THE CYTOGENETICS LABORATORY

细胞遗传学实验室

• 批准号: 7391946
• 负责人: MARK E HASKINS
• 金额: $ 10.07万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-08-01 至 2007-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7391946
• 关键词: CYTOGENETICS; LABORATORY

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Basic studies continue to concentrate primarily on Giemsa banded karyotyping of animals with sexual anomalies as well as syndromic disorders that suggest the presence of a chromosomal anomaly. Since the last progress report, a total of 19 cytogentic studies were performed on canine and feline cases referred to the genetics clinic or following consultations. Canine investigations included DiGeorge Syndrome, os clitoris, azoospermia, ichthyosis, a PCH-1 heterozygote, intersex, hypospadias extreme, and ataxia. A feline case of intersex was also examined. Novel disease presentations included canine soft tissue swelling, ichthyosis, ataxia, extreme joint laxity, perodontal disease, DiGeorge syndrome, cystrophic mineralization in limbs, HD and ED. Feline cases of spinal muscular atrophy, ostogenesis imperfecta, multiple fractures, cutaneous asthemia, and other connective tissue disorders were also examined. Studies include Western blotting using antibodies specific for different collagens and pulse chase assays for collagen type I function and assembly. The Western blots have been challenging as many of the antibodies are specific to species other than the dog and cat. The findings in animals with skin fragility are being summarized as part of a dermatology residency project and will be submitted for publication. Another primary role of the cytogenetic laboratory is tissue culture of models currently under investigation in the Referral Center. Affected cell lines have been added for canine MPS IIIB, MPS VI, PFK, X-linked anhydrotic ectodemal dysplaisa, epidermolysis bullosa, and lethal acrodermatitis in an effort to define cellular pathology, provide RNA and DNA for mutation analysis, and for use in future cross-correction studies. Fibroblast cultures were established from dogs with hypophosphatemia, Labrador myopathy, renal failure, possible MPS III, and pathologic fractures. Fibroblast cultures from cats were established for MPS VI, an undiagnosed lysosomal storage disease, pyruvate kinase deficiency, I-cell disease, portocaval shunt, GM2 gangliosidosis, neuromuscular disease, MPS VII, MPS I, and GM1 gangliosidosis. Methods have been developed for the use of fluorescence in situ hybridization (FISH) to define the physical location of genes on canine chromosomes. This approach uses gene probes labeled with fluorescent dyes. The labeled probes are hybridized to metaphase chromosomes and detected by fluorescence microscopy, allowing cloned genes and other markers to be mapped to specific chromosome locations. The FISH method provides a means of mapping the mutant genes involved in canine genetic diseases to their chromosomal locations, further increasing knowledge of the comparative medical genetics of these important models and enhancing their use as animal models of human genetic disease. In addition, the FISH studies add to the development of the canine genome map, increasing its value as a resource for finding new canine homologs of human genetic diseases. We have collaborated in a worldwide initiative to map the dog genome. As a part of this work, we have FISH-mapped a series of markers that serve as anchors for linkage groups on canine chromosomes. These linkage groups contain 341 mapped markers distributed over 37 canine autosomes and the X Chromosome. These gene markers simultaneously serve as anchor loci for microsatellite markers in the canine linkage map and establish the chromosomal locations of the linkage groups, allowing the canine gene regions to be lined up with the homologous regions of human and mouse chromosomes. This provides access to the rich source of information on evolutionarily conserved arrangements of genes on chromosomes in these other species, greatly facilitating attempts to locate and isolate canine genes by positional cloning methods.

该子项目是利用 NIH/NCRR 资助的中心拨款提供的资源的众多研究子项目之一。子项目和研究者 (PI) 可能已从另一个 NIH 来源获得主要资金，因此可以在其他 CRISP 条目中得到体现。列出的机构是中心的机构，不一定是研究者的机构。基础研究继续主要集中于具有性异常的动物的吉姆萨带状核型分析以及表明存在染色体异常的综合征性疾病。 自上次进展报告以来，总共对转至遗传学诊所或咨询后的犬类和猫科动物病例进行了 19 项细胞遗传学研究。犬类研究包括迪乔治综合症、阴蒂、无精症、鱼鳞病、PCH-1杂合子、雌雄同体、极度尿道下裂和共济失调。还检查了猫科双性人病例。 新的疾病表现包括犬软组织肿胀、鱼鳞病、共济失调、关节极度松弛、牙周病、迪乔治综合征、四肢囊性矿化、HD和ED。还检查了患有脊髓性肌肉萎缩、成骨不全、多发性骨折、皮肤贫血和其他结缔组织疾病的猫科动物病例。研究包括使用针对不同胶原蛋白的特异性抗体进行蛋白质印迹分析，以及针对 I 型胶原蛋白功能和组装的脉冲追踪测定。蛋白质印迹一直具有挑战性，因为许多抗体对狗和猫以外的物种具有特异性。皮肤脆弱动物的研究结果正在作为皮肤科住院医师项目的一部分进行总结，并将提交出版。 细胞遗传学实验室的另一个主要作用是对转诊中心目前正在研究的模型进行组织培养。已添加犬 MPS IIIB、MPS VI、PFK、X 连锁无水性外胚层发育不良、大疱性表皮松解症和致死性肢端皮炎的受影响细胞系，以努力定义细胞病理学，提供用于突变分析的 RNA 和 DNA，并用于未来的交叉校正研究。从患有低磷血症、拉布拉多肌病、肾衰竭、可能的 MPS III 和病理性骨折的狗中建立成纤维细胞培养物。猫的成纤维细胞培养物用于 MPS VI、一种未确诊的溶酶体贮积病、丙酮酸激酶缺乏症、I 细胞病、门腔静脉分流、GM2 神经节苷脂沉积症、神经肌肉疾病、MPS VII、MPS I 和 GM1 神经节苷脂沉积症。 已经开发出使用荧光原位杂交（FISH）来确定犬染色体上基因的物理位置的方法。该方法使用用荧光染料标记的基因探针。标记的探针与中期染色体杂交并通过荧光显微镜检测，从而将克隆的基因和其他标记映射到特定的染色体位置。 FISH方法提供了一种将犬遗传病相关突变基因定位到其染色体位置的方法，进一步增加了对这些重要模型的比较医学遗传学的了解，并增强了它们作为人类遗传病动物模型的用途。此外，FISH 研究促进了犬基因组图谱的开发，提高了其作为寻找人类遗传疾病的新犬同源物的资源的价值。我们合作开展了一项全球性计划，绘制狗基因组图谱。作为这项工作的一部分，我们对一系列标记进行了 FISH 作图，这些标记用作犬染色体上连锁群的锚点。这些连锁群包含分布在 37 个犬常染色体和 X 染色体上的 341 个定位标记。这些基因标记同时充当犬连锁图谱中微卫星标记的锚定位点，并建立连锁群的染色体位置，使犬基因区域与人类和小鼠染色体的同源区域对齐。这提供了有关这些其他物种染色体上进化上保守的基因排列的丰富信息来源，极大地促进了通过位置克隆方法定位和分离犬基因的尝试。