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 border="0" cellpadding="0" cellspacing="0" width="1218">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:441px;">Ethanol</td>
			<td style="width:221px;">Decon (Fisher Scientific)</td>
			<td style="width:260px;">435541</td>
			<td style="width:296px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">50 ml conical tube</td>
			<td>Fisher Scientific</td>
			<td>1443222</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">cotton balls</td>
			<td>Walmart</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">heat mat</td>
			<td>Sunbeam</td>
			<td>0000756-500-000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Holding tray (ice cube tray)</td>
			<td>Walmart</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Electronic stopwatch</td>
			<td>GOGO</td>
			<td>PC396</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Plexiglass box</td>
			<td>constructed in workshop</td>
			<td></td>
			<td>12&#34; by 12&#34;&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vixia HF R400 Camcorder</td>
			<td>Canon</td>
			<td>8155B004</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">9oz Clear Cups</td>
			<td>Walmart</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1/4 inch wire mesh</td>
			<td>Home Depot</td>
			<td>204331884 (online) / 554219 (in store)</td>
			<td>12&#34; by 12&#34;&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bubble wrap</td>
			<td>VWR</td>
			<td>470092-416</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Straight specimen forceps</td>
			<td>VWR</td>
			<td>82027-438</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fine-tip dissecting forceps</td>
			<td>VWR</td>
			<td>82027-408</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fine scissors</td>
			<td>VWR</td>
			<td>82027-578</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Paraformaldehyde 16% solution</td>
			<td>Electron Microscopy Sciences</td>
			<td>15710</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">10x phosphate buffered saline pH 7.4</td>
			<td>American Bioanalytical</td>
			<td>AB11072-04000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sucrose</td>
			<td>JT Baker</td>
			<td>4072-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">superfrost slides</td>
			<td>Fisher Scientific</td>
			<td>12-550-15</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hematoxylin Stain Solution</td>
			<td>Fisher Scientific (Ricca)</td>
			<td>353016</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Eosin Y Stain Solution</td>
			<td>Fisher Scientific (Ricca)</td>
			<td>2845-32</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tris hydrochloride</td>
			<td>Sigma</td>
			<td>T3253</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tris</td>
			<td>American Bioanalytical</td>
			<td>AB02000-01000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nicotinamide adenine dinucleotide, reduced disodium salt hydrate</td>
			<td>Sigma</td>
			<td>N8129</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nitrotetrazolium Blue chloride</td>
			<td>Sigma</td>
			<td>N6876</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Acetone</td>
			<td>JT Baker</td>
			<td>9006-05</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium phosphate monobasic monohydrate</td>
			<td>Sigma</td>
			<td>S9638</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium phosphate dibasic heptahydrate</td>
			<td>Sigma</td>
			<td>S9390</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium succinate dibasic hexahydrate</td>
			<td>Sigma</td>
			<td>S2378</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">VectaMount aqueous mounting medium</td>
			<td>Vector Labs</td>
			<td>H-5501-60</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cover glass</td>
			<td>Fisher Scientific</td>
			<td>12-545-M</td>
			<td>60 x 24 mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">AxioImager A1 microscope</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Video camera tripod</td>
			<td>Amazon</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Optimal Cutting Temperature (OCT)</td>
			<td>Fischer Scientific</td>
			<td>23730571</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cryostat Sectioning&#160; Machine</td>
			<td>Leica&#160;</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

