Name: a phenotyping regimen for genetically modified mice used to study genes implicated in human diseases of aging
Uploaded: 2016-07-14T15:00:00.000+00:00
Duration: 9 min 37 s
Description: Watch this Scientific Journal Video about A Phenotyping Regimen for Genetically Modified Mice Used to Study Genes Implicated in Human Diseases of Aging at JoVE.com

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。

伦理学声明:涉及动物的研究均符合的指南在耶鲁大学护理和实验动物使用和动物管理和使用委员会（IACUC）的健康建议全国学院进行。 1.转基因小鼠的行为测试注意:所有的小鼠应经受相同的测试方案，以习惯的差异限制为处理。测试应该在一天中的各个时间的同时进行。 <ol><li>新生儿后肢测试 注:后肢测试是从出生后一天（P）4每天进行，以P10评价近端后肢肌肉力量，虚弱和疲劳。 <ol><li>交通运输小鼠试验区，并允许休息测试前30分钟。 </li><li>通过清洗的50ml锥形管中，用70％的乙醇，推两（P4-P8）或一（P9-P10）棉球，将n的底部准备测试装置UBE并固定在垂直位置。 </li><li>取下笼和记录重量的一种小狗。保持小狗在隔热垫保持托盘，只要不是测试。 </li><li>轻轻暂停新生儿的后肢在锥形管的边，使得其朝下朝向棉球。伯爵制作拉尝试和措施延迟的数量用秒表下降。 </li><li>重复一次，然后用标签标记的小狗。随后，由脚趾剪裁识别P6幼崽。轻轻用的床上用品从家里笼更换前擦幼仔。 </li><li>擦拭用70％乙醇锥形管中。 </li><li>重复测试旁边的小狗，直到整个垃圾已通过测试。 </li><li>处置棉花球和锥形管。所有设备在70％乙醇。 </li></ol></li><li>刚断奶的试验观察 注:断奶观测试验是由P19每天给予P21得分运动和一般的活动水平。 <ol><li>交通运输小鼠试验区ND允许休息测试前30分钟。 </li><li>制备通过用70％乙醇清洗标明的基础上的2英寸的正方形6×6栅格有机玻璃测试盒。设置视频设备的侧面，使得整个盒子是在视场。保持设置及周边地区的每一天一样，以避免引入新意。 </li><li>在干净的容纳杯取下笼代替一个断奶。记录体重。 </li><li>通过拍摄测试身份证，鼠标的识别号码，年龄，检测日期开始视频录制。从保温杯到中心广场传输鼠标。时间为三分钟，计数在测试过程中通过鼠标的两个前爪走过的行数。 </li><li>返回鼠标到一个干净的笼子，结束视频录制。 </li><li>清洁用70％乙醇的试验装置。 </li><li>重复，直到所有垃圾进行测试，然后返回所有幼崽的家笼。 </li><li>清洗所有的检测设备，用70％的乙醇。 </li><li&gt;使用视频，定量通过目视观察清点后，其中一只老鼠站在明确的同时支持前爪和疏导活动，其中两个前爪从事清洁饲养事件的数量。 </li></ol></li><li>丝网握力测试 注:握力测试是在P22和P26之间的隔日进行，以评估神经肌肉的力量。 <ol><li>交通运输小鼠试验区，并允许在笼休息测试前30分钟。 </li><li>由清洗有机玻璃箱和网格，用70％的乙醇制备装置。将泡沫包装在盒子的底部和纸盖。 </li><li>单独的小鼠分成三个组，在个别保温杯放置。 </li><li>放置第一鼠标到丝网的中心，并慢慢倒转10-20厘米的软表面上方在有机玻璃箱的底部。测量潜伏期用秒表下降。终止检测后，如果鼠标没有60秒已经过去秋季。 </li><li>返回鼠标保温杯，请用70％乙醇网格和更换纸张在泡沫包装。返回到第一前进行该组中剩余的两只小鼠的试验。重复，直到每个鼠标一共有三次被测试。 </li><li>返回组到一个干净的笼子里，并继续剩余组。如果有比在任何一组三只老鼠少了，保证5分钟审间的恢复时间间隔是允许的。 </li><li>返回所有小鼠家笼，用70％乙醇的纸和干净的测试设备处理。 </li></ol></li></ol> 2，视网膜结构检查<ol><li>通过腹腔注射氯胺酮（100毫克/千克）和赛拉嗪（10毫克/千克）麻醉在P30-35动物。按捏脚垫评估麻醉深度和进行只在不存在的响应，在与IACUC准则线。用4％PFA / PBS中48灌注。 </li><li>要去除眼睛，在切口SI清洁皮肤用70％乙醇TE时，在颅底通过皮肤切割前。剥离背部皮肤，以暴露颅骨和干净，用70％的乙醇。使用清洁工具，小心翼翼地颅骨切开到达眼窝并露出眼睛。切断视神经，轻轻地从插座上拔下的眼睛，在PBS冲洗。 </li><li>瞩目在4％PFA / PBS中，在室温下10分钟。 </li><li>将目光中含有PBS的小皮氏培养皿。请使用拆线剪刀角膜中心的小切口。通过切割角膜和巩膜之间的边缘除去角膜。使用镊子去除虹膜。最后，剥离的RPE层，而使视网膜完好和代替透镜。 注:4％PFA / PBS中固定会导致视网膜色素上皮脱离，允许视网膜色素上皮易于从视网膜的外侧剥离。 </li><li>重新修复在4％PFA / 30％蔗糖/ PBS中，在室温下15分钟的眼睛返回到含PBS的培养皿之前。拉出LENS使用镊子，使玻璃体与它一起。 </li><li> Cryoprotect在30％蔗糖过夜，在4℃。 </li><li>外套眼最佳切削温度化合物（OCT），并转移到含有新鲜十月的嵌入模具。定向眼使得矢状面是与切割面平行，用尽可能少的动作尽可能和避免引入气泡。通过在干冰/乙醇浴中浸渍模具闪冻。存储块在-80℃。 </li><li>使用低温恒温器，切割20微米的矢状切面和收集预清洗幻灯片。 </li><li>染色切片与根据标准方法49苏木精和曙红。图片使用配备亮视野显微镜光学显微镜。 </li></ol> 3.组织化学染色<ol><li>样品制备<ol><li>通过开放吸入一滴使用过量的异氟醚（丙二醇30％）在P30-35动物实施安乐死。通过停止评估安乐死呼吸，心跳和缺乏应对捏足垫。确认颈椎脱位和器官摘除，符合IACUC指导方针死亡。 </li><li>以除去脑，清理在切口部位用70％乙醇的皮肤，在颅底通过皮肤切割前。剥离背部皮肤，以暴露颅骨和干净，用70％的乙醇。用干净的乐器，通过头骨小心切割，取出暴露大脑。取出膜，轻轻地从头骨腔内取出大脑。冲洗于PBS中。 </li><li>大衣华侨城大脑，并转移到含有新鲜十月的嵌入模具。定向脑使得矢状面是与切割面平行，用尽可能少的动作尽可能和避免引入气泡。通过在-80℃下浸渍在干冰/乙醇浴中并存储在模具闪冻。 </li><li>使用低温恒温器，切割10微米的矢状切面和收集预清洗幻灯片。 </li></ol></li> &lt;LI&gt; NADH黄递酶染色<ol><li>通过溶解5.72克的Tris-HCl中的1.66克Tris碱在1L去离子水制备的0.05M Tris缓冲液，pH值7.6。储存于4℃多达六个星期。 </li><li>制备NADH溶液（2.25毫摩尔NADH在0.05M Tris缓冲液）。分装和储存在-20°C。 </li><li>制备NBT溶液（2.5毫摩尔硝基蓝四氮唑在0.05M Tris缓冲液）。在玻璃瓶中储存于4℃多达六个星期。 </li><li>结合的NADH溶液和NBT溶液等体积以使染色溶液和1ml添加​​到每个滑动。孵育在37℃下在湿润的腔室30分钟。 </li><li>抽吸染色溶液中并用的dh 2 O洗涤三次</li><li>在上升和丙酮的递减浓度洗三次，每次/ DH 2 O（30％，60％，90％，60％，30％），在室温下2分钟。 </li><li>在蒸馏水中2 O冲洗三次，并用含水介质装入部分。使用光学显微镜图像配明亮˚Field显微镜。蓝染色对应的酶活性。 </li></ol></li><li>琥珀酸脱氢酶组织化学染色<ol><li>准备0.2M的磷酸二氢钠/ DH 2 O和0.2M钠磷酸氢二钠/ DH 2 O. </li><li>由3份磷酸二氢用20份氢二钠磷酸结合使0.2M磷酸缓冲液，pH 7.6。 </li><li>制备染色溶液（0.1M琥珀酸钠，1.25M的NBT在0.2M磷酸缓冲液）和1ml添加​​到每个滑动。孵育在37℃下在湿润的腔室60分钟。 </li><li>抽吸染色液并用蒸馏水2 O洗涤三次</li><li>升序洗三次，每次降丙酮浓度/ DH 2 O（30％，60％，90％，60％，30％），在室温下2分钟。 </li><li>在蒸馏水中2 O冲洗三次，并用含水介质装入部分。图片使用配备亮视野显微镜光学显微镜。蓝染色相当于ENzymatic活动。 </li></ol></li></ol>

