We thank John Olsen (Wisconsin Department of Natural Resources), Tom Cooley, Daniel O'Brien and Steve Schmitt (Michigan Department of Natural Resources) and Dr. Daniel Walsh (USGS National Wildlife Health Center) for assistance with tissue acquisition. We also thank the Wisconsin State Laboratory of Hygiene for diagnostic testing services, and Dr. Tonie Rocke and her staff (USGS National Wildlife Health Center) for use of equipment. Any use of trade, product, or firm names is for descriptive purposes only and does not imply endorsement by the U.S. Government.

在美国地质调查局国家野生动物健康中心进行的动物工作是按照实验动物福利准则NIH办公室和体制下的动物护理和使用委员会的协议＃EP080716执行。从狩猎采集动物组织来自威斯康星州或自然资源密歇根部门礼物。 1.溶液制备注意：下面列出的解决方案都需要准备以下第2的CER检测基材。制备各从较高浓度的储备溶液，这些解决方案;推荐浓度原液将括号中所列各有关化学名后给出。准备好所有的解决方案在基板制备和储存新鲜的在4°C，直到使用。 <ol><li>为裂解缓冲液，制备下列在10mM Tris，pH值7.5（的1M Tris，pH值7.5原液推荐）中的溶液：100mM的s裂果氯化物（氯化钠，1M储备溶液），10毫乙二胺四乙酸（EDTA，500毫摩尔EDTA，pH 8.0的储备溶液），0.5％NP-40（10％储备液），0.5％脱氧胆酸盐（DOC，10％原液）。 </li><li>对转换缓冲，制备0.05％的十二烷基硫酸钠（SDS，10％储备液）和0.5％的Triton X-100（10％储备液）在1磷酸盐缓冲盐水（PBS，10，pH 7.4的储备液）的溶液中。 </li><li>对于离液的解决方案，准备盐酸胍（盐酸胍，8M的股票）在1X PBS（10X原液）两个3米的解决方案。用浓盐酸（HCl）调节的解决方案之一的pH至3.5±0.05。检查第二溶液的pH值保持在pH 7.4±0.05和调整用浓盐酸或氢氧化钠（NaOH）中，如果必要的。 </li></ol> 2.准备CER检测底物双<ol><li>获得健康的，非TSE感染脑组织从感兴趣的物种和制备10％重量每体积（重量/体积）匀浆中使用以下任何一种方法的裂解缓冲液：DOUNCE，珠磨机或研钵和杵均化。进一步细化导致匀浆对他们进行注射器和针头的同质化，利用增加计针，直到基材可以被吸入并通过27G的注射器针头排出自如。 <ol><li>从啮齿动物和其他小型哺乳动物准备基板，均质全脑;（二）从大型哺乳动物准备基板，使用OBEX（脑干）组织准备匀浆。当选择脑匀浆的量，准备在步骤2.6用于蛋白质沉淀离心机的容量不超过50％。 </li><li>如果收购了脑组织的质量是有问题的，评估的PrP C水平通过SDS-PAGE 16和免疫17基板事先准备。 </li></ol></li><li>分割脑匀浆成在分开的标记的塑料锥形管2等体积。加入盐酸胍（的3M溶液中1 PBS）中在任一pH值3.5或7.4至1的每个管：1的体积比向脑匀浆。 </li><li>检查两种溶液的pH值。用浓HCl将pH调节3.5衬底至pH的pH±0.05。确保其他基片的pH值保持在pH7.4±0.05，并根据需要在必要时用HCl或NaOH调节pH。避免通过明智加入酸或碱的过冲的pH值。 </li><li> 5小时旋转，在室温（RT）下在最终过端混合器解决方案。 </li><li>通过加入4体积的甲醇中以在每个锥形管的底物溶液中沉淀的蛋白质，涡旋拌匀，孵育在-20℃进行16-18小时。 </li><li>沉积物样品离心以13,000×g离心30分钟，在4℃。小心倒出并丢弃上清液，并且允许甲醇从粒料蒸发。使用棉尖涂药器，以协助吸收甲醇紧贴于管的内部。不要让小球过度博士y和一旦甲醇不再可见，进行到下一个步骤。 </li><li>悬浮颗粒的蛋白质在转换缓冲。使用的转换缓冲等于脑匀浆的起始原料分配到各管中，在步骤2.2的量的量。 注：该转换缓冲用于从两个pH为3.5和7.4，经处理的衬底准备重悬蛋白质粒料在上面的步骤1.2（其中包含洗涤剂的低含量和生理pH）中定义的溶液，这一点至关重要。 </li><li>在cuphorn超声仪简要超声基板解决方案（10秒，约30％的最大功率），分装成0.5 - 1.0毫升卷标记好的1.5 ml离心管。在每一基底的等分试样进行质量控制的免疫印迹（步骤2.9）。存储其他等分在-80°C，直到准备进行CER检测（第4）。 </li><li>执行关于在使用前CER基板对（ 图2）的质量控制。这些措施确保了CER基板将providË最佳效果转化研究。 <ol><li>确保在两个基板可比数额的PrP 的C。在10-25微升每个衬底通过SDS-PAGE 16和免疫印迹17比较朊病毒水平。如果蛋白水平在pH为7.4和3.5的基材是彼此的10％之内〜，衬底对适合在CER研究用途。 注：朊蛋白免疫印迹不应该表现出广泛的PrP的Ç降解的证据。对朊蛋白的免疫反应应该是主要&gt; 20 kDa的。频带小于这个分子量可以降解产物。带型应该是基板对之间大致相同。 </li><li>作为朊下在两个基板应的PK敏感，治疗10-25微升每个衬底具有的PK在100微克/毫升的最终浓度和消化的样品以1000rpm在37℃下1小时的热振动筛。评估任何剩余的PrP信号通过SDS-PAGE 16和17的免疫印迹。底材具有朊病毒水库不适合在CER研究中使用。 </li></ol></li></ol> 3.准备TSE代理种子<ol><li>获得来自感兴趣物种TSE感染的脑组织及（重量/体积）匀浆中的1×PBS pH 7.4的使用在上面的步骤2.1中列出的方法制备的10％。 注：种子还可以从中枢神经系统以外的其他TSE感染的组织中产生，但是，通过从TSE感染的末梢组织衍生的种子转换脑源性底物的效率尚未被评估实验中公开的文献。 </li><li>等分所得匀浆（S）为100 - 在-80℃，直到准备300微升体积在0.5ml微量离心管并储存以执行CER测定。 </li></ol> 4. CER分析注：CER测定涉及从一种物种加入相对少量的PrP TSE的（设置在“种子”）成相对过剩的PrP C与另一个物种已经部分变性在任一pH值3.5或7.4（设置在未感染脑“衬底”）。以下扩展晃动潜伏期，朊蛋白的C模板驱动的转换由朊病毒TSE每个反应是由朊病毒水库剩余PK消化和免疫检测后的光密度信号进行评估。在先前在pH 3.5 与 7.4变性基板相比较的PrP 水库水平提供了物种屏障的量度。该测定过程的一个实验性的概述在图1中提供。 <ol><li>慢慢解冻未感染的CER基底对通过将它们放置在床上的冰（约1小时解冻时间）的顶部（测定需要预先在两个pH值3.5和7.4变性基板等体积）和TSE感染的种子的解决方案。 </li><li>一旦完全解冻，吸，然后驱逐通过每个底物液27G的注射器针头几次重新匀化它。简要超声仪处理10秒，约30％的最大功率每个TSE感染种子的解决方案。保留所有的解决方案在冰同时准备转化反应。 </li><li>标签5个别低结合，薄壁PCR每TSE试剂管被测试。 </li><li>通过加入5微升10％TSE感染种子溶液以95微升（一）CER基板的在pH7.4和（b）的CER基板在pH 3.5制备制备制备在这些管中转化反应。 <ol><li>对于每一个实验样品，准备在此前双方在pH为3.5和7.4变性CER基板对平行反应。 </li><li>通过实证确定优化TSE剂种子到未受感染的大脑基​​底的比率。常见种子：采用基片比率保持100微升的总反应体积，并包括1:99微升，2:98微升和5:95微升。对于大多数应用，该5:95微升种子：基板的体积比是合适的，并且所显示的内容在第是的协议。 </li></ol></li><li>准备每TSE剂3的对照样品被测试如下：（1）加入5微升10％TSE感染种子溶液以95微升的转换缓冲器作为输入朊病毒水库的控制;添加5μl的转换缓冲到在（B）的pH为3.5和（c）pH为7.4，作为非特异性的，非模板化的转换控制制备95微升底物。 </li><li>短暂涡旋振荡每一样品在其封闭PCR管以混合种子和衬底良好。用瞬时脉冲在低速台式微型离心机遵循以除去反应混合物截留在PCR管盖的任何体积。 </li><li>负载样品中单独标记的PCR管放入台式热振动筛配接受PCR管。摇动样品以1000rpm在37℃下24小时。 </li><li>以下晃动，添加十二烷基肌氨酸钠，以2％的终浓度（重量/体积）和PK为100微克/毫升的最终浓度。消化的样品以1000rpm在37℃下进行1小时的热振动筛。 ñOTE：增加肌氨酸到的朊蛋白的C PK消化样品转换后的辅助工具。非种子选手的样品（步骤4.5）的假阳性信号加入肌氨酸的几乎消除。 </li><li>加入SDS-PAGE样品缓冲液，热样品至95℃5分钟，并解决剩余的PrP 水库通过SDS-PAGE 16随后的免疫印迹17每个样品中。 注：在加入样品缓冲液和加热，样品可以被立即处理或样品可以储存在-20℃或-80℃之前，免疫印迹。使用NOVEX的NuPAGE凝胶和传输系统中生成这里介绍的所有数据。从CER测定各个样品分别用样品缓冲液，并根据制造商的说明还原剂。样品随后被加热到95℃5分钟和30微升各样品装入一个12％的Bis-Tris预制凝胶。 </li><li>计算作为度量的转换效率比（CER）的物种屏障。朊病毒水库的通过光密度17测量水平和由该pH为3.5的样品的划分pH 7.4的样品的总密度。乘以100，以产生一个百分比。 </li><li>从独立的实验重复编译数据，以产生一个平均的CER±标准为一种具有给定TSE剂偏差。 </li></ol>

