In total, 13,406 hours of cytogenetic experiments including analyzes and the use of microscope systems and processing workstations use were undertaken by the Cytogenetic and Microscopy Core in the reporting period to serve the NHGRI scientific community. The Cytogenetic Section experiments include cell culture, harvest, slide making and processing of all these techniques and microscope analyses related with cytogenetic, molecular cytogenetic and RNA scope research projects. The Microscopy Section work included the training of investigators and institute trainees in how to use Confocal Laser Scanning Microscopy in studies that comprise of 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.), along with live-cell, digitizing slides and super resolution microscopy. Microscopy usage is described by the metric of hours logged by Principal Investigators or their trainees. The Core maintains two confocal systems (Zeiss LSM 880 + Airyscan and Spinning disk), one long-term live-cell system, one automated slide scanner, two epi-fluorescence microscopes all fitted with CCD cameras and five computer workstations. Below is an abbreviated list of projects that the Core collaborated in the past year: The laboratory of Dr. Gahl (MGB) 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, atypical 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 region in several patients. The laboratory of Dr. Liu (GMBB) is performing studies to determine if a specific genetic alteration occurs in mammary tumors that develop in MMTV-PPARdelta transgenic mice (Cancer Res. 73:4349, 2013). This is the first transgenic model to demonstrate that activation of the nuclear receptor PPARdelta elicits oncogenesis in mammary epithelial tissue. The development of infiltrating ductal carcinomas in these mice has a phenotype similar to luminal B breast cancer and is associated with metabolic activation of the mTOR pathway. We are determining if this tumor phenotype is associated with genomic instability and a specific genetic lesion. The laboratory of Dr. Muenke (MGB) study is investigating adults with sex chromosome variants (including 45,X; 47,XXY; 47,XYY; 47,XXX) to assess and characterize phenotypes from clinical to molecular level. Our hypothesis is that intrinsic factors genetic differences between XX and XY cells have unappreciated biological consequences throughout the body and contribute to sex differences in disease incidence and severity. We collaborate with David Page, MD, director of the Whitehead Institute, on this study. The Page lab has identified homologous genes on the X and Y chromosome that are dosage-sensitive, expressed throughout the body, and encode regulators of chromatin modification, transcription, translation, and protein stability. All the patients are evaluated at the NIH Clinical Center with a comprehensive clinical evaluation and collection of numerous biospecimens (blood, buccal and skin). We are actively recruiting patients and enroll approximately 20-30 participants per year. The Page lab will conduct deep, genome-wide transcriptional and epigenetic profiling of these individuals to learn how X and Y gene dosage influences the molecular genetic landscape of cells. The project of Dr. Sood (GMBB) is evaluating the function of CECR1 using zebrafish. Using morpholino knockdown technology they generate embryos with transient loss of function for cecr1b. These experiments are performed in transgenic fish where blood vessels are marked by GFP and red blood cells are marked by DsRed. The core supports the project using the confocal microscope to image the developing blood vessels in the head and trunk regions of the knockdown embryos. Dr. Kastner lab is studying the mechanism of inflammation in patients with trisomy 8 mosaicism. In some of their patients we found constitutional trisomy 8 mosaicism (diagnosed by blood karyotype) and is fresh tissue from the esophagus, stomach and buccal cells. They were originally planning to look at the copy number of chromosome 8 genes in the bulk tissue, but also, they would like to know if non-hematopoietic cells in the GI tract also have trisomy 8. Performing FISH may be a way to clarify which type of cells carry trisomy 8. Dr. Biesecker (MGMGB) lab is studying Proteus syndrome that is a rare, progressive overgrowth disorder caused by a somatic activating variant c.49G>A, p.(E17K) in AKT1. To address limitations of human studies, they created mouse model with endogenously-regulated expression of a conditional allele containing the Proteus syndrome variant. To test the Happle hypothesis, which states that the Proteus syndrome variant only survives by mosaicism, we activated the variant allele by crossing Akt1 conditional and ACTB-Cre mice which express CRE ubiquitously. Embryos from this cross were not viable and died between E11.5 and E17.5, supporting the Happle hypothesis. Mutant embryos were pale, had fewer visible blood vessels and increased hemorrhage in the skin. Whole-mount immunostaining of E14.5 and E15.5 mutant skin showed no mature arteries or veins in the vasculature, suggesting a vascular remodeling defect. To determine the extent of the vascular defects in these embryos, we used the iDISCO clearing method to perform whole mount immunolabeling and volume imaging on mutant and wild type embryos. Endothelial and smooth muscle cells were labeled with antibodies to PECAM-1 and smooth muscle actin (SMA), respectively, to visualize the vasculature in embryos collected from E11.5 to E14.5. Cleared and stained embryos were imaged in the NHGRI Cytogenetic and Microscopy Core using the Super Resolution Fast Acquisition Mode for Zeiss LSM 880 with Airyscan, followed by Airyscan processing and visualization using Imaris x64 software. Examination of E12.5 embryos showed that the vertebral arteries failed to complete formation with heterogeneous, abnormal connections of the basilar artery to the aorta. In E13.5 mutant embryos, several vessels including the thoracic and external carotid arteries were missing, underdeveloped, or had abnormal patterning. Studies to fully characterize the vascular anomalies in mutant embryos are ongoing and will be used to investigate the mechanism that leads to disruption of the vasculature and to develop quantitative outcome measures for therapeutic trials in these mice. In Dr. McGuire (MGMGB) lab studies include the Mitochondria that are ubiquitous organelles that generate ATP through the process of oxidative phosphorylation (OxPhos) which is required for various cell processes. Organs such as the brain require many mitochondria to meet their energy demand. This is why it is most commonly affected by mitochondrial disease (MD). Patients with MD are a central cause of neurodegeneration that is characterized by behavioral, motor, and co
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