The authors have no competing interests to disclose.

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?...

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5​​0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&gt;。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&lt;EM&gt; 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。

<table><tbody><tr><td>Ethanol</td><td>Decon (Fisher Scientific)</td><td>435541</td><td></td></tr><tr><td>50 ml conical tube</td><td>Fisher Scientific</td><td>1443222</td><td></td></tr><tr><td>cotton balls</td><td>Walmart</td><td></td><td></td></tr><tr><td>heat mat</td><td>Sunbeam</td><td>0000756-500-000</td><td></td></tr><tr><td>Holding tray (ice cube tray)</td><td>Walmart</td><td></td><td></td></tr><tr><td>Electronic stopwatch</td><td>GOGO</td><td>PC396</td><td></td></tr><tr><td>Plexiglass box</td><td>constructed in workshop</td><td></td><td>12&quot; by 12&quot;&amp;nbsp;</td></tr><tr><td>Vixia HF R400 Camcorder</td><td>Canon</td><td>8155B004</td><td></td></tr><tr><td>9 oz Clear Cups</td><td>Walmart</td><td></td><td></td></tr><tr><td>1/4 inch wire mesh</td><td>Home Depot</td><td>204331884 (online) / 554219 (in store)</td><td>12&quot; by 12&quot;&amp;nbsp;</td></tr><tr><td>Bubble wrap</td><td>VWR</td><td>470092-416</td><td></td></tr><tr><td>Straight specimen forceps</td><td>VWR</td><td>82027-438</td><td></td></tr><tr><td>Fine-tip dissecting forceps</td><td>VWR</td><td>82027-408</td><td></td></tr><tr><td>Fine scissors</td><td>VWR</td><td>82027-578</td><td></td></tr><tr><td>Paraformaldehyde 16% solution</td><td>Electron Microscopy Sciences</td><td>15710</td><td></td></tr><tr><td>10x phosphate buffered saline pH 7.4</td><td>American Bioanalytical</td><td>AB11072-04000</td><td></td></tr><tr><td>Sucrose</td><td>JT Baker</td><td>4072-01</td><td></td></tr><tr><td>superfrost slides</td><td>Fisher Scientific</td><td>12-550-15</td><td></td></tr><tr><td>Hematoxylin Stain Solution</td><td>Fisher Scientific (Ricca)</td><td>353016</td><td></td></tr><tr><td>Eosin Y Stain Solution</td><td>Fisher Scientific (Ricca)</td><td>2845-32</td><td></td></tr><tr><td>Tris hydrochloride</td><td>Sigma</td><td>T3253</td><td></td></tr><tr><td>Tris</td><td>American Bioanalytical</td><td>AB02000-01000</td><td></td></tr><tr><td>Nicotinamide adenine dinucleotide, reduced disodium salt hydrate</td><td>Sigma</td><td>N8129</td><td></td></tr><tr><td>Nitrotetrazolium Blue chloride</td><td>Sigma</td><td>N6876</td><td></td></tr><tr><td>Acetone</td><td>JT Baker</td><td>9006-05</td><td></td></tr><tr><td>Sodium phosphate monobasic monohydrate</td><td>Sigma</td><td>S9638</td><td></td></tr><tr><td>Sodium phosphate dibasic heptahydrate</td><td>Sigma</td><td>S9390</td><td></td></tr><tr><td>Sodium succinate dibasic hexahydrate</td><td>Sigma</td><td>S2378</td><td></td></tr><tr><td>VectaMount aqueous mounting medium</td><td>Vector Labs</td><td>H-5501-60</td><td></td></tr><tr><td>Cover glass</td><td>Fisher Scientific</td><td>12-545-M</td><td>60 mm x 24 mm</td></tr><tr><td>AxioImager A1 microscope</td><td>Zeiss</td><td></td><td></td></tr><tr><td>Video camera tripod</td><td>Amazon</td><td></td><td></td></tr><tr><td>Optimal Cutting Temperature (OCT)</td><td>Fischer Scientific</td><td>23730571</td><td></td></tr><tr><td>Cryostat Sectioning&amp;nbsp; Machine</td><td>Leica&amp;nbsp;</td><td>CM1900</td><td>Discontinued but since replaced by CM1950</td></tr></tbody></table>