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M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lesion+Profiling+and+Subcellular+Prion+Localization+of+Cervid+Chronic+Wasting+Disease+in+Domestic+Cats.">Lesion Profiling and Subcellular Prion Localization of Cervid Chronic Wasting Disease in Domestic Cats.</a> Vet Pathol. , (2014).</li><li>Stewart, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+predictions+of+prion+disease+susceptibility+in+carnivore+species+based+on+variability+of+the+prion+gene+coding+region.">Genetic predictions of prion disease susceptibility in carnivore species based on variability of the prion gene coding region.</a> PLoS One. 7 (12), e50623 (2012).</li><li>Russell, W. M., Burch, R. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Principles of Humane Experimental Technique. Metheun. , (1959).</li></ol>

The authors have nothing to disclose.

为了顺利完成本协议的，应注意所用的基板准备（步骤2.1.2）和种子底物比对转化反应（步骤4.4.2）未受感染的脑组织付给的PrP C水平。根据我们的经验，大脑可以提取用作CER基板大幅期间验尸后，只要朊病毒C是本通过免疫印迹（ 图4）。事实上，一些自溶观察用于CER研究山猫的大脑。然而，来自这些大脑衍生基板孵育仍然导致朊病毒水库的形成。我们推测，?...

传染性海绵状脑病（传染性海绵状脑病，朊病毒病）是一组的致命的神经变性疾病与影响各种动物和人的延长潜伏期。的TSE的推定病原体包括主机朊病毒蛋白（PRP）的错误折叠的异构体，其能够自我繁殖由朊病毒（PrP的C）的正常细胞形式的模板驱动转换成感染性的，疾病相关的形式（PRP TSE）积聚在受感染的主机1的中枢神经系统组织。传染性朊病毒哺乳动物一般从主机到主机传输的物种特异性的方式，这已经引起了“TSE物种屏障”限间传播的事件2的概念。朊病毒物种屏障的生物因素没有得到很好地理解。传染性的PrP TSE和主机的PrP C C之间的氨基酸序列相似性一个强烈影响是否发生转换3-5，但仍不足以解释在体内 6,7观察到的所有朊病毒传播事件。 因此，TSE物种屏障特性在很大程度上依赖于动物攻击研究：曝光从给定的物种幼稚动物朊病毒从另一个并测量产生的温育时间，疾病发作和发作率的传输效率的指标。小鼠表达的PrP C来自异源物种也可用于这种类型的研究8。用转基因小鼠生产和长期朊病毒生物测定，以及使用动物的伦理方面的考虑相关的成本，是阻碍了TSE物种界限的实验研究。人类物种屏障传染性海绵状脑病的评估依赖于设计，以表达人类的PrP C小鼠。这些小鼠需要很长的潜伏期屈从于人类的传染性海绵状脑病或修改吨他的人朊蛋白C分子迅速发病9。对非人类的TSE在这些小鼠中的潜伏期可能超出正常小鼠寿命使得有挑战性阴性结果的解释。非人灵长类动物也已被用于作为代理为研究人类物种的障碍，但这些研究是充满其他类型的动物实验和非人类灵长类动物的相同的挑战可能无法精确地概括疾病，因为它进入在人类宿主。 动物生物测定仍是“金标准”方法测量物种一个TSE的易感性，但障碍，成本和这些活的动物研究的伦理学已迫使调查的替代品。许多在体外实验的基础上，评估主机的PrP C中的转换蛋白酶K（PK）耐药状态（PRP 水库 ）时，由朊病毒TSE的种子，已经开发并用于研究TSE SPECIES障碍10-12。 在体外测定法的实例包括无细胞转化测定法，蛋白质错误折叠环扩增（PMCA），以及转换效率比率（CER）测定10-14。而没有这些测定的考虑涉及自然感染后的物种障碍帐户外围因素的影响，均可能是有用的，以确定潜在易感宿主为传染性海绵状脑病。 这里，我们提出的协议的CER测定，其中从正常脑匀浆衍生2变性的PrP C底物在台式朊转化反应中使用（ 图1）13。朊病毒下在变性在pH7.4基板只能被转换为朊病毒水库由朊病毒龄接种到在没有一个物种屏障14进行反应。与此相反，朊病毒C在另一基底变性，在pH 3.5允许它被转换为朊病毒水库以下孵化与朊病毒TSE来自任何物种，并作为转换的控制。到朊病毒水库 pH 7.4的衬底相在该pH值3.5底物转化的PrP 的C的比率提供了物种屏障的量度。我们已发现，CER测定所预言的已知的实验室小鼠的物种障碍，各种传染性海绵状脑病，并已用于测定在努力预测众多哺乳动物种，包括大角羊，慢性消耗性疾病（CWD）和其他的TSE 14的物种障碍， 15。研究者感兴趣的一个工具，允许TSE物种界限或PrP的Ç-to-RES朊病毒转化评估快速筛选会发现这种方法非常有用。