Modified Technique for Coronary Artery Ligation in Mice

Myocardial infarction (MI) is one of the most important causes of mortality in humans1-3. In order to improve morbidity and mortality in patients with MI we need better knowledge about pathophysiology of myocardial ischemia. This knowledge may be valuable to define new therapeutic targets for innovative cardiovascular therapies4. Experimental MI model in mice is an increasingly popular small-animal model in preclinical research in which MI is induced by means of permanent or temporary ligation of left coronary artery (LCA)5. In this video, we describe the step-by-step method of how to induce experimental MI in mice.
The animal is first anesthetized with 2% isoflurane. The unconscious mouse is then intubated and connected to a ventilator for artificial ventilation. The left chest is shaved and 1.5 cm incision along mid-axillary line is made in the skin. The left pectoralis major muscle is bluntly dissociated until the ribs are exposed. The muscle layers are pulled aside and fixed with an eyelid-retractor. After these preparations, left thoracotomy is performed between the third and fourth ribs in order to visualize the anterior surface of the heart and left lung. The proximal segment of LCA artery is then ligated with a 7-0 ethilon suture which typically induces an infarct size ~40% of left ventricle. At the end, the chest is closed and the animals receive postoperative analgesia (Temgesic, 0.3 mg/50 ml, ip). The animals are kept in a warm cage until spontaneous recovery.

The surgical procedure used to induce experimental myocardial infarction in mice begins with left thoracotomy between the third and the fourth ribs in order to visualize the anterior surface of the heart and left lung. The left coronary artery is ligated, the chest is closed and the mouse is allowed to recover spontaneously.

改性技术在小鼠冠状动脉结扎

Myocardial infarction (MI) is one of the most important causes of mortality in humans1-3. In order to improve morbidity and mortality in patients with MI ...

科研

医学

The Goeckerman Regimen for the Treatment of Moderate to Severe Psoriasis

Psoriasis is a chronic, immune-mediated inflammatory skin disease affecting approximately 2-3% of the population. The Goeckerman regimen consists of exposure to ultraviolet B (UVB) light and application of crude coal tar (CCT). Goeckerman therapy is extremely effective and relatively safe for the treatment of psoriasis and for improving a patient's quality of life. In the following article, we present our protocol for the Goeckerman therapy that is utilized specifically at the University of California, San Francisco. This protocol details the preparation of supplies, administration of phototherapy and application of topical tar. This protocol also describes how to assess the patient daily, monitor for adverse effects (including pruritus and burning), and adjust the treatment based on the patient's response. Though it is one of the oldest therapies available for psoriasis, there is an absence of any published videos demonstrating the process in detail. The video is beneficial for healthcare providers who want to administer the therapy, for trainees who want to learn more about the process, and for prospective patients who want to undergo treatment for their cutaneous disease.

Psoriasis is a chronic, immune-mediated inflammatory skin disease. The Goeckerman regimen, formulated for the treatment of psoriasis, consists of exposure to ultraviolet B (UVB) light and application of crude coal tar (CCT). The following protocol is for the administration of Goeckerman therapy for the treatment of moderate-to-severe psoriasis.

Goeckerman方案用于治疗中度至重度牛皮癣

Psoriasis is a  chronic, immune-mediated inflammatory skin disease affecting approximately 2-3%  of the population. The Goeckerman regimen consists of ...

Measuring Frailty in HIV-infected Individuals. Identification of Frail Patients is the First Step to Amelioration and Reversal of Frailty

A simple, validated protocol consisting of a battery of tests is available to identify elderly patients with frailty syndrome. This syndrome of decreased reserve and resistance to stressors increases in incidence with increasing age. In the elderly, frailty may pursue a step-wise loss of function from non-frail to pre-frail to frail. We studied frailty in HIV-infected patients and found that ~20% are frail using the Fried phenotype using stringent criteria developed for the elderly1,2. In HIV infection the syndrome occurs at a younger age.
HIV patients were checked for 1) unintentional weight loss; 2) slowness as determined by walking speed; 3) weakness as measured by a grip dynamometer; 4) exhaustion by responses to a depression scale; and 5) low physical activity was determined by assessing kilocalories expended in a week's time. Pre-frailty was present with any two of five criteria and frailty was present if any three of the five criteria were abnormal.
The tests take approximately 10-15 min to complete and they can be performed by medical assistants during routine clinic visits. Test results are scored by referring to standard tables. Understanding which of the five components contribute to frailty in an individual patient can allow the clinician to address relevant underlying problems, many of which are not evident in routine HIV clinic visits.

