NHGRI/DIR Cytogenetics and Microscopy Core

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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).