NHGRI/DIR Cytogenetics and Microscopy Core

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Summary: In total, 7,341 hours of 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 6,176 hours in all microscopes systems and workstations. The Core maintains two confocal systems (Zeiss LSM 880 +Airyscan 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. Crawford (CGB) 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 (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, 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 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) 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. The core performed FISH analyses using BAC clones to do fine mapping of 18p for a subset of patients with partial 18p deletions. Also with Dr. Muenke we study the chromosomalabonormalities that are related to medical conditions in people with sex chromosomes variants. The karyotypes are important to confirm the patients self reported karyotype (many patients dont have the original karyotype report) and ensure that there are no chromosomal abnormalities that could contribute to the phenotype that doctors are seeing in the clinic. 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.