NHGRI/DIR Cytogenetics and Microscopy Core

总结：在本报告期内，共进行了7，341小时的细胞遗传学实验。显微镜服务包括培训研究人员和机构学员如何在研究中使用共聚焦激光扫描显微镜，包括光漂白后荧光恢复（FRAP），荧光共振能量转移（FRET），绿色荧光蛋白（PA-GFP）的光活化，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（药物）的反应，定量分析（荧光，面积，计数等），活细胞和深层组织成像（多光子显微镜）。通过主要研究者或其受训者记录的小时数指标描述显微镜使用情况。在本报告所述期间，所有显微镜系统和工作站的使用时间为6，176小时。 核心拥有两个共聚焦系统（蔡司LSM 880 +Airyscan和旋转盘），一个长期活细胞系统，两个落射荧光显微镜都配有CCD相机和四个计算机工作站。以下是核心在过去一年中合作的项目的简要清单： Crawford博士（CGB）的实验室正在研究RRP 1 B（核糖体RNA加工1同源物B），这是一种位于1号染色体上的人类基因，被发现是一种新的转移抑制因子，可调节基因表达。使用NLO共聚焦显微镜，他们正在通过免疫荧光验证RRP 1B的潜在结合伴侣，其范围从染色质相关因子到mRNA剪接因子。 Gahl博士（MGB）的实验室正在研究Smith-Magenis综合征，这是一种复杂的疾病，其特征是多种先天性异常和行为问题，包括颅面和骨骼异常，各种智力残疾，自我伤害和寻求注意力的行为，言语和运动延迟以及睡眠障碍。 该综合征主要由染色体17p11.2的从头间质性缺失引起。最常见的3.7 Mb缺失发生在约75%的患者中。然而，RAI 1基因的典型缺失（大小范围为1.5至9 Mb）和杂合点突变也与表型相关。缺失可通过细胞遗传学G显带和/或荧光原位杂交（FISH）分析检测。该中心正在进行实验，以确定几名患者的确切缺失区域。 Liu博士（GMBB）的实验室正在进行研究，以确定在MMTV-PPARdelta转基因小鼠中发展的乳腺肿瘤中是否发生特定的遗传改变（Cancer Res. 73：4349，2013）。这是第一个证明核受体PPARdelta的激活会导致乳腺上皮组织肿瘤发生的转基因模型。 这些小鼠中浸润性导管癌的发展具有与腔型B乳腺癌相似的表型，并且与mTOR途径的代谢活化相关。我们正在确定这种肿瘤表型是否与基因组不稳定性和特定的遗传病变有关。 Muenke博士（MGB）的实验室有一个潜在的候选基因，扭曲的胃构化同源物1（TWSG 1），以前被认为是人类复杂遗传学的贡献者。（前脑无裂畸形）HPE基于18 p缺失患者的细胞遗传学研究、TWSG 1缺陷小鼠的动物研究以及TWSG 1与骨形态发生蛋白（BMP）信号传导的关系，其调节HPE中涉及的主要途径，Sonic Hedgehog（SHH）信号传导。核心使用BAC克隆进行FISH分析，对部分18 p缺失的患者子集进行18 p精细定位。我们还与Muenke博士一起研究了与性染色体变异患者的医疗状况有关的染色体异常。核型对于确认患者自我报告的核型很重要（许多患者没有原始核型报告），并确保没有可能导致医生在临床上看到的表型的染色体异常。 Sood博士（GMBB）的项目正在使用斑马鱼评估CECR 1的功能。利用吗啉敲除技术，他们产生了cecr 1b功能暂时丧失的胚胎。这些实验在转基因鱼中进行，其中血管由GFP标记，红细胞由DsRed标记。核心支持使用共聚焦显微镜对敲除胚胎头部和躯干区域的血管发育进行成像的项目。

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.