<table border="0" cellpadding="0" cellspacing="0" width="944">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Name of Material/ Equipment</td>
			<td style="width:205px;">Company</td>
			<td style="width:157px;">Catalog Number</td>
			<td style="width:287px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">12% Bis-Tris SDS-PAGE gels</td>
			<td style="width:205px;">Life Technologies</td>
			<td style="width:157px;">NP0342</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:296px;">0.5 mm zirconium oxide beads</td>
			<td style="width:205px;">Next Advance</td>
			<td style="width:157px;">ZROB05&#160;</td>
			<td style="width:287px;">Other varieties of beads are also effective</td>
		</tr>
		<tr height="66">
			<td height="66" style="height:66px;width:296px;">Antibodies</td>
			<td style="width:205px;">various suppliers</td>
			<td style="width:157px;"></td>
			<td style="width:287px;">Select appropriate primary and secondary antibodies for immunoblot detection of PrPres from species of interest</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Bead homogenizer</td>
			<td style="width:205px;">Next Advance</td>
			<td style="width:157px;">BBY24M</td>
			<td style="width:287px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Centrifuge&#160;</td>
			<td style="width:205px;">Beckman Coulter</td>
			<td style="width:157px;">369434</td>
			<td style="width:287px;">High speed with temperature control</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Conical tubes</td>
			<td style="width:205px;">any brand</td>
			<td style="width:157px;"></td>
			<td style="width:287px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Cotton-tipped applicator</td>
			<td style="width:205px;">Uline</td>
			<td style="width:157px;">S-18991</td>
			<td style="width:287px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:296px;">Cuphorn sonicator</td>
			<td style="width:205px;">Heat Systems-Ultrasonics</td>
			<td style="width:157px;">W-380</td>
			<td style="width:287px;">Heat Systems-Ultrasonics, Inc. is now Qsonica, LLC</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:296px;">Densitometry software program</td>
			<td style="width:205px;">UVP</td>
			<td style="width:157px;">Vision Works LS Image Acquisition and Analysis software</td>
			<td style="width:287px;">Other programs, such as NIH ImageJ, will also work</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Dounce homogenizer</td>
			<td style="width:205px;">Kimble Chase</td>
			<td style="width:157px;">885300</td>
			<td style="width:287px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">End-over-end mixer</td>
			<td style="width:205px;">Labnet International</td>
			<td style="width:157px;">H5600</td>
			<td style="width:287px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ethylenediaminetetra acetic acid</td>
			<td style="width:205px;">Boston Bioproducts</td>
			<td style="width:157px;">P-770</td>
			<td style="width:287px;">Hazardous chemical: eye irritation</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Guanidine hydrochloride, 8M</td>
			<td style="width:205px;">Thermo Fisher Scientific</td>
			<td style="width:157px;">24115</td>
			<td style="width:287px;">Hazardous chemical: acute toxicity, skin irritation, eye irritation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Heating block</td>
			<td style="width:205px;">Fisher Scientific</td>
			<td style="width:157px;">11-718</td>
			<td style="width:287px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hydrochloric acid</td>
			<td style="width:205px;">Sigma-Aldrich&#160;</td>
			<td style="width:157px;">435570</td>
			<td style="width:287px;">Hazardous chemical: strong acid</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">Lithium dodecyl sulfate sample buffer, 4X</td>
			<td style="width:205px;">Life Technologies</td>
			<td style="width:157px;">NP0008</td>
			<td style="width:287px;">Hazardous chemical: skin &#38; respiratory irritation, serious eye damage, flammable solid</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Methanol</td>
			<td style="width:205px;">Fisher Scientific</td>
			<td style="width:157px;">A454-4</td>
			<td style="width:287px;">Hazardous chemical: acute toxicity, flammable liquid</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microcentrifuge tubes</td>
			<td style="width:205px;">any brand</td>
			<td style="width:157px;"></td>
			<td style="width:287px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mini-centrifuge</td>
			<td style="width:205px;">Labnet International</td>
			<td style="width:157px;">C1301</td>
			<td style="width:287px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">N-lauroyl-sarcosine (sarkosyl)</td>
			<td style="width:205px;">Sigma-Aldrich&#160;</td>
			<td style="width:157px;">L-5125</td>
			<td style="width:287px;">Hazardous chemical: acute toxicity, skin irritation, eye damage</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:296px;">Nonidet P-40</td>
			<td style="width:205px;">Amresco</td>
			<td style="width:157px;">M158</td>
			<td style="width:287px;">Hazardous chemical: skin irritation, eye damage</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:296px;">SDS-PAGE gel system</td>
			<td style="width:205px;">Life Technologies</td>
			<td style="width:157px;">NuPAGE electrophoresis system</td>
			<td style="width:287px;">Other SDS-PAGE systems will also work</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">PCR tubes (low-binding)</td>
			<td style="width:205px;">Axygen</td>
			<td style="width:157px;">PCR-02-L-C</td>
			<td style="width:287px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Pestle homogenizer</td>
			<td style="width:205px;">Fisher Scientific</td>
			<td style="width:157px;">03-392-106</td>
			<td style="width:287px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">pH meter</td>
			<td style="width:205px;">Sentron</td>
			<td style="width:157px;">SI600</td>
			<td style="width:287px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Polyvinyldifluoride membrane</td>
			<td style="width:205px;">Millipore</td>
			<td style="width:157px;">IPVH00010</td>
			<td style="width:287px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:296px;">Proteinase K</td>
			<td style="width:205px;">Promega</td>
			<td style="width:157px;">V3021</td>
			<td style="width:287px;">Hazardous chemical: skin &#38; eye irritation, respiratory sensitisation, organ toxicity</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:296px;">Reducing agent for SDS-PAGE samples, 10X</td>
			<td style="width:205px;">Life Technologies</td>
			<td style="width:157px;">NP0009</td>
			<td style="width:287px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Sodium Chloride</td>
			<td style="width:205px;">Fisher Scientific</td>
			<td style="width:157px;">7647-14-5</td>
			<td style="width:287px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Sodium deoxycholate</td>
			<td style="width:205px;">Sigma-Aldrich&#160;</td>
			<td style="width:157px;">D6750</td>
			<td style="width:287px;">Hazardous chemical: acute toxicity</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:296px;">Sodium dodecyl sulfate</td>
			<td style="width:205px;">Thermo Fisher Scientific</td>
			<td style="width:157px;">28364</td>
			<td style="width:287px;">Hazardous chemical: acute toxicity, skin irritation, eye damage, flammable solid</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Sodium hydroxide</td>
			<td style="width:205px;">Sigma-Aldrich&#160;</td>
			<td style="width:157px;">S5881</td>
			<td style="width:287px;">Hazardous chemica; strong base</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:296px;">Syringe</td>
			<td style="width:205px;">BD Biosciences</td>
			<td style="width:157px;">various</td>
			<td style="width:287px;">Use syringe size appropriate to volumes of substrate to be homogenized</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Syringe needles</td>
			<td style="width:205px;">BD Biosciences</td>
			<td style="width:157px;">various</td>
			<td style="width:287px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:296px;">Thermoshaker (PCR tube shaker)</td>
			<td style="width:205px;">Hangzhou All Sheng Instruments</td>
			<td style="width:157px;">MS-100</td>
			<td style="width:287px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:296px;">Tris base</td>
			<td style="width:205px;">Bio Basic&#160;</td>
			<td style="width:157px;">77-86-1</td>
			<td style="width:287px;">Hazardous chemical: skin, eye, respiratory irritation</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:296px;">Triton X-100</td>
			<td style="width:205px;">Integra Chemical Company</td>
			<td style="width:157px;">T756.30.30</td>
			<td style="width:287px;">Hazardous chemical: acute toxicity, eye irritation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Vortexer</td>
			<td style="width:205px;">Fisher Scientific</td>
			<td style="width:157px;">12-812</td>
			<td style="width:287px;"></td>
		</tr>
	</tbody>
</table>

assessing transmissible spongiform encephalopathy species barriers with an in vitro prion protein conversion assay

