一个表型养生为用于研究衰老的人类疾病基因牵连转基因小鼠
Summary
A reverse-genetics approach to understanding gene families associated with human disease is presented, using mouse as a model system, and the subsequent mouse phenotyping schedule is described. Because mice defective in a gene of interest, HtrA2, manifested Parkinsonian symptoms, the phenotyping regimen is focused on identifying neurological defects.
Abstract
Age-related diseases are becoming increasingly prevalent and the burden continues to grow as our population ages. Effective treatments are necessary to lessen the impact of debilitating conditions but remain elusive in many cases. Only by understanding the causes and pathology of diseases associated with aging, can scientists begin to identify potential therapeutic targets and develop strategies for intervention. The most common age-related conditions are neurodegenerative disorders such as Parkinson’s disease and blindness. Age-related macular degeneration (AMD) is the leading cause of blindness in the elderly. Genome wide association studies have previously identified loci that are associated with increased susceptibility to this disease and identified two regions of interest: complement factor H (CFH) and the 10q26 locus, where the age-related maculopathy susceptibility 2 (ARMS2) and high-temperature requirement factor A1 (HtrA1) genes are located. CFH acts as a negative regulator of the alternative pathway (AP) of the complement system while HtrA1 is an extracellular serine protease. ARMS2 is located upstream of HtrA1 in the primate genome, although the gene is absent in mice. To study the effects of these genes, humanized knock-in mouse lines of Cfh and ARMS2, knockouts of Cfh, HtrA1, HtrA2, HtrA3 and HtrA4 as well as a conditional neural deletion of HtrA2 were generated. Of all the genetically engineered mice produced only mice lacking HtrA2, either systemically or in neural tissues, displayed clear phenotypes. In order to examine these mice thoroughly and systematically, an initial phenotyping schedule was established, consisting of a series of tests related to two main diseases of interest: AMD and Parkinson’s. Genetically modified mice can be subjected to appropriate experiments to identify phenotypes that may be related to the associated diseases in humans. A phenotyping regimen with a mitochondrial focus is presented here alongside representative results from the tests of interest.
Introduction