Frailty syndrome is commonly seen in the aged and reflects multi-system physiological change. However, with reduced functional reserve and resilience frailty is also known to be common in the HIV infected population. This study outlined an easily administered screening test to identify HIV patients with frailty. When significant components of frailty are identified, clinicians will be able to focus on amelioration of the problem and promote reversion to the pre-frail state.

在感染艾滋病毒的个体的测量弱者。体弱患者的识别是第一步改良与弱者拨回

A simple, validated protocol consisting of a battery of tests is available to identify elderly patients with frailty syndrome. This syndrome of decreased ...

Collecting Saliva and Measuring Salivary Cortisol and Alpha-amylase in Frail Community Residing Older Adults via Family Caregivers

Salivary measures have emerged in bio-behavioral research that are easy-to-collect, minimally invasive, and relatively inexpensive biologic markers of stress. This article we present the steps for collection and analysis of two salivary assays in research with frail, community residing older adults-salivary cortisol and salivary alpha amylase. The field of salivary bioscience is rapidly advancing and the purpose of this presentation is to provide an update on the developments for investigators interested in integrating these measures into research on aging. Strategies are presented for instructing family caregivers in collecting saliva in the home, and for conducting laboratory analyses of salivary analytes that have demonstrated feasibility, high compliance, and yield quality specimens. The protocol for sample collection includes: (1) consistent use of collection materials; (2) standardized methods that promote adherence and minimize subject burden; and (3) procedures for controlling certain confounding agents. We also provide strategies for laboratory analyses include: (1) saliva handling and processing; (2) salivary cortisol and salivary alpha amylase assay procedures; and (3) analytic considerations.

We demonstrate: (1) procedures for collection of salivary samples in cognitive impaired older adults by family caregivers in the home setting, (2) procedures for measuring stress activity via salivary cortisol and alpha amylase, and (3) representative profiles. Protocols that allow researchers to study stress-linked processes advance our understanding of biological sensitivity and susceptibility.

Salivary measures have emerged in bio-behavioral research  that are easy-to-collect, minimally invasive, and relatively inexpensive  biologic markers of ...

A Modified Precipitation Method to Isolate Urinary Exosomes

Identification of biomarkers that allow early detection of kidney diseases in urine and plasma has been an area of active interest for several years. Urinary exosome vesicles, 40-100 nm in size, are released into the urine under normal conditions by cells from all nephron segments and may contain protein, mRNA and microRNA representative of their cell type of origin. Under conditions of renal dysfunction or injury, exosomes may contain altered proportions of these components, which may serve as biomarkers for disease. There are currently several methods available for isolation of urinary exosomes, and we have previously conducted an experimental comparison of each of these approaches, including three based on ultracentrifugation, one using a nanomembrane ultrafiltration concentrator, one using a commercial precipitation reagent and one using a modification of the precipitation technique using ExoQuick reagent that we developed in our laboratory. We found the modified precipitation method produced the highest yield of exosome particles, miRNA, and mRNA, making this approach suitable for the isolation of exosomes for subsequent RNA profiling. We conclude that the modified exosome precipitation method offers a quick, scalable, and effective alternative for the isolation of exosomes from urine. In this report, we describe our modified precipitation technique using ExoQuick reagent for isolating exosomes from human urine.

This manuscript describes a protocol for isolating exosomes from human urine using a modified precipitation technique.

一种改进沉淀法来隔离尿外来体

Identification of biomarkers that allow early detection of kidney diseases in urine and plasma has been an area of active interest for several years. ...

