We thank Sue Lindquist, Aaron Gitler, and Martin Duennwald for kindly sharing reagents. Our studies were supported by: an American Heart Association Post-Doctoral Fellowship (M.E.J); NIH Director's New Innovator Award DP2OD002177, NIH grants R21NS067354, R21HD074510, and R01GM099836, a Muscular Dystrophy Association Research Award (MDA277268), Packard Center for ALS Research at Johns Hopkins University, Target ALS, and an Ellison Medical Foundation New Scholar in Aging Award (J.S.).

1.生成库<ol><li>构造的Hsp104利用特定于域的易错PCR文库，首先扩增感兴趣与易出错的DNA聚合酶20的域。 </li><li>纯化PCR产物通过凝胶提取。 </li><li>使用标准的定点诱变协议进行大引物延伸步骤：结合为50ng模板质粒，250ng的大引物，200μMdNTPs和高保真DNA聚合酶在PCR缓冲液，并稀释至50μl总体积PCR级水20 。运行一个标准的PCR程序。 注意：用于将基于要被扩增的基因的特定区域而变化的特定的引物。 <ol><li>下述PCR，在37℃下消化亲代DNA模板与1μl的DPN限制性内切酶处理2小时。 </li></ol></li><li>通过电穿孔或其他方法在ultracompetent 大肠杆菌转化库大肠杆菌和小量净化它。 注：库代差异极大基于给定实验的目的。的Hsp104是一个非常大的蛋白质，因此它是不现实的随机化的完整基因，这就是为什么我们利用特定于域的易错PCR。此外，的Hsp104的结构仍然知之甚少，使得定向库挑战21的设计。库可以用定向或随机的方法来诱变来构建，唯一的限制是，该模板质粒骨架应包含URA3基因，以允许在葡萄糖介质5-FOA反选择。 </li></ol> 2.转型的Hsp104图书馆<ol><li>使用标准的醋酸锂/ PEG转化协议22集成的疾病相关的基板为W303aΔHSP104酵母。选择单菌落，筛选它们的毒性隔离关联基板23该病的高毒性菌株。 注：使用集成的应变和IsolaTE的一种单菌落，以确保在所有细胞中表达的平等。克隆病基底到质粒允许整合下比尿嘧啶以外的任何标记（我们使用组氨酸）和所述的Hsp104文库的基因导入pAG416GAL质粒（尿嘧啶标记）24。为了让5-FOA反选择，确保库是从与尿嘧啶标记的质粒表达。其他的酵母菌株也可使用。我们注意到使用W303a和BY4741酵母菌株在WT和ΔHSP104背景（MEJ和JS，未发表意见）相似的毒性抑制的表型。 </li><li>使用相同的醋酸锂/ PEG转化协议22改造图书馆的Hsp104这个压力。向上扩展变换适当地保持库的序列空间和保存预测的文库大小。 </li><li>制版用足够板的转化混合物到非诱导，选择性平板（ 例如 SD-他浦）确保大量菌落的生长。使用大陪替氏培养皿（150毫米），最小化所需的板的数量。回收利用板的转化使转化效率进行评估。 </li><li>变换的Hsp104 WT和载体的阴性对照平行。 </li></ol> 3.筛选抑制Proteotoxicity的<ol><li>利用棉子糖补充丢失的介质（ 如 SRaff，他的浦）洗掉板的殖民地。用血清学移液器和无菌敷料木从板松开殖民地。转移的液体洗涤至50ml锥形管中，涡旋彻底分离的细胞中的任何团块。稀释至轻微浑浊的文化，外径600〜0.025。 注：这里的棉子糖的作用是缓解诱导葡萄糖介导的抑制的细胞，从而引发细胞的半乳糖介导的诱导。 </li><li>振荡在3一夜长大稀释文化，棉子糖辍学媒体0ºC。成长WT的Hsp104和矢量控制并行。 </li><li>第二天早晨，制版浓度范围到个别半乳糖（ 例如 SGal-他浦）的板（1微升至2毫升浓培养于400μl总体积）。镀了广泛的浓度范围内可以保证一板将具有单个菌落。所需的实际浓度取决于所关注的疾病衬底的毒性。 </li><li>归一化矢量和的Hsp104 野生型培养物到库中的OD 600和板等体积的比较相对于所述控件库的增长。 </li><li>为了评估选择严格，板块葡萄糖库（抑制）的媒体。孵育平板2-3天，在30°C以上，直至菌落出现（ 图2）。评估所产生的菌落并比较对照。 注：通常大型和小型的殖民地获得，但菌落大小和行为之间无相关性ivity观察。筛选使用5-FOA计数器选择技术（步骤4），以尽量减少结转测序误报这些潜在的命中。 </li></ol> 4. 5-FOA反选择和定位，以消除误报<ol><li>连胜出单菌落重复到双人和单人辍学介质（ 如 SD-他的浦和SD-他的车牌），并在30ºC成长过夜。重复使用矢量和WT的Hsp104控制。镀上的SD-他之前的5-FOA，增加的可能性，细胞就会失去了质粒的Hsp104当它们被镀上5-FOA增加了5-FOA步骤的效率。 </li><li>为了防止板变干，敷SD-他的浦板块的​​封口膜，并储存在4ºC，同时进行5-FOA屏幕。这些板最终将被用于测序。 </li><li>条纹从菌落中的SD-他的板，以单菌落对5-FOA平板（YPD培养基+1克/ L的5-FOA）并孵育1-2天，在30＆＃186; C，直到单菌落出现。这里，5-FOA转化成毒性产物（5-氟脱氧尿苷）在包含URA3基因的细胞。因此，任何细胞仍窝藏质粒的Hsp104会死。 </li><li>连胜从5-FOA平板式两份的单菌落到SD浦和SD-他的板。测试3个菌落为每个命中的可能性增加，至少一个集落的每个命中会失去了的Hsp104质粒。孵育1-2天，在30℃下。已经失去了质粒的Hsp104将生长在SD-他的板，但没有SD浦板块的殖民地。 </li><li>成长已经失去了质粒的Hsp104用于探测试验菌株。长株棉子糖辍学媒体饱和度（ 如 SRaff-他）在96深孔2毫升板过夜30ºC震荡。一定要包括5-FOA处理的对照菌株。 </li><li>准备板的斑点检测。使用多道移液器，分装200微升棉子糖辍学介质（ 如 SRaff-他）每以及一个96孔板的，保留列1和7为纯的培养物。等份加入250μl各16的饱和培养物的成列1和板7。 </li><li>连续稀释的培养物5倍到板的各列中，用多通道移液管充分混合。从柱6和12去除50微升稀释的培养物，以确保每个孔中的最终体积为200微升。这是必要的，以确保均匀的斑点。 </li><li>用96螺栓复制工具（青蛙），发现一式两份的培养到SD-他和SGal，他的板。孵育所述板在30°C以上为2-3天。 </li><li>选择用于测序显示出对SGal-他板类似于对照的毒性菌株。放弃误报菌株，但并不像控制有毒。 5-FOA也是对自己往往有毒的，所以放弃，表现出对SD-他的片生长不良或表现出显着毒性较大比单独的疾病基板株（ <strong&gt;的图3）。 </li></ol> 5.测序的变体的Hsp104菌落PCR <ol><li>从SD-他浦板刮〜10微升酵母在3微升的20mM的NaOH中的PCR条管和冻融裂解细胞。放置在-80ºC冷冻带状管10分钟，或在液氮中，然后孵育99ºC在热循环仪进行10分钟。稀释该菌株以100微升用PCR级水。 </li><li>准备PCR反应：5μLPCR模板，0.5微升100μM的引物各0.5微升10毫的dNTP，在PCR缓冲液0.25微升的DNA聚合酶，并进行多达25微升总体积PCR级水。设计引物来扩增随机区域加上两端约100bp。最小化的放大区域的尺寸，以增加成功的扩增的可能性。运行一个标准的PCR程序。 </li><li>分析通过琼脂糖凝胶电泳的样品，以确认适当的SIZ的PCR产物的扩增Ë。重复PCR，如果没有产品存在。 注：PCR失败通常是由于过多的酵母在步骤5.1被使用。 </li><li>制备样品用于DNA测序。除去PCR引物或者通过PCR纯化或使用PCR产物的清除剂，如外切核酸酶I和虾碱性磷酸酶的酶（ExoSAP-IT）的降解单链DNA。此试剂酶促降解未掺入的引物和dNTP，从而允许更高的吞吐量。 </li><li>序列的PCR产物。一定要深入分析测序图谱的基因突变。突变可以被观察到的两个核苷酸在给定的位点（ 图4）的混合物，如酵母经常怀有多种质粒。 </li></ol>

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The authors have nothing to disclose.