Studies to understanding interspecies transmission of transmissible spongiform encephalopathies (TSEs, prion diseases) are challenging in that they typically rely upon lengthy and costly in vivo animal challenge studies. A number of in vitro assays have been developed to aid in measuring prion species barriers, thereby reducing animal use and providing quicker results than animal bioassays. Here, we present the protocol for a rapid in vitro prion conversion assay called the conversion efficiency ratio (CER) assay. In this assay cellular prion protein (PrPC) from an uninfected host brain is denatured at both pH 7.4 and 3.5 to produce two substrates. When the pH 7.4 substrate is incubated with TSE agent, the amount of PrPC that converts to a proteinase K (PK)-resistant state is modulated by the original host&#8217;s species barrier to the TSE agent. In contrast, PrPC in the pH 3.5 substrate is misfolded by any TSE agent. By comparing the amount of PK-resistant prion protein in the two substrates, an assessment of the host&#8217;s species barrier can be made. We show that the CER assay correctly predicts known prion species barriers of laboratory mice and, as an example, show some preliminary results suggesting that bobcats (Lynx rufus) may be susceptible to white-tailed deer (Odocoileus virginianus) chronic wasting disease agent.

成功使用CER测定的在很大程度上取决于在转化反应中使用的底物对的质量。出于这个原因，下面的过程以制备CER基板对，一个质量控制免疫应执行（ 图2）。对于两个基板，测定10-25微升通过免疫印迹。的PrP C应该容易检测在每个衬底和朊病毒C水平必须接近两者是相同的。免疫反应性将主要​​高于20 kDa的可见光，但较小频带也可以是显而易见的。根据我们的经验，更...

Watch this Scientific Journal Video about Assessing Transmissible Spongiform Encephalopathy Species Barriers with an In Vitro Prion Protein Conversion Assay at JoVE.com

Assessing Transmissible Spongiform Encephalopathy Species Barriers with an In Vitro Prion Protein Conversion Assay

Measuring the barrier to the interspecies transmission of prion diseases is challenging and typically involves animal challenges or biochemical assays. Here, we present an in vitro prion protein conversion assay with the ability to predict species barriers.

评估传染性海绵状脑病物种界限与<em&gt;在体外</em&gt;朊蛋白转化测定法

Studies to understanding interspecies transmission of transmissible spongiform encephalopathies (TSEs, prion diseases) are challenging in that they ...

assessing-transmissible-spongiform-encephalopathy-species-barriers

Research

JoVE Journal

Medicine

Assessing Transmissible Spongiform Encephalopathy Species Barriers with an In Vitro Prion Protein Conversion Assay

7.7K 次观看.  USGS National Wildlife Health Center. Measuring the barrier to the interspecies transmission of prion diseases is challenging and typically involves animal challenges or biochemical assays. Here, we present an in vitro prion protein conversion assay with the ability to predict species barriers.

视频: Assessing Transmissible Spongiform Encephalopathy Species Barriers with an In Vitro Prion Protein Conversion Assay

Biomarkers in an Animal Model for Revealing Neural, Hematologic, and Behavioral Correlates of PTSD

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for objective diagnosis but also for the evaluation of therapeutic efficacy and resilience to trauma. Ongoing research is directed at identifying molecular biomarkers for PTSD, including traumatic stress induced proteins, transcriptomes, genomic variances and genetic modulators, using biologic samples from subjects' blood, saliva, urine, and postmortem brain tissues. However, the correlation of these biomarker molecules in peripheral or postmortem samples to altered brain functions associated with psychiatric symptoms in PTSD remains unresolved. Here, we present an animal model of PTSD in which both peripheral blood and central brain biomarkers, as well as behavioral phenotype, can be collected and measured, thus providing the needed correlation of the central biomarkers of PTSD, which are mechanistic and pathognomonic but cannot be collected from people, with the peripheral biomarkers and behavioral phenotypes, which can.
Our animal model of PTSD employs restraint and tail shocks repeated for three continuous days - the inescapable tail-shock model (ITS) in rats. This ITS model mimics the pathophysiology of PTSD 17, 7, 4, 10. We and others have verified that the ITS model induces behavioral and neurobiological alterations similar to those found in PTSD subjects 17, 7, 10, 9. Specifically, these stressed rats exhibit (1) a delayed and exaggerated startle response appearing several days after stressor cessation, which given the compressed time scale of the rat's life compared to a humans, corresponds to the one to three months delay of symptoms in PTSD patients (DSM-IV-TR PTSD Criterian D/E 13), (2) enhanced plasma corticosterone (CORT) for several days, indicating compromise of the hypothalamopituitary axis (HPA), and (3) retarded body weight gain after stressor cessation, indicating dysfunction of metabolic regulation.
The experimental paradigms employed for this model are: (1) a learned helplessness paradigm in the rat assayed by measurement of acoustic startle response (ASR) and a charting of body mass; (2) microdissection of the rat brain into regions and nuclei; (3) enzyme-linked immunosorbent assay (ELISA) for blood levels of CORT; (4) a gene expression microarray plus related bioinformatics tools 18. This microarray, dubbed rMNChip, focuses on mitochondrial and mitochondria-related nuclear genes in the rat so as to specifically address the neuronal bioenergetics hypothesized to be involved in PTSD.

We describe a rat model of post traumatic stress disorder (PTSD) that reveals the persistent alterations in neuroendocrine function and the delayed long-term, exaggerated fear response, characteristic of PTSD patients. The animal model and methods described here are useful for correlating biomarkers in brain nuclei, which are mechanistic but cannot be measured in patients, with biomarkers in peripheral white blood cells, which can.