Construction of Vapor Chambers Used to Expose Mice to Alcohol During the Equivalent of all Three Trimesters of Human Development

Exposure to alcohol during development can result in a constellation of morphological and behavioral abnormalities that are collectively known as Fetal Alcohol Spectrum Disorders (FASDs). At the most severe end of the spectrum is Fetal Alcohol Syndrome (FAS), characterized by growth retardation, craniofacial dysmorphology, and neurobehavioral deficits. Studies with animal models, including rodents, have elucidated many molecular and cellular mechanisms involved in the pathophysiology of FASDs. Ethanol administration to pregnant rodents has been used to model human exposure during the first and second trimesters of pregnancy. Third trimester ethanol consumption in humans has been modeled using neonatal rodents. However, few rodent studies have characterized the effect of ethanol exposure during the equivalent to all three trimesters of human pregnancy, a pattern of exposure that is common in pregnant women. Here, we show how to build vapor chambers from readily obtainable materials that can each accommodate up to six standard mouse cages. We describe a vapor chamber paradigm that can be used to model exposure to ethanol, with minimal handling, during all three trimesters. Our studies demonstrate that pregnant dams developed significant metabolic tolerance to ethanol. However, neonatal mice did not develop metabolic tolerance and the number of fetuses, fetus weight, placenta weight, number of pups/litter, number of dead pups/litter, and pup weight were not significantly affected by ethanol exposure. An important advantage of this paradigm is its applicability to studies with genetically-modified mice. Additionally, this paradigm minimizes handling of animals, a major confound in fetal alcohol research.

We demonstrate the construction of alcohol vapor chambers using readily available materials that simultaneously house 6 mouse cages. We further describe their use in a mouse model of fetal alcohol exposure equivalent to all 3&#160;trimesters of human pregnancy. This paradigm exposes animals during gestation and postnatal days 1-12.

蒸汽腔体建设用于公开小鼠酒精在所有三个学期人类发展的等效

Exposure to alcohol during development can result in a constellation of morphological and behavioral abnormalities that are collectively known as Fetal ...

A Guide to Generating and Using hiPSC Derived NPCs for the Study of Neurological Diseases

Post-mortem studies of neurological diseases are not ideal for identifying the underlying causes of disease initiation, as many diseases include a long period of disease progression prior to the onset of symptoms. Because fibroblasts from patients and healthy controls can be efficiently reprogrammed into human induced pluripotent stem cells (hiPSCs), and subsequently differentiated into neural progenitor cells (NPCs) and neurons for the study of these diseases, it is now possible to recapitulate the developmental events that occurred prior to symptom onset in patients. We present a method by which to efficiently differentiate hiPSCs into NPCs, which in addition to being capable of further differentiation into functional neurons, can also be robustly passaged, freeze-thawed or transitioned to grow as neurospheres, enabling rapid genetic screening to identify the molecular factors that impact cellular phenotypes including replication, migration, oxidative stress and/or apoptosis. Patient derived hiPSC NPCs are a unique platform, ideally suited for the empirical testing of the cellular or molecular consequences of manipulating gene expression.

This protocol describes how neural progenitor cells can be differentiated from human induced pluripotent stem cells, in order to yield a robust and replicative neural cell population, which may be used to identify the developmental pathways contributing to disease pathogenesis in many neurological disorders.

指南生成和使用hiPSC派生的NPC的神经系统疾病的研究

Post-mortem studies of neurological diseases are not ideal for identifying the underlying causes of disease initiation, as many diseases include a long ...

Induction of Accelerated Atherosclerosis in Mice: The "Wire-Injury" Model

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. Moreover, by rupture of the defective vascular wall, atherosclerosis induces occlusive thrombus formation, which represents the main cause of myocardial infarction or stroke and the most frequent cause of death. Despite the advances in the cardiovascular field, many questions remain unanswered, and additional basic research is essential to improve our understanding of the molecular mechanisms during atherosclerosis and its effects. Due to limited clinical studies, there is a need for representative animal models recreating atherosclerotic conditions such as neointima formation after stent implantation, balloon angioplasty, or endarterectomy. Since the mouse presents many advantages and is the most frequently used model for studying molecular processes, the current study proposes an invasive procedure of endothelial denudation, also known as the wire-injury model, which is representative of the human condition of neointima formation in arteries after revascularization procedures.