a phenotyping regimen for genetically modified mice used to study genes implicated in human diseases of aging

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.

本节描述了使用这些方法获得的结果的例子。在后肢试验，拉尝试次数所作和延迟落在相加超过每天两次连续测试。这种测试可用于比较基因不同的组来区分具有降低的神经肌肉强度HtrA2的tm1jhoh小鼠（HTRA2 KO）小鼠在图1A - B表明在拉和延迟的数量没有变化，从P4-6落入随后减小在P7-10神经肌肉强度，相比于同窝（HTRA2 WT）。随着年龄的...

Watch this Scientific Journal Video about A Phenotyping Regimen for Genetically Modified Mice Used to Study Genes Implicated in Human Diseases of Aging at JoVE.com

A Phenotyping Regimen for Genetically Modified Mice Used to Study Genes Implicated in Human Diseases of Aging

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.

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

Age-related diseases are becoming increasingly prevalent and the burden continues to grow as our population ages. Effective treatments are necessary to ...

a-phenotyping-regimen-for-genetically-modified-mice-used-to-study

Research

JoVE Journal

Medicine

8.2K 次观看.  Yale University School of Medicine. 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.

视频: A Phenotyping Regimen for Genetically Modified Mice Used to Study Genes Implicated in Human Diseases of Aging

Assessment of Sensorimotor Function in Mouse Models of Parkinson's Disease

Sensitive and reliable behavioral outcome measures are essential to the evaluation of potential therapeutic treatments in preclinical trials for many neurodegenerative diseases. In Parkinson's disease, sensorimotor tests sensitive to varying degrees of nigrostriatal dysfunction are fundamental for testing the efficacy of potential therapeutics. Reliable and quite elegant sensorimotor measures exist for rats, however many of these tests measure sensorimotor asymmetry within the rat and are not entirely suitable for the newer genetic mouse models of PD. We have put together a battery of sensorimotor tests inspired by the sensitive tests in rats and adapted for mice. The test battery highlighted in this study is chosen for a) its sensitivity in a wide variety of mouse models of PD, b) its ease in implementing into a study, and c) its low expense. These tests have proven useful in characterizing novel genetic mouse models of PD as well as in testing potential disease-modifying therapies.

In Parkinson's disease and movement disorders in general, sensitive and reliable behavioral assays are essential for testing novel potential therapeutics. Here, we describe a manageable battery of sensorimotor tests for mice that are sensitive to varying degrees of injury to the nigrostriatal system and useful for preclinical studies.

帕金森氏症小鼠模型的感觉功能评估

Sensitive and  reliable behavioral outcome measures are essential to the evaluation of  potential therapeutic treatments in preclinical trials for many  ...

科研

行为学

A Genetically Engineered Mouse Model of Sporadic Colorectal Cancer

Despite the advantages of easy applicability and cost-effectiveness, colorectal cancer mouse models based on tumor cell injection have severe limitations and do not accurately simulate tumor biology and tumor cell dissemination. Genetically engineered mouse models have been introduced to overcome these limitations; however, such models are technically demanding, especially in large organs such as the colon in which only a single tumor is desired.
As a result, an immunocompetent, genetically engineered mouse model of colorectal cancer was developed which develops highly uniform tumors and can be used for tumor biology studies as well as therapeutic trials. Tumor development is initiated by surgical, segmental infection of the distal colon with adeno-cre virus in compound conditionally mutant mice. The tumors can be easily detected and monitored via colonoscopy. We here describe the surgical technique of segmental adeno-cre infection of the colon, the surveillance of the tumor via high-resolution colonoscopy and present the resulting colorectal tumors.