在这里，我们提出我们的方法来隔离的抑制用酵母proteinopathy车型疾病相关基材的毒性增效的Hsp104变种。使用这种方法，变体大文库可以筛选在高吞吐量，唯一的限制是对所有通过5-FOA二次筛选变体的数量。通过在96孔格式这些步骤，我们例行公事地检查了200个命中在5-FOA步过1-2周的课程时间。在最初的筛选步骤中求出的匹配个数将变化很大程度上取决于衬底毒性和每一特定库的组成。当测序命中，...

酵母proteinopathy模型已经开发了用于蛋白质错误折叠疾病，包括肌萎缩侧索硬化症（ALS）和帕金森氏病（PD）1-3。蛋白质TDP-43和FUS，在ALS患者其错误折叠的表达，是有毒的，mislocalize形成细胞质聚集在酵母1,2。类似地，α突触核蛋白（α-顺式），其牵涉在PD的表达，是有毒的和mislocalizes形成细胞质聚集体在酵母3。这些特性概括在表型的患者患有这些疾病4,5。因此，酵母模型提供了一个有益的平台，筛选，以防止或逆转这些表型2,6-13蛋白质或小分子。我们感兴趣的蛋白，其能够可逆聚集和毒性由于TDP-43，FUS，和α - 顺式的发展。我们注重的Hsp104，一个AAA +蛋白酵母是唯一能解聚蛋白的两个FROM无定形聚集和淀粉样蛋白在酵母中，但它没有人类同源14,15。的Hsp104被精细地调谐到分解的内源性酵母朊病毒，并具有唯一的解聚与人类神经变性疾病的底物，它从不通常遇到16,17的能力有限。因此，我们的目标是工程师的Hsp104的增强版本，它们能够efficaciously分解这些人的基板。要做到这一点，我们构建大库使用易错PCR变种的Hsp104;这些库可以利用酵母proteinopathy 17款进行筛选。我们采用了域针对性的方法来构建和筛选库，为的Hsp104是非常大的17。我们最初集中在中间域的Hsp104 17的纵向（MD），但类似的方法可以用于筛选其他领域。这些模型可以筛选disaggregase活动直接，而不是如表面显示，这是剩下的替代技术ricted用于监测结合18。 我们的协议是基于两个筛选步骤（ 图1）。首先，抑制酵母病衬底的毒性的Hsp104变体被选择。这样做，所述变体的Hsp104和疾病相关的衬底被共转化进ΔHSP104酵母。我们采用ΔHSP104酵母探索在没有野生型（WT）17的Hsp104的Hsp104序列空间。重要的是，缺失的Hsp104不影响α-顺，FUS，或TDP-43的毒性在酵母和的Hsp104 WT的表达提供了最小的救援1,13,17。酵母，然后铺于诱导培养基来诱导这两种蛋白质的表达。酵母窝藏的压制殖民地的疾病相关基板胙增长毒性变异的Hsp104。这些变体被选择用于进一步分析，而菌落保持变体不抑制毒性死亡。不过，误报是在该屏幕一个很大的问题。 TDP-43，FUS，和α - 顺式的表达是高度有毒的，它创建了对于毒性无关的Hsp104变体的自发遗传抑制器的外观强烈的选择压力被表达。因此，我们已经使用了一个辅助屏幕也相对高吞吐量，以消除这些非特异性毒性抑制器17。在此二次筛选，选择的酵母与5- Fluorootic酸（5-FOA）处理以对抗选择为的Hsp104质粒19。的菌株，然后通过点样测定，以确保所述衬底的毒性的Hsp104质粒丢失后恢复评估衬底（TDP-43，FUS，或α - 顺式）的毒性。因此，酵母，其中毒性在本二次筛选恢复推测最初显示毒性抑制因的Hsp104变体的存在。这些酵母被指定为“命中”，然后将质粒的Hsp104应回收并测序，以确定在所述的Hsp104基因17（ 图1）中的突变。任何命中应该然后通过构建突变独立地使用定点诱变，然后再测试的毒性抑制作确认。该协议的潜在应用是广泛的。使用这些方法，任何类型的蛋白质的文库可以进行筛选，即抑制任何底物蛋白质是在酵母中的有毒的毒性变异体。

