THE CYTOGENETICS LABORATORY

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。基础研究继续主要集中在有性异常的动物的Giemsa显带核型分析，以及暗示存在染色体异常的综合征障碍。自上次进展报告以来，总共对转诊到遗传学诊所或会诊后的犬类和猫科病例进行了19项细胞遗传学研究。犬的检查包括DiGeorge综合征、阴蒂、无精子症、鱼鳞病、PCH-1杂合子、双性、极端尿道下裂和共济失调。还检查了一起猫的双性病例。新的疾病表现包括犬软组织肿胀、鱼鳞病、共济失调、极度关节松弛、牙周病、DiGeorge综合征、四肢囊性矿化、HD和ED。还检查了脊髓性肌萎缩、成骨不全、多发性骨折、皮肤干燥和其他结缔组织疾病的猫病例。研究包括使用针对不同胶原蛋白的特异性抗体的Western blotting，以及I型胶原功能和组装的脉冲追踪法。西方印迹法一直在挑战，因为许多抗体是针对狗和猫以外的物种的。在皮肤脆弱的动物身上的发现正在作为皮肤科住院医生项目的一部分进行总结，并将提交发表。细胞遗传学实验室的另一个主要作用是组织培养目前在转诊中心进行研究的模型。犬MPS IIIB、MPS VI、PFK、X连锁脱水外胚层发育不良、大疱性表皮松解症和致死性肢端皮炎的受影响细胞系已被添加，以努力定义细胞病理学，为突变分析提供RNA和DNA，并用于未来的交叉校正研究。成纤维细胞培养取自患有低磷血症、拉布拉多肌病、肾功能衰竭、可能的MPS III和病理性骨折的狗。对未确诊的溶酶体储存性疾病MPS VI、丙酮酸激酶缺乏症、I细胞病、门腔分流、GM2神经节苷脂沉积症、神经肌肉疾病、MPS VII、MPS I和GM1神经节苷脂沉积症建立了猫成纤维细胞培养体系。已开发出使用荧光原位杂交(FISH)来确定基因在犬类染色体上的物理位置的方法。这种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，允许克隆的基因和其他标记被定位到特定的染色体位置。FISH方法提供了一种方法，将涉及犬类遗传病的突变基因定位到它们的染色体位置，进一步增加了对这些重要模型的比较医学遗传学的了解，并加强了它们作为人类遗传病动物模型的使用。此外，FISH研究对犬类基因组图谱的发展做出了贡献，增加了其作为寻找人类遗传病新的犬类同源物的资源的价值。我们在一项绘制狗基因组图谱的全球倡议中进行了合作。作为这项工作的一部分，我们对一系列标记进行了FISH定位，这些标记作为犬类染色体上的连锁群的锚。这些连锁群包含341个定位标记，分布在37个犬常染色体和X染色体上。这些基因标记同时作为犬连锁图谱中微卫星标记的锚点，并建立连锁群的染色体位置，使犬基因区域与人和鼠染色体的同源区域排成一条线。这为这些物种提供了丰富的进化保守的基因在染色体上排列的信息来源，极大地方便了通过位置克隆方法定位和分离犬基因的尝试。

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.