A protocol for the establishment of a genetically engineered mouse model of colorectal cancer by segmental adeno-cre infection and its surveillance via high-resolution colonoscopy is presented.

遗传工程小鼠模型的散发性结肠直肠癌

Despite the advantages of easy applicability and cost-effectiveness, colorectal cancer mouse models based on tumor cell injection have severe limitations ...

癌症研究

Forward Genetic Approach to Uncover Stress Resistance Genes in Mice &#8212; A High-throughput Screen in ES Cells

Phenotype-driven genetic screens in mice is a powerful technique to uncover gene functions, but are often hampered by extremely high costs, which severely limits its potential. We describe here the use of mouse embryonic stem (ES) cells as surrogate cells to screen for a phenotype of interest and subsequently introduce these cells into a host embryo to develop into a living mouse carrying the phenotype. This method provides (1) a cost effective, high-throughput platform for genetic screen in mammalian cells; (2) a rapid way to identify the mutated genes and verify causality; and (3) a short-cut to develop mouse mutants directly from these selected ES cells for whole animal studies. We demonstrated the use of paraquat (PQ) to select resistant mutants and identify mutations that confer oxidative stress resistance. Other stressors or cytotoxic compounds may also be used to screen for resistant mutants to uncover novel genetic determinants of a variety of cellular stress resistance.

Stress resistance is one of the hallmarks for longevity and is known to be genetically governed. Here, we developed an unbiased high-throughput method to screen for mutations that confer stress resistance in ES cells with which to develop mouse models for longevity studies.

正向遗传学的方法来揭开抗逆基因小鼠 - 在ES细胞高通量筛选

Phenotype-driven genetic screens in mice is a powerful technique to uncover gene functions, but are often hampered by extremely high costs, which severely ...

发育生物学

Morris Water Maze Test: Optimization for Mouse Strain and Testing Environment

The Morris water maze (MWM) is a commonly used task to assess hippocampal-dependent spatial learning and memory in transgenic mouse models of disease, including neurocognitive disorders such as Alzheimer&#8217;s disease. However, the background strain of the mouse model used can have a substantial effect on the observed behavioral phenotype, with some strains exhibiting superior learning ability relative to others. To ensure differences between transgene negative and transgene positive mice can be detected, identification of a training procedure sensitive to the background strain is essential. Failure to tailor the MWM protocol to the background strain of the mouse model may lead to under- or over- training, thereby masking group differences in probe trials. Here, a MWM protocol tailored for use with the F1 FVB/N x 129S6 background is described. This is a frequently used background strain to study the age-dependent effects of mutant P301L tau (rTg(TauP301L)4510 mice) on the memory deficits associated with Alzheimer&#8217;s disease. Also described is a strategy to re-optimize, as dictated by the particular testing environment utilized.

This manuscript describes a Morris water maze (MWM) protocol tailored for use with a commonly used mouse model of Alzheimer's disease. The MWM is widely used in transgenic mouse models. Implementation of a procedure sensitive to the background strain of the mouse model is essential for detecting group differences.

Morris水迷宫实验:优化小鼠品系和测试环境

 The Morris water maze (MWM) is a commonly used task to assess hippocampal-dependent spatial learning and memory in transgenic mouse models of disease, ...

Using Mouse Oocytes to Assess Human Gene Function During Meiosis I

Embryonic aneuploidy is the major genetic cause of infertility in humans. Most of these events originate during female meiosis, and albeit positively correlated with maternal age, age alone is not always predictive of the risk of generating an aneuploid embryo. Therefore, gene variants might account for incorrect chromosome segregation during oogenesis. Given that access to human oocytes is limited for research purposes, a series of assays were developed to study human gene function during meiosis I using mouse oocytes. First, messenger RNA (mRNA) of the gene and gene variant of interest are microinjected into prophase I-arrested mouse oocytes. After allowing time for expression, oocytes are synchronously released into meiotic maturation to complete meiosis I. By tagging the mRNA with a sequence of a fluorescent reporter, such as green fluorescent protein (Gfp), the localization of the human protein can be assessed in addition to the phenotypic alterations. For example, gain or loss of function can be investigated by establishing experimental conditions that challenge the gene product to fix meiotic errors. Although this system is advantageous in investigating human protein function during oogenesis, adequate interpretation of results should be undertaken given that protein expression is not at endogenous levels and, unless controlled for (i.e. knocked out or down), murine homologs are also present in the system.