This study describes an invasive procedure for the induction of accelerated atherosclerosis in mice. In comparison to other methods using electric- or cryo-induced injury, mechanical-induced injury mimics the human condition of restenosis after revascularization therapies and is ideal for the study of the molecular mechanisms involved.

小鼠加速动脉硬化的诱导:"电线损伤"模型

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. ...

Phosphorus-31 Magnetic Resonance Spectroscopy: A Tool for Measuring In Vivo Mitochondrial Oxidative Phosphorylation Capacity in Human Skeletal Muscle

Skeletal muscle mitochondrial oxidative phosphorylation (OXPHOS) capacity, which is critically important in health and disease, can be measured in vivo and noninvasively in humans via phosphorus-31 magnetic resonance spectroscopy (31PMRS). However, the approach has not been widely adopted in translational and clinical research, with variations in methodology and limited guidance from the literature. Increased optimization, standardization, and dissemination of methods for in vivo 31PMRS would facilitate the development of targeted therapies to improve OXPHOS capacity and could ultimately favorably impact cardiovascular health. 31PMRS produces a noninvasive, in vivo measure of OXPHOS capacity in human skeletal muscle, as opposed to alternative measures obtained from explanted and potentially altered mitochondria via muscle biopsy. It relies upon only modest additional instrumentation beyond what is already in place on magnetic resonance scanners available for clinical and translational research at most institutions. In this work, we outline a method to measure in vivo skeletal muscle OXPHOS. The technique is demonstrated using a 1.5 Tesla whole-body MR scanner equipped with the suitable hardware and software for 31PMRS, and we explain a simple and robust protocol for in-magnet resistive exercise to rapidly fatigue the quadriceps muscle. Reproducibility and feasibility are demonstrated in volunteers as well as subjects over a wide range of functional capacities.

Phosphorus-31 Magnetic Resonance Spectroscopy: A Tool for Measuring In Vivo Mitochondrial Oxidative Phosphorylation Capacity in Human Skeletal Muscle

This work demonstrates the feasibility of an in vivo phosphorus-31 magnetic resonance spectroscopy (31PMRS) technique to quantify mitochondrial oxidative phosphorylation (OXPHOS) capacity in human skeletal muscle.

磷31磁共振波谱分析:一种测量工具<em&gt;在体内</em&gt;线粒体氧化磷酸化能力人骨骼肌

Skeletal muscle mitochondrial oxidative phosphorylation (OXPHOS) capacity, which is critically important in health and disease, can be measured in vivo ...

In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice

Mitochondria are the powerhouses of cells and produce cellular energy in the form of ATP. Mitochondrial dysfunction contributes to biological aging and a wide variety of disorders including metabolic diseases, premature aging syndromes, and neurodegenerative diseases such as Alzheimer's disease (AD) and Parkinson's disease (PD). Maintenance of mitochondrial health depends on mitochondrial biogenesis and the efficient clearance of dysfunctional mitochondria through mitophagy. Experimental methods to accurately detect autophagy/mitophagy, especially in animal models, have been challenging to develop. Recent progress towards the understanding of the molecular mechanisms of mitophagy has enabled the development of novel mitophagy detection techniques. Here, we introduce several versatile techniques to monitor mitophagy in human cells, Caenorhabditis elegans (e.g., Rosella and DCT-1/ LGG-1 strains), and mice (mt-Keima). A combination of these mitophagy detection techniques, including cross-species evaluation, will improve the accuracy of mitophagy measurements and lead to a better understanding of the role of mitophagy in health and disease.

In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice

Mitophagy, the process of clearing damaged mitochondria, is necessary for mitochondrial homeostasis and health maintenance. This article presents some of the latest mitophagy detection methods in human cells, Caenorhabditis elegans, and mice.

体外和体内检测人类细胞中的 Mitophagy,线虫和小鼠

Mitochondria are the powerhouses of cells and produce cellular energy in the form of ATP. Mitochondrial dysfunction contributes to biological aging and a ...