NHGRI/DIR Cytogenetics and Microscopy Core

点击翻译按钮获取中文摘要

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.