<table border="0" cellpadding="0" cellspacing="0" width="766">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:320px;">Table of Specific Materials/Equipment</td>
			<td style="width:227px;"></td>
			<td style="width:132px;"></td>
			<td style="width:87px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">GeneMorphII EZClone Domain Mutagenesis Kit</td>
			<td>Agilent</td>
			<td>200552</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">150mm Petri dishes</td>
			<td>Falcon</td>
			<td>351058</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">5-Fluorootic Acid</td>
			<td>Research Products International</td>
			<td>f10501-5.0</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">96-DeepWell 2mL Plates</td>
			<td>Eppendorf</td>
			<td>0030 502.302</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">96 bold replicator tool</td>
			<td>V&#38;P Scientific</td>
			<td>vp-404</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">ExoSAP-IT</td>
			<td>Affymetrix</td>
			<td>78200</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolating potentiated hsp104 variants using yeast proteinopathy models

Many protein-misfolding disorders can be modeled in the budding yeast Saccharomyces cerevisiae. Proteins such as TDP-43 and FUS, implicated in amyotrophic lateral sclerosis, and &#945;-synuclein, implicated in Parkinson&#8217;s disease, are toxic and form cytoplasmic aggregates in yeast. These features recapitulate protein pathologies observed in patients with these disorders. Thus, yeast are an ideal platform for isolating toxicity suppressors from libraries of protein variants. We are interested in applying protein disaggregases to eliminate misfolded toxic protein conformers. Specifically, we are engineering Hsp104, a hexameric AAA+ protein from yeast that is uniquely capable of solubilizing both disordered aggregates and amyloid and returning the proteins to their native conformations. While Hsp104 is highly conserved in eukaryotes and eubacteria, it has no known metazoan homologue. Hsp104 has only limited ability to eliminate disordered aggregates and amyloid fibers implicated in human disease. Thus, we aim to engineer Hsp104 variants to reverse the protein misfolding implicated in neurodegenerative disorders. We have developed methods to screen large libraries of Hsp104 variants for suppression of proteotoxicity in yeast. As yeast are prone to spontaneous nonspecific suppression of toxicity, a two-step screening process has been developed to eliminate false positives. Using these methods, we have identified a series of potentiated Hsp104 variants that potently suppress the toxicity and aggregation of TDP-43, FUS, and &#945;-synuclein. Here, we describe this optimized protocol, which could be adapted to screen libraries constructed using any protein backbone for suppression of toxicity of any protein that is toxic in yeast.

我们已经构建了随机的中间领域的Hsp104变体库，并筛选其用于抑制TDP-43毒性。库进行转化并接种到葡萄糖和半乳糖的板（ 图2），以评估在屏幕的严格性。单菌落，选择并且该菌株，使用5-FOA消除的Hsp104质粒计数器选中。将这些菌株再评估以确认毒性是由于TDP-43的单独，没有的Hsp104变体。在初始屏幕中选择的变体的一个子集的点样分析表明，所选择的4菌落，2显示的Hsp104介导的TDP-43的?...

Watch this Scientific Journal Video about Isolating Potentiated Hsp104 Variants Using Yeast Proteinopathy Models at JoVE.com

Isolating Potentiated Hsp104 Variants Using Yeast Proteinopathy Models

Yeast proteinopathy models are valuable tools to assess the toxicity and aggregation of proteins implicated in disease. Here, we present methods for screening Hsp104 variant libraries for toxicity suppressors. This protocol could be adapted to screen any protein library for toxicity suppressors of any protein that is toxic in yeast.

隔离增效变种的Hsp104利用酵母Proteinopathy模型

Many protein-misfolding disorders can be modeled in the budding yeast Saccharomyces cerevisiae. Proteins such as TDP-43 and FUS, implicated in amyotrophic ...

isolating-potentiated-hsp104-variants-using-yeast-proteinopathy-models

Research

JoVE Journal

Biology

8.1K 次观看.  Perelman School of Medicine at the University of Pennsylvania. Yeast proteinopathy models are valuable tools to assess the toxicity and aggregation of proteins implicated in disease. Here, we present methods for screening Hsp104 variant libraries for toxicity suppressors. This protocol could be adapted to screen any protein library for toxicity suppressors of any protein that is toxic in yeast.