年龄相关的疾病正在成为现代社会越来越普遍。随着医疗科学的进步和平均寿命的延长，人口不断老化和这些疾病负担的增加。有效的治疗是必要的，以减轻衰弱的状况的影响，但在许多情况下，仍然难以实现。只有了解的原因和与衰老相关的疾病病理上的科学家开始识别潜在的治疗目标和制定战略的干预。常见年龄相关病症包括神经变性疾病如帕金森氏病（PD）和年龄相关性黄斑变性（AMD）。 PD为在人类中引起的神经变性的最普遍的运动障碍。多数PD患者表现出症状，如后50岁静止性震颤，运动迟缓和刚度。早发性也已病例的约10％观察到。
AMD是失明的主要原因老年人，逐渐损害感光体，并在眼睛的视网膜色素上皮细胞（RPE）。中央视力受损，但周边视力一般不受影响。有两种形式AMD的。在“干”的形式，被称为RPE和Bruch膜（BM）之间的玻璃膜疣形式外蛋白沉积，导致地理萎缩和中央视力模糊。更严重的“湿”的形式从新生血管形成的结果，从对面的BM脉络膜入RPE和光感受器层，并可能导致hamorrhaging导致视网膜组织永久损坏视网膜下方。全基因组关联研究先前已经确定了与增加的易感性相关的这种疾病，并确定感兴趣的两个区域的位点：1号染色体上和10q26基因座，其中，与年龄相关的黄斑病的易感性2（ARMS2）和补体因子H（CFH）高温需求因素A1（HTRA1）基因位于1-5 </sup>。这些等位基因的组合增加的AMD的可能性以剂量依赖的方式和具体的SNP可与AMD的3-6的湿或干的形式被优先相关联。
CFH作为通过抑制C3的激活补体系统的替代途径（AP）的负调节物。单核苷酸多态性（SNP）已被链接到的AMD的增加的风险，导致外显子9与组氨酸酪氨酸402的交换，由于为T到C的取代1。在AMD据信该AP是由于CFH功能丧失，但是否与SNP起因果作用尚不清楚misregulated。一种假设是，带正电荷的组氨酸被认为是否定CFH结合至相互作用的蛋白质的C-反应蛋白和硫酸肝素1,7的能力。 在 CFH Y402H的体外研究提供相互矛盾的结果在变体之间的功能差异，并在体内工作<EM> CFH – / –表达人源化的CFH正在进行8只小鼠。 ARMS2位于灵长类基因组HTRA1的上游，尽管基因在小鼠中不存在。 HTRA1是丝氨酸蛋白酶，但ARMS2的特征较差。在AMD相关基因座的SNP之间的连锁不平衡使得难以确定在这个区域中的基因的单个突变的风险的贡献，但最近的工作已表明，它是HTRA1的过表达，而不是ARMS2，导致新血管形成和视网膜下蛋白沉积物9-11。然而，在该基因座的基因的紧密接近可允许不能使用随机插入转基因进行研究的相互作用。
除了AMD，丝氨酸蛋白酶将hTRA家族已经与许多人类疾病有关。所有将hTRA蛋白含有丝氨酸蛋白酶结构域，随后被至少一个C末端PDZ结构域。 HTRA1，HtrA3和HtrA4共享GReatest同源性，包括一个信号肽，胰岛素样生长因子结合结构域，Kazal蛋白酶抑制剂域，丝氨酸蛋白酶结构域和PDZ结构域的。 HtrA2的具有不同的N-末端，线粒体定位序列，跨膜结构域的组成和凋亡抑制结合结构域，随后通过蛋白酶和PDZ结构域12-16。哺乳动物HTRA1由底物诱导重构在其蛋白酶结构域17-20的活性位点调节，HtrA2的也可通过丝氨酸蛋白酶和PDZ结构域，抑制蛋白酶活性21之间的交互调制。有趣的是，PDZ结构域不会出现类似赋予调控HtrA3 16。将hTRA蛋白酶也可以由外在因素调节：它最近证实存在HTRA1之间调节相互作用和protoporphyrins 22和HtrA2的可通过磷酸化后的p38MAP激酶的激活来调节通路在一个PINK1依赖性23。将hTRA家族在小鼠的个别成员的缺失已经被记录在案，但机械效应大多不清楚部分是由于缺乏明显的表型。
HTRA1在蛋白质的质量控制的一个重要的功能和它的错误调节或突变已经与许多不同的人类疾病，包括关节炎，癌症和AMD 3,4,24-32的风险增加相关联。在神经组织HtrA2的功能的丧失已在人类和小鼠的PD表型相关联，而其从非神经组织的结果在加速损失老化33-37。 HtrA3失调已经与疾病，包括先兆子痫和某些类型的癌症38,39相关联。上调HtrA4已子痫前期患者，但敲除小鼠的胎盘被观察到不显示明显的表型40,41。在一些基因敲除小鼠中观察到的缺乏表型的已假定为的将hTRA家族成员之间的补偿的结果：它被认为是既HtrA4和HTRA1与TGF-β家族蛋白的相互作用，在HtrA4 41的缺失允许补偿由HTRA1。同样地，它被认为是由于HTRA1和HtrA3具有高度域同源性的，他们可能具有互补功能42。然而，已经提出，将hTRA蛋白可以具有部分拮抗作用，竞争来调节公共目标43。
为了进一步研究这些风险因素生成3人性化敲入鼠标线。在CFH TM1（CFH * 9）jhoh和CFH TM2（CFH * 9）jhoh，所述CFH基因的外显子9被替换为人类同源物的外显子9。CFH TM1（CFH * 9）jhoh编码非疾病相关的酪氨酸残基402位置，而CFH TM2（CFH * 9）jhoh携带Y402H，风险相关的SNP。在ARMS2 tm1jhoh人类ARMS2序列靶向HTRA1的上游的区域。放置在基因序列，但包括UBIC启动子的下游的上游A loxP的侧翼STOP序列通过杂交到OzCre小鼠，这表示ROSA26启动子的控制下Cre重组酶，如前所述34切除。除了 这些敲入线，生成对CFH和HTRA1（CFH tm1jhoh和HTRA1 tm1jhoh），以及其他已知的将hTRA家族成员条件性敲除等位基因：HtrA2的 （HtrA2的tm1jhoh），HtrA3（HtrA3 tm1jhoh）和HtrA4（ HtrA4 tm1jhoh）。外显子3，HTRA1;种系击倒了通过杂交OzCre小鼠动物改造以侧翼与loxP位点，使得缺失导致活性域（CFH的移码和/或缺失特定外显子的外显子在2-3，HtrA2的 ：外显子2-4，HtrA3;外显子3，HtrA4;外显子4-6）34,41。 HtrA2的的神经缺失，使用下的巢蛋白启动子控制Cre重组酶删除（HtrA2的FLOX;的Tg（NES-CRE）1Kln / J），也被描述34。唯一欠缺的HtrA2的小鼠，无论是全身或神经组织，清晰的显示表型，与帕金森表型呈现。