As the genetic variants associated with human disease begin to become uncovered, it is becoming increasingly important to develop systems with which to rapidly evaluate the biological significance of those identified variants. This protocol describes methods for evaluating human gene function during female meiosis I using mouse oocytes.

小鼠卵母细胞在减数分裂过程中对人类基因功能的评估

Embryonic aneuploidy is the major genetic cause of infertility in humans. Most of these events originate during female meiosis, and albeit positively ...

遗传学

Automated, Long-term Behavioral Assay for Cognitive Functions in Multiple Genetic Models of Alzheimer's Disease, Using IntelliCage

Multiple factors&#8212;such as aging and genes&#8212;are frequently associated with cognitive decline. Genetically modified mouse models of cognitive decline, such as Alzheimer&#39;s disease (AD), have become a promising tool to elucidate the underlying mechanisms and promote the therapeutic advances. An important step is the validation and characterization of expected behavioral abnormality in the models, in the case of AD, cognitive decline. The long-term behavioral investigations of laboratory animals to study the effect of aging demand substantial efforts from researchers. The IntelliCage system is a high-throughput and cost-effective test battery for mice that eliminates the need for daily human handling. Here, we describe how the system is utilized in the long-term phenotyping of a genetic Alzheimer&#39;s disease model, specifically focusing on the cognitive functions. The experiment employs repeated battery of tests that assess spatial learning and executive functions. This cost-effective age-dependent phenotyping allows us to identify the transient and/or permanent effects of genes on various cognitive aspects.

This paper describes a protocol for cognitive assessments for genetic models of the Alzheimer&#39;s disease using the IntelliCage system, which is a high throughput automated behavioral monitoring system with operant conditioning.

基于 IntelliCage 的阿尔茨海默病多基因模型认知功能的自动、长期行为检测

Multiple factors&#8212;such as aging and genes&#8212;are frequently associated with cognitive decline. Genetically modified mouse models of cognitive ...

Gait Analysis of Age-dependent Motor Impairments in Mice with Neurodegeneration

Motor behavior tests are commonly used to determine the functional relevance of a rodent model and to test newly developed treatments in these animals. Specifically, gait analysis allows recapturing disease relevant phenotypes that are observed in human patients, especially in neurodegenerative diseases that affect motor abilities such as Parkinson&#39;s disease (PD), Alzheimer&#39;s disease (AD), amyotrophic lateral sclerosis (ALS), and others. In early studies along this line, the measurement of gait parameters was laborious and depended on factors that were hard to control (e.g., running speed, continuous running). The development of ventral plane imaging (VPI) systems made it feasible to perform gait analysis at a large scale, making this method a useful tool for the assessment of motor behavior in rodents. Here, we present an in-depth protocol of how to use kinematic gait analysis to examine the age-dependent progression of motor deficits in mouse models of neurodegeneration; mouse lines with decreased levels of endophilin, in which neurodegenerative damage progressively increases with age, are used as an example.

In this study, we demonstrate the use of kinematic gait analysis based on ventral plane imaging to monitor the subtle changes in motor coordination as well as the progression of neurodegeneration with advancing age in mouse models (e.g., endophilin mutant mouse lines).

变性小鼠年龄依赖性运动障碍的步态分析

Motor behavior tests are commonly used to determine the functional relevance of a rodent model and to test newly developed treatments in these animals. ...

A Protocol to Evaluate and Quantify Retinal Pigmented Epithelium Pathologies in Mouse Models of Age-Related Macular Degeneration

Age-related macular degeneration (AMD) is a debilitating retinal disorder in aging populations. It is widely believed that dysfunction of the retinal pigmented epithelium (RPE) is a key pathobiological event in AMD. To understand the mechanisms that lead to RPE dysfunction, mouse models can be utilized by researchers. It has been established by previous studies that mice can develop RPE pathologies, some of which are observed in the eyes of individuals diagnosed with AMD. Here, we describe a phenotyping protocol to assess RPE pathologies in mice. This protocol includes the preparation and evaluation of retinal cross-sections using light microscopy and transmission electron microscopy, as well as that of RPE flat mounts by confocal microscopy. We detail the common types of murine RPE pathologies observed by these techniques and ways to quantify them through unbiased methods for statistical testing. As proof of concept, we use this RPE phenotyping protocol to quantify the RPE pathologies observed in mice overexpressing transmembrane protein 135 (Tmem135) and aged wild-type C57BL/6J mice. The main goal of this protocol is to present standard RPE phenotyping methods with unbiased quantitative assessments for scientists using mouse models of AMD.