视频: Isolating Potentiated Hsp104 Variants Using Yeast Proteinopathy Models

A Quantitative Assessment of The Yeast Lipidome using Electrospray Ionization Mass Spectrometry

Lipids are one of the major classes of biomolecules and play important roles membrane dynamics, energy storage, and signalling1-4. The budding yeast Saccharomyces cerevisiae, a genetically and biochemically manipulable unicellular eukaryote with annotated genome and very simple lipidome, is a valuable model for studying biological functions of various lipid species in multicellular eukaryotes2,3,5. S. cerevisiae has 10 major classes of lipids with chain lengths mainly of 16 or 18 carbon atoms and either zero or one degree of unsaturation6,7. Existing methods for lipid identification and quantification - such as high performance liquid chromatography, thin-layer chromatography, fluorescence microscopy, and gas chromatography followed by MS - are well established but have low sensitivity, insufficiently separate various molecular forms of lipids, require lipid derivitization prior to analysis, or can be quite time consuming. Here we present a detailed description of our experimental approach to solve these inherent limitations by using survey-scan ESI/MS for the identification and quantification of the entire complement of lipids in yeast cells. The described method does not require chromatographic separation of complex lipid mixtures recovered from yeast cells, thereby greatly accelerating the process of data acquisition. This method enables lipid identification and quantification at the concentrations as low as g/ml and has been successfully applied to assessing lipidomes of whole yeast cells and their purified organelles. Lipids extraction from whole yeast cells for using this method of lipid analysis takes two to three hours. It takes only five to ten minutes to run each sample of extracted and dried lipids on a Q-TOF mass spectrometer equipped with a nano-electrospray source.

We describe a new quantitative lipidomics method for identifying numerous lipid species in yeast using survey-scan electrospray ionization mass spectrometry (ESI/MS). This method exceeds currently available methods for lipid identification and quantification in the ability to resolve various molecular forms of lipids, sensitivity, and speed.

一个定量评估的酵母使用电喷雾质谱法Lipidome

Lipids are one of the major classes of biomolecules and play important roles membrane dynamics, energy storage, and signalling1-4. The budding yeast ...

科研

生物学

Genetic Studies of Human DNA Repair Proteins Using Yeast as a Model System 

Understanding the roles of human DNA repair proteins in genetic pathways is a formidable challenge to many researchers. Genetic studies in mammalian systems have been limited due to the lack of readily available tools including defined mutant genetic cell lines, regulatory expression systems, and appropriate selectable markers. To circumvent these difficulties, model genetic systems in lower eukaryotes have become an attractive choice for the study of functionally conserved DNA repair proteins and pathways. We have developed a model yeast system to study the poorly defined genetic functions of the Werner syndrome helicase-nuclease (WRN) in nucleic acid metabolism. Cellular phenotypes associated with defined genetic mutant backgrounds can be investigated to clarify the cellular and molecular functions of WRN through its catalytic activities and protein interactions. The human WRN gene and associated variants, cloned into DNA plasmids for expression in yeast, can be placed under the control of a regulatory plasmid element. The expression construct can then be transformed into the appropriate yeast mutant background, and genetic function assayed by a variety of methodologies. Using this approach, we determined that WRN, like its related RecQ family members BLM and Sgs1, operates in a Top3-dependent pathway that is likely to be important for genomic stability. This is described in our recent publication [1] at <a href="http://www.impactaging.com">www.impactaging.com</a>. Detailed methods of specific assays for genetic complementation studies in yeast are provided in this paper.

Genetic Studies of Human DNA Repair Proteins Using Yeast as a Model System

Genetic studies in yeast can be employed to investigate the molecular and cellular functions of human genes in cellular DNA metabolism. Methods are described for the genetic characterization of the human WRN gene product defective in the premature aging disorder Werner syndrome in functionally conserved pathways using yeast as a tractable model system.

人类DNA修复蛋白的遗传研究利用酵母作为模型系统

Understanding the roles of human DNA repair proteins in genetic pathways is a formidable challenge to many researchers.  Genetic studies in mammalian ...