在动物模型揭示神经，血液，创伤后应激障碍和行为相关因素的生物标志物

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for ...

科研

医学

Assessing Teratogenic Changes in a Zebrafish Model of Fetal Alcohol Exposure

Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, including mental retardation due to disordered and damaged brain development. Fetal alcohol spectrum disorder (FASD) is a term used to cover a continuum of birth defects that occur due to maternal alcohol consumption, and occurs in approximately 4% of children born in the United States. With 50% of child-bearing age women reporting consumption of alcohol, and half of all pregnancies being unplanned, unintentional exposure is a continuing issue2. In order to best understand the damage produced by ethanol, plus produce a model with which to test potential interventions, we developed a model of developmental ethanol exposure using the zebrafish embryo. Zebrafish are ideal for this kind of teratogen study3-8. Each pair lays hundreds of eggs, which can then be collected without harming the adult fish. The zebrafish embryo is transparent and can be readily imaged with any number of stains. Analysis of these embryos after exposure to ethanol at different doses and times of duration and application shows that the gross developmental defects produced by ethanol are consistent with the human birth defect. Described here are the basic techniques used to study and manipulate the zebrafish FAS model.

In order to understand the molecular mechanisms of the ethanol-induced developmental damage, we have developed a zebrafish model of ethanol exposure and are exploring the physical, cellular, and genetic alterations that occur after ethanol exposure1. We then seek to find potential interventions and rapidly test them in this animal model.

在斑马鱼模型的胎儿酒精暴露评估致畸的变化

Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, ...

Isolation of Soluble and Insoluble PrP Oligomers in the Normal Human Brain

The central event in the pathogenesis of prion diseases involves a conversion of the host-encoded cellular prion protein PrPC into its pathogenic isoform PrPSc 1. PrPC is detergent-soluble and sensitive to proteinase K (PK)-digestion, whereas PrPSc forms detergent-insoluble aggregates and is partially resistant to PK2-6. The conversion of PrPC to PrPSc is known to involve a conformational transition of &alpha;-helical to &beta;-sheet structures of the protein. However, the in vivo pathway is still poorly understood. A tentative endogenous PrPSc, intermediate PrP* or "silent prion", has yet to be identified in the uninfected brain7.
Using a combination of biophysical and biochemical approaches, we identified insoluble PrPC aggregates (designated iPrPC) from uninfected mammalian brains and cultured neuronal cells8, 9. Here, we describe detailed procedures of these methods, including ultracentrifugation in detergent buffer, sucrose step gradient sedimentation, size exclusion chromatography, iPrP enrichment by gene 5 protein (g5p) that specifically bind to structurally altered PrP forms10, and PK-treatment. The combination of these approaches isolates not only insoluble PrPSc and PrPC aggregates but also soluble PrPC oligomers from the normal human brain. Since the protocols described here have been used to isolate both PrPSc from infected brains and iPrPC from uninfected brains, they provide us with an opportunity to compare differences in physicochemical features, neurotoxicity, and infectivity between the two isoforms. Such a study will greatly improve our understanding of the infectious proteinaceous pathogens. The physiology and pathophysiology of iPrPC are unclear at present. Notably, in a newly-identified human prion disease termed variably protease-sensitive prionopathy, we found a new PrPSc that shares the immunoreactive behavior and fragmentation with iPrPC 11, 12. Moreover, we recently demonstrated that iPrPC is the main species that interacts with amyloid-&beta; protein in Alzheimer disease13. In the same study, these methods were used to isolate Abeta aggregates and oligomers in Alzheimer's disease13, suggesting their application to non-prion protein aggregates involved in other neurodegenerative disorders.

A new species of cellular prion protein (PrPC) has recently been identified in uninfected human brains using the methods described here. These methods can be used to isolate various PrP species, while some of them are also useful in isolating other misfolded protein aggregates from human brains.

在正常人脑分离的可溶性和不溶性朊蛋白低聚物

The central event in the pathogenesis of prion diseases involves a conversion of the host-encoded cellular prion protein PrPC into its pathogenic isoform ...

Simulating Pancreatic Neuroplasticity: In Vitro Dual-neuron Plasticity Assay

Neuroplasticity is an inherent feature of the enteric nervous system and gastrointestinal (GI) innervation under pathological conditions. However, the pathophysiological role of neuroplasticity in GI disorders remains unknown. Novel experimental models which allow simulation and modulation of GI neuroplasticity may enable enhanced appreciation of the contribution of neuroplasticity in particular GI diseases such as pancreatic cancer (PCa) and chronic pancreatitis (CP). Here, we present a protocol for simulation of pancreatic neuroplasticity under in vitro conditions using newborn rat dorsal root ganglia (DRG) and myenteric plexus (MP) neurons. This dual-neuron approach not only permits monitoring of both organ-intrinsic and -extrinsic neuroplasticity, but also represents a valuable tool to assess neuronal and glial morphology and electrophysiology. Moreover, it allows functional modulation of supplied microenvironmental contents for studying their impact on neuroplasticity. Once established, the present neuroplasticity assay bears the potential of being applicable to the study of neuroplasticity in any GI organ.

Simulating Pancreatic Neuroplasticity: In Vitro Dual-neuron Plasticity Assay

Neuronal plasticity is an increasingly recognized, but insufficiently understood feature of the gastrointestinal (GI) tract. Here, in the example of human pancreatic disorders, we present an in vitro neuroplasticity assay for the study of neuronal plasticity in the GI tract at both morphological and functional level.

模拟胰腺神经可塑性：<em&gt;体外</em&gt;双神经元可塑性分析

Neuroplasticity is an inherent feature of the enteric nervous system and gastrointestinal (GI) innervation under pathological conditions. However, the ...

