失的患者进行18p的精细图表 Myung博士（GMBB）的实验室已经确定了人类蛋白ELG1，该蛋白对多种DNA损害剂反应，并在DNA破裂的位置定位于染色体上。 核心通过染色体破裂分析，姐妹染色单体交换以及带有端粒探针的鱼进行了中期染色体的光谱核分型实验和基因组不稳定性的研究。此外，核心进行了分析端粒动力学，细胞中蛋白质运动以及损伤DNA研究的定位的实验。 Pavan博士（GDRB）的实验室正在分析参与黑色素瘤疾病进展的鼠基因座的染色体扩增。我们通过在Sox10基因产物的原位图像进行后处理分析中建立亚细胞定位和像素定量来协助SOX10突变的效果。用于RPS7突变的核型分析。 Yang Group博士（GDRB）的实验室正在研究人工耳蜗区域的毛细胞取向。我们通过提供膜蛋白的特征，Wnt和BMP途径的相互作用以及骨发育中的刺猬信号来协助它们。 除了这些主要项目外，核心还与NHGRI和其他一些NIH调查人员一起在许多其他项目中都进行了或协助。 These include: polydactyly mapping (Biesecker, GDRB), NF1 deletion mapping (Stewart GDRB), 2D and Time lapse imaging in Wiskott-Aldrich Syndrome in a mouse model (Candiotti, GMBB), FISH, 2D imaging and post processing in the cooperation of mutations between leukemia phenotypes in a mouse model, mapping markers in Zebra- fish chromosomes and screening genes involved in normal hematopoiesis and leukemia and studying acute myeloid leukemia protein, Cbfb-MYH11 (Liu, GMBB), development of an assay to identify bacterial strains in mouse skin sections and a mouse model of atopic dermatitis, and imaging protein expression in embryonic mouse epidermal sections (Segre, GMBB), 2D and Time lapse in methylmalonic acidemia, cobalamin disorders (Venditti, GMBB), FISH mapping in the Progerin mouse model (Nabel, GTB), co-localization studies on the influence of GBA mutations in the development of parkinsonism and gene dosage analysis in patient with Gaucher disease (Sidransky, MGB), 2D imaging, co-localization and Time Lapse in monitoring endogenous (Yap65) translocation from the nucleus to the cytoplasm and colocalization of polycystin-2 and interacting proteins, the effect of microtubules on Polycystin-2 localization and the role of FAK on cell-cell adhesion (Milgram, adjunct NHLBI) and 3D imaging in motor neuron innervations of the Drosophila thoracic ganglion and absence of the Cdk5激活剂p35导致果蝇（Giniger辅助Ninds）中的神经退行性变化。