Yeast Colony Embedding Method

Patterning of different cell types in embryos is a key mechanism in metazoan development. Communities of microorganisms, such as colonies and biofilms also display patterns of cell types. For example, in the yeast S. cerevisiae, sporulated cells and pseudohyphal cells are not uniformly distributed in colonies. The functional importance of patterning and the molecular mechanisms that underlie these patterns are still poorly understood.
One challenge with respect to investigating patterns of cell types in fungal colonies is that unlike metazoan tissue, cells in colonies are relatively weakly attached to one another. In particular, fungal colonies do not contain the same extensive level of extracellular matrix found in most tissues . Here we report on a method for embedding and sectioning yeast colonies that reveals the interior patterns of cell types in these colonies. The method can be used to prepare thick sections (0.5 &mu;) useful for light microscopy and thin sections (0.1 &mu;) suitable for transmission electron microscopy. Asci and pseudohyphal cells can easily be distinguished from ovoid yeast cells by light microscopy , while the interior structure of these cells can be visualized by EM.
The method is based on surrounding colonies with agar, infiltrating them with Spurr's medium, and then sectioning. Colonies with a diameter in the range of 1-2 mm are suitable for this protocol. In addition to visualizing the interior of colonies, the method allows visualization of the region of the colony that invades the underlying agar.

A method for embedding yeast colonies allowing sectioning for light and electron microscopy. This protocol allows determination of the distribution of sporulated cells and pseudohyphal cells within colonies providing a new tool toward understanding the organization of cell types within a fungal community.

酵母殖民地嵌入方法

Patterning of  different cell types in embryos is a key mechanism in metazoan development. Communities of microorganisms, such as colonies and biofilms ...

Principles of Rodent Surgery for the New Surgeon

For both scientific and animal welfare reasons, training in basic surgical concepts and techniques should be undertaken before ever seeking to perform surgery on a rodent. Students, post-doctoral scholars, and others interested in performing surgery on rodents as part of a research protocol may not have had formal surgical training as part of their required coursework. Surgery itself is a technical skill, and one that will improve with practice. The principles of aseptic technique, however, often remain unexplained or untaught. For most new surgeons, this vital information is presented in piecemeal fashion or learned on the job, neither of which is ideal. It may also make learning how to perform a particular surgery difficult, as the new surgeon is learning both a surgical technique and the principles of asepsis at the same time. This article summarizes and makes recommendations for basic surgical skills and techniques necessary for successful rodent surgery. This article is designed to supplement hands-on training by the user's institution.

Before attempting surgery, a new surgeon should have training in basic surgical techniques and concepts. This article will present basic surgical considerations with an emphasis on rodents.

鼠类外科的原则，为新的外科医生

For both scientific and animal welfare reasons, training in basic surgical concepts and techniques should be undertaken before ever seeking to perform ...

Manual Restraint and Common Compound Administration Routes in Mice and Rats

Being able to safely and effectively restrain mice and rats is an important part of conducting research. Working confidently and humanely with mice and rats requires a basic competency in handling and restraint methods. This article will present the basic principles required to safely handle animals. One-handed, two-handed, and restraint with specially designed restraint objects will be illustrated. Often, another part of the research or testing use of animals is the effective administration of compounds to mice and rats. Although there are a large number of possible administration routes (limited only by the size and organs of the animal), most are not used regularly in research. This video will illustrate several of the more common routes, including intravenous, intramuscular, subcutaneous, and oral gavage. The goal of this article is to expose a viewer unfamiliar with these techniques to basic restraint and substance administration routes. This video does not replace required hands-on training at your facility, but is meant to augment and supplement that training.

Working safely and humanely with research rodents requires a core competency in handling and restraint methods. This article will present the basic principles required to safely handle and effectively administer compounds to mice and rats.

手动约束和常见的化合物在小鼠和大鼠的给药途径

Being able to safely and effectively restrain mice and rats is an important part of conducting research.  Working confidently and humanely with mice and ...

High-throughput Yeast Plasmid Overexpression Screen

The budding yeast, Saccharomyces cerevisiae, is a powerful model system for defining fundamental mechanisms of many important cellular processes, including those with direct relevance to human disease. Because of its short generation time and well-characterized genome, a major experimental advantage of the yeast model system is the ability to perform genetic screens to identify genes and pathways that are involved in a given process. Over the last thirty years such genetic screens have been used to elucidate the cell cycle, secretory pathway, and many more highly conserved aspects of eukaryotic cell biology 1-5. In the last few years, several genomewide libraries of yeast strains and plasmids have been generated 6-10. These collections now allow for the systematic interrogation of gene function using gain- and loss-of-function approaches 11-16. Here we provide a detailed protocol for the use of a high-throughput yeast transformation protocol with a liquid handling robot to perform a plasmid overexpression screen, using an arrayed library of 5,500 yeast plasmids. We have been using these screens to identify genetic modifiers of toxicity associated with the accumulation of aggregation-prone human neurodegenerative disease proteins. The methods presented here are readily adaptable to the study of other cellular phenotypes of interest.