由于一些感兴趣的这些基因都假定被定位于线粒体11,44-47，和产生HtrA2的缺失帕金森表型，具有线粒体和神经焦点的表型方案在此描述并提供从感兴趣的试验的代表性结果。为了检查产生的彻底调查人，年龄相关的疾病，系统建立的初始表型时间表遗传工程小鼠，由一系列的相关的两个主要的测试感兴趣的疾病：AMD和帕金森氏。
Protocol
Representative Results
Discussion
强大的治疗是需要限制衰弱与人类老化条件的影响，但他们仍然是难以捉摸的许多条件。为了识别潜在的治疗目标和制定干预策略的原因和与衰老相关疾病的病理首先必须了解。不是所有的转基因小鼠立即表现为，与相关的所关注的疾病，即使那些基因先前已挂在人类研究中的条件明确表型。因此更系统的调查是必需的，转基因小鼠中应当进行适当的实验，以确定可能在人类中进行相关的相?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
资助这项研究来自Rosebay医学基金会和耶鲁大学医学院院长的研究基金（JH）。我们感谢克莱尔·柯尼希博士与行为实验的帮助。基因是在Ozgene的（澳大利亚珀斯）生成的工程小鼠线。
Materials
| Ethanol | Decon (Fisher Scientific) | 435541 | |
| 50 ml conical tube | Fisher Scientific | 1443222 | |
| cotton balls | Walmart | ||
| heat mat | Sunbeam | 0000756-500-000 | |
| Holding tray (ice cube tray) | Walmart | ||
| Electronic stopwatch | GOGO | PC396 | |
| Plexiglass box | constructed in workshop | 12" by 12" | |
| Vixia HF R400 Camcorder | Canon | 8155B004 | |
| 9oz Clear Cups | Walmart | ||
| 1/4 inch wire mesh | Home Depot | 204331884 (online) / 554219 (in store) | 12" by 12" |
| Bubble wrap | VWR | 470092-416 | |
| Straight specimen forceps | VWR | 82027-438 | |
| Fine-tip dissecting forceps | VWR | 82027-408 | |
| Fine scissors | VWR | 82027-578 | |
| Paraformaldehyde 16% solution | Electron Microscopy Sciences | 15710 | |
| 10x phosphate buffered saline pH 7.4 | American Bioanalytical | AB11072-04000 | |
| Sucrose | JT Baker | 4072-01 | |
| superfrost slides | Fisher Scientific | 12-550-15 | |
| Hematoxylin Stain Solution | Fisher Scientific (Ricca) | 353016 | |
| Eosin Y Stain Solution | Fisher Scientific (Ricca) | 2845-32 | |
| Tris hydrochloride | Sigma | T3253 | |
| Tris | American Bioanalytical | AB02000-01000 | |
| Nicotinamide adenine dinucleotide, reduced disodium salt hydrate | Sigma | N8129 | |
| Nitrotetrazolium Blue chloride | Sigma | N6876 | |
| Acetone | JT Baker | 9006-05 | |
| Sodium phosphate monobasic monohydrate | Sigma | S9638 | |
| Sodium phosphate dibasic heptahydrate | Sigma | S9390 | |
| Sodium succinate dibasic hexahydrate | Sigma | S2378 | |
| VectaMount aqueous mounting medium | Vector Labs | H-5501-60 | |
| Cover glass | Fisher Scientific | 12-545-M | 60 x 24 mm |
| AxioImager A1 microscope | Zeiss | ||
| Video camera tripod | Amazon | ||
| Optimal Cutting Temperature (OCT) | Fischer Scientific | 23730571 | |
| Cryostat Sectioning Machine | Leica | CM1900 | Discontinued but since replaced by CM1950 |
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