Using Adeno-associated Virus as a Tool to Study Retinal Barriers in Disease

M&#252;ller cells are the principal glial cells of the retina. Their end-feet form the limits of the retina at the outer and inner limiting membranes (ILM), and in conjunction with astrocytes, pericytes and endothelial cells they establish the blood-retinal barrier (BRB). BRB limits material transport between the bloodstream and the retina while the ILM acts as a basement membrane that defines histologically the border between the retina and the vitreous cavity. Labeling M&#252;ller cells is particularly relevant to study the physical state of the retinal barriers, as these cells are an integral part of the BRB and ILM. Both BRB and ILM are frequently altered in retinal disease and are responsible for disease symptoms.
There are several well-established methods to study the integrity of the BRB, such as the Evans blue assay or fluorescein angiography. However these methods do not provide information on the extent of BRB permeability to larger molecules, in nanometer range. Furthermore, they do not provide information on the state of other retinal barriers such as the ILM. To study BRB permeability alongside retinal ILM, we used an AAV based method that provides information on permeability of BRB to larger molecules while indicating the state of the ILM and extracellular matrix proteins in disease states. Two AAV variants are useful for such study: AAV5 and ShH10. AAV5&#160;has a natural tropism for photoreceptors but it cannot get across to the outer retina when administered into the vitreous when the ILM is intact (i.e., in wild-type retinas). ShH10 has a strong tropism towards glial cells and will selectively label M&#252;ller glia in both healthy and diseased retinas. ShH10 provides more efficient gene delivery in retinas where ILM is compromised. These viral tools coupled with immunohistochemistry and blood-DNA analysis shed light onto the state of retinal barriers in disease.

To investigate the blood-retinal barrier permeability and the inner limiting membrane integrity in animal models of retinal disease, we used several adeno-associated virus (AAV) variants as tools to label retinal neurons and glia. Virus mediated reporter gene expression is then used as an indicator of retinal barrier permeability.

使用腺相关病毒作为一种工具来研究疾病中的视网膜障碍

M&#252;ller cells are the principal glial cells of the retina. Their end-feet form the limits of the retina at the outer and inner limiting membranes ...

A Piglet Model of Neonatal Hypoxic-Ischemic Encephalopathy

Birth asphyxia, which causes hypoxic-ischemic encephalopathy (HIE), accounts for 0.66 million deaths worldwide each year, about a quarter of the world&#8217;s 2.9 million neonatal deaths. Animal models of HIE have contributed to the understanding of the pathophysiology in HIE, and have highlighted the dynamic process that occur in brain injury due to perinatal asphyxia. Thus, animal studies have suggested a time-window for post-insult treatment strategies. Hypothermia has been tested as a treatment for HIE in pdiglet models and subsequently proven effective in clinical trials. Variations of the model have been applied in the study of adjunctive neuroprotective methods and piglet studies of xenon and melatonin have led to clinical phase I and II trials1,2. The piglet HIE model is further used for neonatal resuscitation- and hemodynamic studies as well as in investigations of cerebral hypoxia on a cellular level. However, it is a technically challenging model and variations in the protocol may result in either too mild or too severe brain injury. In this article, we demonstrate the technical procedures necessary for establishing a stable piglet model of neonatal HIE. First, the newborn piglet (&#60; 24 hr old, median weight 1500 g) is anesthetized, intubated, and monitored in a setup comparable to that found in a neonatal intensive care unit. Global hypoxia-ischemia is induced by lowering the inspiratory oxygen fraction to achieve global hypoxia, ischemia through hypotension and a flat trace amplitude integrated EEG (aEEG) indicative of cerebral hypoxia. Survival is promoted by adjusting oxygenation according to the aEEG response and blood pressure. Brain injury is quantified by histopathology and magnetic resonance imaging after 72 hr.

Hypoxic-ischemic encephalopathy following perinatal asphyxia can be studied using animal models. We demonstrate the procedures necessary for establishing a piglet model of neonatal hypoxic-ischemic encephalopathy.

新生儿缺氧缺血性脑病的小猪模型

Birth asphyxia, which causes hypoxic-ischemic encephalopathy (HIE), accounts for 0.66 million deaths worldwide each year, about a quarter of the ...

Modeling Encephalopathy of Prematurity Using Prenatal Hypoxia-ischemia with Intra-amniotic Lipopolysaccharide in Rats

Encephalopathy of prematurity (EoP) is a term that encompasses the central nervous system (CNS) abnormalities associated with preterm birth. To best advance translational objectives and uncover new therapeutic strategies for brain injury associated with preterm birth, preclinical models of EoP must include similar mechanisms of prenatal global injury observed in humans and involve multiple components of the maternal-placental-fetal system. Ideally, models should produce a similar spectrum of functional deficits in the mature animal and recapitulate multiple aspects of the pathophysiology. To mimic human systemic placental perfusion defects, placental underperfusion and/or chorioamnionitis associated with pathogen-induced inflammation in early preterm birth, we developed a model of prenatal transient systemic hypoxia-ischemia (TSHI) combined with intra-amniotic lipopolysaccharide (LPS). In pregnant Sprague Dawley rats, TSHI via uterine artery occlusion on embryonic day 18 (E18) induces a graded placental underperfusion defect associated with increasing CNS damage in the fetus. When combined with intra-amniotic LPS injections, placental inflammation is increased and CNS damage is compounded with associated white matter, gait and imaging abnormalities. Prenatal TSHI and TSHI+LPS prenatal insults meet several of the criteria of an EoP model including recapitulating the intrauterine insult, causing loss of neurons, oligodendrocytes and axons, loss of subplate, and functional deficits in adult animals that mimic those observed in children born extremely preterm. Moreover, this model allows for the dissection of inflammation induced by divergent injury types.

