NHGRI/DIR Cytogenetics and Microscopy Core

摘要：在本报告所述期间，总共进行了4663小时的细胞遗传学实验。显微镜服务包括培训研究人员和研究所受训人员如何使用共聚焦激光扫描显微镜进行下列研究：光漂白后荧光恢复(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、对刺激的反应(药物)、定量分析(荧光、面积、计数等)以及活细胞和深层组织成像(使用多光子显微镜)。显微镜的使用由首席调查员或他们的受训人员记录的小时数来描述。在本报告所述期间，使用包括2967.5个PI和受训人员共同使用小时、952个长期活细胞小时、468.5个电子荧光小时和546.5个在核心计算机工作站上的后处理小时。核心拥有两个共焦系统(蔡司NLO和旋转磁盘)、一个长期活细胞系统、两个均装有CCD摄像机的外荧光显微镜和四个计算机工作站。以下是核心部门在过去一年中合作的项目的简短清单： 克劳福德博士(CGB)的实验室正在研究RRP1B(核糖体RNA处理1同源B)，这是一种位于染色体1上的人类基因，被发现是一种新的转移抑制因子，被发现可以调节基因的表达。使用NLO共聚焦显微镜，他们正在通过免疫荧光验证RRP1B的潜在结合伙伴，范围从染色质相关因子到mRNA剪接因子。 Gahl博士的实验室正在研究Smith-Magenis综合征，这是一种以多种先天性异常和行为问题为特征的复杂疾病，包括头面部和骨骼异常、各种智力残疾、自残和寻求注意的行为、言语和运动延迟以及睡眠障碍。该综合征主要是由染色体17p11.2的从头开始间质缺失引起的。最常见的3.7Mb缺失发生在大约75%的患者中。然而，RAI1基因大小在1.5-9Mb之间的典型缺失和杂合点突变也与表型相关。这些缺失可以通过细胞遗传学G显带和/或荧光原位杂交(FISH)分析来检测。Core正在进行实验，以确定几名患者的确切缺失区域。 刘博士(GMBB)的实验室正在进行研究，以确定在MMTV-PPAR Delta转基因小鼠中发生的乳腺肿瘤是否发生了特定的基因变化(癌症研究报告73：4349,2013)。这是第一个证明核受体PPARDelta激活会导致乳腺上皮组织发生肿瘤的转基因模型。在这些小鼠中，浸润性导管癌的发展具有与管腔B乳腺癌相似的表型，并与mTOR途径的代谢激活有关。我们正在确定这种肿瘤表型是否与基因组不稳定和特定的遗传损伤有关。 Muenke(MGB)博士的实验室有一个潜在的候选基因，Twisted原肠形成同源基因1(TWSG1)，该基因先前被认为是人类(Holoproserey)HPE复杂遗传学的贡献者，其基础是对18p缺失患者的细胞遗传学研究，对TWSG1缺陷小鼠的动物研究，以及TWSG1与骨形态发生蛋白(BMP)信号的关系，BMP信号调节HPE中涉及的主要途径，Sonic Hedgehog(SHH)信号。CORE使用BAC克隆进行FISH分析，以对部分18P缺失的患者子集进行18P精细定位。 Myung博士(GMBB)的实验室已经确定了一种人类蛋白质ELG1，它能对多种DNA损伤剂做出反应，并定位在DNA断裂的染色体上。该中心协助进行中期染色体的光谱核型实验，并通过染色体断裂分析、姐妹染色单体交换和用端粒探针进行FISH来研究基因组不稳定性。此外，Core还进行了显微镜实验，分析了端粒动力学、蛋白质在细胞中的运动以及DNA损伤的定位。 Sood博士(GMBB)的项目是用斑马鱼来评估CECR1的功能。使用吗啉基因敲除技术，他们产生了cecr1b功能短暂丧失的胚胎。这些实验是在转基因鱼身上进行的，其中血管用GFP标记，红细胞用DsRed标记。该核心支持使用共聚焦显微镜对击倒胚胎头部和躯干区域发育中的血管进行成像的项目。 杨博士(GDRB)的实验室正在利用先进的核心成像技术追踪胚胎肢芽中的单个细胞，研究长骨在发育过程中的伸长。这使他们能够跟随自己的行为和命运，从而使他们能够理解这种定向增长是如何发生的。

Summary: In total, 4,663 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 2,967.5 Confocal hours of PI and trainee usage, 952 long-term live-cell hours, 468.5 epi-fluorescent hours and 546.5 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. 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 fluorrrescence 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. 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, and FISH with telomeric probes. Also, the Core performed microscopy experiments analyzing telomere dynamics, protein movement in cells and localization of damage DNA studies. 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. 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 using advanced imaging techniques in the core. This allows them to follow their behavior and fate and thus enables them to understand how this directional growth occurs.