Mouse models can be useful tools for investigating the biology of the retinal pigmented epithelium (RPE). It has been established that mice can develop an array of RPE pathologies. Here, we describe a phenotyping protocol to elucidate and quantify RPE pathologies in mice using light, transmission electron, and confocal microscopy.

评估和量化年龄相关性黄斑变性小鼠模型中视网膜色素上皮病理的协议

Age-related macular degeneration (AMD) is a debilitating retinal disorder in aging populations. It is widely believed that dysfunction of the retinal ...

Rapid Genotyping of Animals Followed by Establishing Primary Cultures of Brain Neurons

High-resolution analysis of the morphology and function of mammalian neurons often requires the genotyping of individual animals followed by the analysis of primary cultures of neurons. We describe a set of procedures for: labeling newborn mice to be genotyped, rapid genotyping, and establishing low-density cultures of brain neurons from these mice. Individual mice are labeled by tattooing, which allows for long-term identification lasting into adulthood. Genotyping by the described protocol is fast and efficient, and allows for automated extraction of nucleic acid with good reliability. This is useful under circumstances where sufficient time for conventional genotyping is not available, e.g., in mice that suffer from neonatal lethality. Primary neuronal cultures are generated at low density, which enables imaging experiments at high spatial resolution. This culture method requires the preparation of glial feeder layers prior to neuronal plating. The protocol is applied in its entirety to a mouse model of the movement disorder DYT1 dystonia (&#916;E-torsinA knock-in mice), and neuronal cultures are prepared from the hippocampus, cerebral cortex and striatum of these mice. This protocol can be applied to mice with other genetic mutations, as well as to animals of other species. Furthermore, individual components of the protocol can be used for isolated sub-projects. Thus this protocol will have wide applications, not only in neuroscience but also in other fields of biological and medical sciences.

We describe procedures for labeling and genotyping newborn mice and generating primary neuronal cultures from them. The genotyping is rapid, efficient and reliable, and allows for automated nucleic-acid extraction. This is especially useful for neonatally lethal mice and their cultures that require prior completion of genotyping.

动物的基因分型快速其次是建立大脑神经元原代培养

High-resolution analysis of the morphology and function of mammalian neurons often requires the genotyping of individual animals followed by the analysis ...

神经科学

Assessment of Spontaneous Alternation, Novel Object Recognition and Limb Clasping in Transgenic Mouse Models of Amyloid-&#946; and Tau Neuropathology

Here we describe a staged, behavioral testing approach that can be used to screen for compounds that exhibit in vivo efficacy on cognitive and functional motor behaviors in transgenic mouse models of &#946;-amyloidosis and tauopathy. The paradigm includes tests for spontaneous alternation in a Y-maze, novel object recognition, and limb clasping. These tests were selected because they: 1) interrogate function of cognitive or motor domains and the correlate neural circuitry relevant to the human disease state, 2) have clearly defined endpoints, 3) have easily implementable quality control checks, 4) can be run in a moderate throughput format, and 5) require little intervention by the investigator. These methods are designed for investigators looking to screen compounds for activity in short-term and working memory tasks, or functional motor behaviors associated with Alzheimer's disease mouse models. The methods described here use behavioral tests that engage a number of different brain regions including hippocampus and various cortical areas. Investigators that desire cognitive tests that specifically assess cognition mediated by a single brain region could use these techniques to supplement other behavioral tests.

Described here is a staged, behavioral screening approach that can be used to screen for compounds that exhibit in vivo efficacy on cognitive and functional motor behaviors in transgenic mouse models of &#946;-amyloidosis and tauopathy. These methods are optimized to screen compounds for activity in short-term and working memory tasks.

自发交替评估，淀粉样蛋白β和脑神经病理学转基因小鼠模型中的新物体识别和肢体紧扣

Here we describe a staged, behavioral testing approach that can be used to screen for compounds that exhibit in vivo efficacy on cognitive and functional ...