Encephalopathy of prematurity encompasses the central nervous system abnormalities associated with injury from preterm birth. This report describes a clinically relevant rat model of in utero transient systemic hypoxia-ischemia and intra-amniotic lipopolysaccharide administration (LPS) that mimics chorioamnionitis, and the related impact of infectious stimuli and placental underperfusion on CNS development.

早产儿脑病建模使用产前缺氧缺血大鼠羊膜腔内毒素

Encephalopathy of prematurity (EoP) is a term that encompasses the central nervous system (CNS) abnormalities associated with preterm birth. To best ...

Assessing Specificity of Anticancer Drugs In Vitro

A procedure for assessing specificity of anticancer drugs in vitro using cultures containing both tumor and non-tumor cells is demonstrated. The key element is the quantitative determination of a tumor-specific genetic alteration in relation to a universal sequence using a dual-probe digital PCR assay and the subsequent calculation of the proportion of tumor cells. The assay is carried out on a culture containing tumor cells of an established line and spiked-in non-tumor cells. The mixed culture is treated with a test drug at various concentrations. After the treatment, DNA is prepared directly from the survived adhesive cells in wells of 96-well plates using a simple and inexpensive method, and subjected to a dual-probe digital PCR assay for measuring a tumor-specific genetic alteration and a reference universal sequence. In the present demonstration, a heterozygous deletion of the NF1 gene is used as the tumor-specific genetic alteration and a RPP30 gene as the reference gene. Using the ratio NF1/RPP30, the proportion of tumor cells was calculated. Since the dose-dependent change of the proportion of tumor cells provides an in vitro indication for specificity of the drug, this genetic and cell-based in vitro assay will likely have application potential in drug discovery. Furthermore, for personalized cancer-care, this genetic- and cell-based tool may contribute to optimizing adjuvant chemotherapy by means of testing efficacy and specificity of candidate drugs using primary cultures of individual tumors.

Assessing Specificity of Anticancer Drugs In Vitro

The goal of this protocol is to assess specificity of anticancer drugs in vitro using mixed cultures containing both tumor and non-tumor cells.

评估抗癌药物的特异性<em&gt;体外</em

A procedure for assessing specificity of anticancer drugs in vitro using cultures containing both tumor and non-tumor cells is demonstrated. The key ...

Forskolin-induced Swelling in Intestinal Organoids: An In Vitro Assay for Assessing Drug Response in Cystic Fibrosis Patients

Recently-developed cystic fibrosis transmembrane conductance regulator (CFTR)-modulating drugs correct surface expression and/or function of the mutant CFTR channel in subjects with cystic fibrosis (CF). Identification of subjects that may benefit from these drugs is challenging because of the extensive heterogeneity of CFTR mutations, as well as other unknown factors that contribute to individual drug efficacy. Here, we describe a simple and relatively rapid assay for measuring individual CFTR function and response to CFTR modulators in vitro. Three dimensional (3D) epithelial organoids are grown from rectal biopsies in standard organoid medium. Once established, the organoids can be bio-banked for future analysis. For the assay, 30-80 organoids are seeded in 96-well plates in basement membrane matrix and are then exposed to drugs. One day later, the organoids are stained with calcein green, and forskolin-induced swelling is monitored by confocal live cell microscopy at 37 &#176;C. Forskolin-induced swelling is fully CFTR-dependent and is sufficiently sensitive and precise to allow for discrimination between the drug responses of individuals with different and even identical CFTR mutations. In vitro swell responses correlate with the clinical response to therapy. This assay provides a cost-effective approach for the identification of drug-responsive individuals, independent of their CFTR mutations. It may also be instrumental in the development of future CFTR modulators.

Forskolin-induced Swelling in Intestinal Organoids: An In Vitro Assay for Assessing Drug Response in Cystic Fibrosis Patients

This protocol describes an assay for measuring CFTR function and CFTR modulator responses in cultured tissue from subjects with cystic fibrosis (CF). Biopsy-derived intestinal organoids swell in a cAMP-driven fashion, a response that is defective (or strongly reduced) in CF organoids and can be restored by exposure to CFTR modulators.

毛喉素诱导在肠组织体溶胀：一个<em&gt;体外</em&gt;检测方法的评估囊性纤维化患者对药物的反应

Recently-developed cystic fibrosis transmembrane conductance regulator (CFTR)-modulating drugs correct surface expression and/or function of the mutant ...

Neonatal Murine Cochlear Explant Technique as an In Vitro Screening Tool in Hearing Research

While there have been remarkable advances in hearing research over the past few decades, there is still no cure for Sensorineural Hearing Loss (SNHL), a condition that typically involves damage to or loss of the delicate mechanosensory structures of the inner ear. Sophisticated in vitro and ex vivo assays have emerged in recent years, enabling the screening of an increasing number of potentially therapeutic compounds while minimizing resources and accelerating efforts to develop cures for SNHL. Though homogenous cultures of certain cell types continue to play an important role in current research, many scientists now rely on more complex organotypic cultures of murine inner ears, also known as cochlear explants. The preservation of organized cellular structures within the inner ear facilitates the in situ evaluation of various components of the cochlear infrastructure, including inner and outer hair cells, spiral ganglion neurons, neurites, and supporting cells. Here we present the preparation, culture, treatment, and immunostaining of neonatal murine cochlear explants. The careful preparation of these explants facilitates the identification of mechanisms that contribute to SNHL and constitutes a valuable tool for the hearing research community.

Neonatal Murine Cochlear Explant Technique as an In Vitro Screening Tool in Hearing Research

The goal of this protocol is to demonstrate the preparation, culture, treatment, and immunostaining of neonatal murine cochlear explants. The technique can be utilized as an in vitro screening tool in hearing research.

新生儿鼠耳蜗外科手术技术<em&gt;体外</em&gt;听力研究筛选工具

While there have been remarkable advances in hearing research over the past few decades, there is still no cure for Sensorineural Hearing Loss (SNHL), a ...