Chromosome 21 Elimination In A New Mouse Model of Down Syndrome

项目摘要 唐氏综合征（DS）是由人类21号染色体三体（Hsa 21）引起的最复杂的疾病之一 遗传干扰与人类生存期一致; 80%的21三体妊娠流产。 CA的交互作用。Hsa 21上的600个转录序列直接影响细胞，通过改变Hsa 21的作用， 个体细胞在发育过程中，通过对邻近细胞的次级影响。由此产生的结果 包括80多个影响身体每个系统的临床特征，尽管只有这些特征的一个子集 在任何21三体个体中观察到。分子遗传学在DS研究中的出现带来了 用新发现的Hsa 21基因制成的单基因转基因小鼠; 还原论者的观点认为，DS表型是独立作用的单基因效应的总和。 Davisson的Ts 65 Dn小鼠从根本上改变了DS研究的范式， 对于Hsa 21直系同源物的大子集。对这些小鼠的分析表明，它们表现出三体性， 与人类21三体综合征的某些结果相当。Ts 65 Dn已经是两个多世纪以来的工作马了。 然而，几十年来，基因组计划等进展显示出这种模式的弱点。鉴于关键的 由于小鼠在开发改善DS特征的疗法中的作用，需要更好的遗传模型。我们 将表征新建立的携带Hsa 21作为小鼠人工染色体的模型“MAC 21”。它 包含比任何现存模型更好的Hsa 21遗传表示，并且与具有Hsa 21的其他模型不同， 这些老鼠不是马赛克因此，MAC 21避免了现有DS模型的许多限制（具体目标1）。 虽然三体的所有影响最终都是特定细胞中错误表达的产物，但我们可以有效地 区分三体表型是发育不良的产物， 三体性对细胞功能影响。例如，面中部骨骼再生主要负责 患有DS和Ts 65 Dn小鼠的人的特征性面部外观是由于颅骨的减少而出现的。 神经嵴细胞在从神经管分层、迁移和增殖的所有阶段，形成神经嵴细胞。 第一咽弓，因此细胞太少，无法正确形成Meckel软骨。因此，“扁平”骨架 方面是由于细胞过少，作为发育异常的结果，由于三体性，而细胞 组成这些结构的人似乎功能正常。同样，DS和 Ts 65 Dn是颗粒细胞前体增殖不足的产物，但出现了成体细胞的功能 基本正常。在特定阶段的特定细胞中使用基于CRISPR的Hsa 21消融，我们将寻求 区别这些影响。这种区别似乎是学术性的，但实际上， 以及DS表型的性质对于动物模型在临床中的成功应用至关重要。 发展复杂遗传病的产前治疗（具体目标2）。

Project Summary Down syndrome (DS), caused by trisomy for human chromosome 21 (Hsa21), is among the most complex genetic perturbations compatible with human survival past term; 80% of conceptuses with trisomy 21 miscarry. The interactions of ca. 600 transcribed sequences on Hsa21 affect cells directly and, by altering the actions of individual cells during development, via secondary effects on neighboring cells. The resulting outcomes comprise 80+ clinical features affecting every system in the body, although only a subset of these features is observed in any individual with trisomy 21. The advent of molecular genetics in DS research brought with it single gene transgenic mice made with newly discovered Hsa21 genes; phenotypes were interpreted from the reductionist perspective that the DS phenotype is the sum of independently acting single gene effects. Davisson’s Ts65Dn mouse fundamentally changed the paradigm for DS research, providing a model trisomic for a large subset of Hsa21 orthologs. Analysis of these mice showed that they manifest trisomy in ways that are comparable to some outcomes of trisomy 21 in people. Ts65Dn has been the work horse for more than two decades, however, advances such as the genome project show weaknesses in that model. Given the critical role for the mouse in developing therapies to ameliorate features of DS, better genetic models are needed. We will characterize a newly established model, “MAC21”, carrying Hsa21 as a mouse artificial chromosome. It contains a better genetic representation of Hsa21 than any extant model and unlike other models with Hsa21, these mice are not mosaic. Thus MAC21 avoids many limitations of existing DS models (Specific Aim 1). While all effects of trisomy are ultimately a product of misexpression in specific cells, we can usefully differentiate trisomic phenotypes that are a product of maldevelopment from those which represent an ongoing impact of trisomy on cell function. For example, midface skeletal retrusion that is substantially responsible for the characteristic facial appearance of people with DS and Ts65Dn mice arises due to a reduction of cranial neural crest cells at all stages of delamination from the neural tube, migration and proliferation that forms the first pharyngeal arch, thus there are too few cells to properly form Meckel’s cartilage. Hence the “flat” skeletal aspect is due to hypocellularity as a consequence of a developmental anomaly due to trisomy, while the cells composing these structures appear to be function normally. Similarly, hypoplastic cerebellum in DS and Ts65Dn is a product of inadequate proliferation of granule cell precursors, but function of adult cells appears largely normal. Using CRISPR-based ablation of Hsa21 in specific cells at specific stages, we will seek to differentiate these effects. The distinction may seem academic, but in fact, specific knowledge of the timing as well as the nature of DS phenotypes is critical to the successful application of animal models in the development of prenatal therapies for complex genetic disease (Specific Aim 2).