Here we describe a plasmid overexpression screen in Saccharomyces cerevisiae, using an arrayed plasmid library and a high-throughput yeast transformation protocol with a liquid handling robot.

高通量酵母质粒过表达屏幕

The budding yeast, Saccharomyces cerevisiae, is a powerful model system for defining fundamental mechanisms of many important cellular processes, ...

Purification of Hsp104, a Protein Disaggregase

Hsp104 is a hexameric AAA+ protein1 from yeast, which couples ATP hydrolysis to protein disaggregation2-10 (Fig. 1). This activity imparts two key selective advantages. First, renaturation of disordered aggregates by Hsp104 empowers yeast survival after various protein-misfolding stresses, including heat shock3,5,11,12. Second, remodeling of cross-beta amyloid fibrils by Hsp104 enables yeast to exploit myriad prions (infectious amyloids) as a reservoir of beneficial and heritable phenotypic variation13-22. Remarkably, Hsp104 directly remodels preamyloid oligomers and amyloid fibrils, including those comprised of the yeast prion proteins Sup35 and Ure223-30. This amyloid-remodeling functionality is a specialized facet of yeast Hsp104. The E. coli orthologue, ClpB, fails to remodel preamyloid oligomers or amyloid fibrils26,31,32.
Hsp104 orthologues are found in all kingdoms of life except, perplexingly, animals. Indeed, whether animal cells possess any enzymatic system that couples protein disaggregation to renaturation (rather than degradation) remains unknown33-35. Thus, we and others have proposed that Hsp104 might be developed as a therapeutic agent for various neurodegenerative diseases connected with the misfolding of specific proteins into toxic preamyloid oligomers and amyloid fibrils4,7,23,36-38. There are no treatments that directly target the aggregated species associated with these diseases. Yet, Hsp104 dissolves toxic oligomers and amyloid fibrils composed of alpha-synuclein, which are connected with Parkinson's Disease23 as well as amyloid forms of PrP39. Importantly, Hsp104 reduces protein aggregation and ameliorates neurodegeneration in rodent models of Parkinson's Disease23 and Huntington's disease38. Ideally, to optimize therapy and minimize side effects, Hsp104 would be engineered and potentiated to selectively remodel specific aggregates central to the disease in question4,7. However, the limited structural and mechanistic understanding of how Hsp104 disaggregates such a diverse repertoire of aggregated structures and unrelated proteins frustrates these endeavors30,40-42.
To understand the structure and mechanism of Hsp104, it is essential to study the pure protein and reconstitute its disaggregase activity with minimal components. Hsp104 is a 102kDa protein with a pI of ~5.3, which hexamerizes in the presence of ADP or ATP, or at high protein concentrations in the absence of nucleotide43-46. Here, we describe an optimized protocol for the purification of highly active, stable Hsp104 from E. coli. The use of E. coli allows simplified large-scale production and our method can be performed quickly and reliably for numerous Hsp104 variants. Our protocol increases Hsp104 purity and simplifies His6-tag removal compared to a previous purification method from E. coli47. Moreover, our protocol is more facile and convenient than two more recent protocols26,48.

Here, we describe a protocol for the purification of highly active Hsp104, a hexameric AAA+ protein from yeast, which couples ATP hydrolysis to protein disaggregation. This scheme exploits a His6-tagged construct for affinity purification from E. coli followed by anion-exchange chromatography, His6-tag removal with TEV protease, and size-exclusion chromatography.

Hsp104纯化，蛋白质Disaggregase

Hsp104 is a hexameric AAA+ protein1 from yeast, which couples ATP hydrolysis to protein disaggregation2-10 (Fig. 1). This activity imparts two key ...

