NHGRI/DIR Cytogenetics and Microscopy Core

NHGRI/DIR 细胞遗传学和显微镜核心

• 批准号: 8565588
• 负责人: Leslie Biesecker
• 金额: $ 113.69万
• 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/8565588
• 关键词: 17p11.2; 18p; Abnormal Karyotype; Allergens; Animals; Area; Asthma; Attention; Base Sequence; Behavior; Binding; Biological; Biological Assay; Bone Morphogenetic Proteins; Candidate Disease Gene; Cell Culture Techniques; Cell Nucleus; Cell model; Cells; Cellular Morphology; Chromatin; Chromatin Structure; Chromosome Breakage; Chromosome Deletion; Chromosomes; Collection; Complementary DNA; Complex; Computer Workstations; Confocal Microscopy; Craniofacial Abnormalities; Cytogenetic Analysis; Cytogenetics; Cytoplasmic Organelle; DNA; DNA Damage; DNA mapping; Data; Data Set; Defect; Development; Disease; Embryo; Fiber; Fluorescence; Fluorescence Microscopy; Fluorescence Resonance Energy Transfer; Fluorescent in Situ Hybridization; Four-dimensional; G-Banding; Gene Expression; Genes; Genetic Predisposition to Disease; Genomic Instability; Glucokinase; Goals; Green Fluorescent Proteins; Growth; Hepatocyte; Hereditary Disease; Holoprosencephaly; Homologous Gene; Hour; Human; Human Genetics; Image; Imagery; Immunofluorescence Immunologic; Individual; Institutes; Intellectual functioning disability; Karyotype determination procedure; Label; Laboratories; Laser Scanning Confocal Microscopy; Life; Limb Bud; Lung; Maps; Measures; Messenger RNA; Metaphase; Methodology; Metric; Microscope; Microscopy; Mission; Mitosis; Mitotic; Motor; Movement; Mucins; Mus; National Human Genome Research Institute; Neoplasm Metastasis; Nuclear; Nucleic acid sequencing; Pathway interactions; Patients; Pharmaceutical Preparations; Phenotype; Photons; Physiology; Point Mutation; Preparation; Principal Investigator; Problem behavior; Procedures; Process; Production; Progeria; Protein Dynamics; Protein Family; Proteins; RNA Processing; RNA Splicing; Recovery; Reporting; Research Personnel; Resolution; Resources; Ribosomal RNA; Role; Scientist; Series; Services; Signal Transduction; Sister Chromatid Exchange; Sleep disturbances; Smith Magenis syndrome; Sorting - Cell Movement; Specimen; Spectral Karyotyping; Speech; Stimulus; Stretching; Syndrome; System; Technology; Thick; Time; Tissues; Transgenes; Transgenic Mice; Variant; Work; Yang; base; blood glucose regulation; cell type; charge coupled device camera; fluorescence microscope; follow-up; gastrulation; genetic regulatory protein; human disease; insight; interstitial; investigator training; long bone; member; multi-photon; nexin; novel; research study; response; skeletal abnormality; smoothened signaling pathway; telomere; two-dimensional

Summary: In total, 1,655 (3,075 hours) cytogenetic experiments were performed in the reporting period. 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 is described by the metric of hours logged by Principal Investigators or their trainees. For this reporting period, usage involved 1,328 Confocal hours of PI and trainee usage, 433 long-term live-cell hours, 786 epi-fluorescent hours and 849 post-processing hours on the Core's computer workstation. The Core maintains two confocal systems (Zeiss NLO and Spinning disk), one long-term live-cell system, two epi-fluorescence microscopes all fitted with CCD cameras and four computer workstations. Below is an abbreviated list of projects that the Core collaborated in the past year: The laboratory of Dr. Collins (GTB) is collaborating with the Core in several projects: 1-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. 2- 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. 3- Dr. Collin's lab scientists are using Core microscopes to measure the intensity of mucin production in the lungs of allergen-challenged mice. These data are being used to gain important insights about the role of genetic susceptibility to asthma. 4- Also they are working in the interaction of human glucokinase (GCK) with its regulatory protein (GKRP) in the hepatocyte nucleus that is critical for glucose homeostasis, with misregulation of either protein associated with human disease. They have used fluorescence microscopy to quantitatively show differences for the first time in cellular localization and interaction with glucokinase for the common disease-associated GKRP P446L variant in a model cellular system. The laboratory of Dr. Crawford is working with RRP1B (ribosomal RNA processing 1 homolog B), a human gene located in chromosme 1 found to be a novel metastasis suppressor that was found to regulate gene expression. Using NLO confocal microscopy they are validating potential binding partners of RRP1B, which range from chromatin-associated factors to mRNA splicing factors, through immunofluorescence. The laboratory of Dr. Gahl is studying Smith-Magenis syndrome a complex disorder characterized by multiple congenital anomalies and behavior problems, including craniofacial and skeletal abnormalities, variable intellectual disability, self-injurious and attention-seeking behaviors, speech and motor delay, and sleep disturbance. The syndrome is primarily caused by de novo interstitial deletions of chromosome 17p11.2. The most common 3.7 Mb deletion occurs in approximately 75% of the patients. However, a typical deletions that can range from 1.5 to 9 Mb in size and heterozygous point mutations of the RAI1 gene are also associated with the phenotype. The deletions are detectable by cytogenetic G-banding and/or by fluorescence in situ hybridization (FISH) analyses. The Core is working on experiments to define the exact deleted reagion in several patients. Dr. Milgram's laboratory is studying a protein called Sorting Nexin 27 (SNX27), which is a unique member of this protein family as it contains a PDZ domain. The PDZ domain is involved in interactions between SNX27 and other proteins. They have identified many novel interactions involving SNX27 and have used detailed microscopy procedures in the Core, to demonstrate the co-localization of SNX27 with these interacting proteins in multiple cell types. The laboratory of Dr. Muenke has a potential candidate gene, Twisted Gastrulation Homolog 1 (TWSG1), that was previously suggested as a contributor to the complex genetics of human (Holoprosencephaly) HPE based on cytogenetic studies of patients with 18p deletions, animal studies of TWSG1 deficient mice, and 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 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 microscopy experiments analyzing telomere dynamics, protein movement in cells and localization of damage DNA studies. The laboratory of Dr. Yang (GDRB) is studying the elongation of the long bones during development by tracing individual cells within the embryonic limb bud. This allows them to follow their behavior and fate and thus enables us to understand how this directional growth occurs.

摘要：报告期内共进行了1,655次（3,075小时）细胞遗传学实验。显微镜服务包括培训研究人员和研究所学员如何使用共聚焦激光扫描显微镜进行研究，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、二维（2D）、三维（3D）和四维（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（多光子显微镜）。显微镜使用情况是由主要研究人员或他们的受训者记录的小时数来描述的。在本报告所述期间，使用了1328个PI共聚焦小时，433个长期活细胞小时，786个外延荧光小时和849个核心计算机工作站的后处理小时。“核心”拥有两个共聚焦系统（蔡司NLO和旋转磁盘），一个长期活细胞系统，两个配备CCD相机的外显荧光显微镜和四个计算机工作站。以下是过去一年Core合作项目的简要列表：