Mouse Sperm Cryopreservation and Recovery using the I&middot;Cryo Kit

Thousands of new genetically modified (GM) strains of mice have been created since the advent of transgenesis and knockout technologies. Many of these valuable animals exist only as live animals, with no backup plan in case of emergency. Cryopreservation of embryos can provide this backup, but is costly, can be a lengthy procedure, and generally requires a large number of animals for success. Since the discovery that mouse sperm can be successfully cryopreserved with a basic cryoprotective agent (CPA) consisting of 18&#37; raffinose and 3&#37; skim milk, sperm cryopreservation has become an acceptable and cost-effective procedure for archiving, distributing and recovery of these valuable strains.
 Here we demonstrate a newly developed I&bull;Cryo kit for mouse sperm cryopreservation. Sperm from five commonly-used strains of inbred mice were frozen using this kit and then recovered. Higher protection ratios of sperm motility (&gt; 60&#37;) and rapid progressive motility (&gt; 45&#37;) compared to the control (basic CPA) were seen for sperm frozen with this kit in 5 inbred mouse strains. Two cell stage embryo development after IVF with the recovered sperm was improved consistently in all 5 mouse strains examined. Over a 1.5 year period, 49 GM mouse lines were archived by sperm cryopreservation with the I&bull;Cryo kit and later recovered by IVF.

Mouse Sperm Cryopreservation and Recovery using the I&amp;middot;Cryo Kit

Here we demonstrate the newly developed I&#x2022;Cryo kit for mouse sperm cryopreservation. Two-cell stage embryo development with frozen-thawed sperm was improved consistently in 5 mouse strains with the use of this kit. Over a 1.5 year period, 49 genetically modified mouse lines were archived by sperm cryopreservation with the I&#x2022;Cryo kit and later successfully recovered by IVF.

精子冷冻保存和恢复使用I ·冷冻包

Thousands of new genetically modified (GM) strains of mice have been created since the advent of transgenesis and knockout technologies.  Many of these ...

Detection of Rare Genomic Variants from Pooled Sequencing Using SPLINTER

As DNA sequencing technology has markedly advanced in recent years2, it has become increasingly evident that the amount of genetic variation between any two individuals is greater than previously thought3. In contrast, array-based genotyping has failed to identify a significant contribution of common sequence variants to the phenotypic variability of common disease4,5. Taken together, these observations have led to the evolution of the Common Disease / Rare Variant hypothesis suggesting that the majority of the "missing heritability" in common and complex phenotypes is instead due to an individual's personal profile of rare or private DNA variants6-8. However, characterizing how rare variation impacts complex phenotypes requires the analysis of many affected individuals at many genomic loci, and is ideally compared to a similar survey in an unaffected cohort. Despite the sequencing power offered by today's platforms, a population-based survey of many genomic loci and the subsequent computational analysis required remains prohibitive for many investigators.
To address this need, we have developed a pooled sequencing approach1,9 and a novel software package1 for highly accurate rare variant detection from the resulting data. The ability to pool genomes from entire populations of affected individuals and survey the degree of genetic variation at multiple targeted regions in a single sequencing library provides excellent cost and time savings to traditional single-sample sequencing methodology. With a mean sequencing coverage per allele of 25-fold, our custom algorithm, SPLINTER, uses an internal variant calling control strategy to call insertions, deletions and substitutions up to four base pairs in length with high sensitivity and specificity from pools of up to 1 mutant allele in 500 individuals. Here we describe the method for preparing the pooled sequencing library followed by step-by-step instructions on how to use the SPLINTER package for pooled sequencing analysis (<a href="http://www.ibridgenetwork.org/wustl/splinter" target="_blank">http://www.ibridgenetwork.org/wustl/splinter</a>). We show a comparison between pooled sequencing of 947 individuals, all of whom also underwent genome-wide array, at over 20kb of sequencing per person. Concordance between genotyping of tagged and novel variants called in the pooled sample were excellent. This method can be easily scaled up to any number of genomic loci and any number of individuals. By incorporating the internal positive and negative amplicon controls at ratios that mimic the population under study, the algorithm can be calibrated for optimal performance. This strategy can also be modified for use with hybridization capture or individual-specific barcodes and can be applied to the sequencing of naturally heterogeneous samples, such as tumor DNA.

Pooled DNA sequencing is a fast and cost-effective strategy to detect rare variants associated with complex phenotypes in large cohorts. Here we describe the computational analysis of pooled, next-generation sequencing of 32 cancer-related genes using the SPLINTER software package. This method is scalable, and applicable to any phenotype of interest.

罕见的基因变异检测从集资使用斯普林特测序

As DNA sequencing technology has markedly advanced in recent years2, it has become increasingly evident that the amount of genetic variation between any ...

Screening for Functional Non-coding Genetic Variants Using Electrophoretic Mobility Shift Assay (EMSA) and DNA-affinity Precipitation Assay (DAPA)

Population and family-based genetic studies typically result in the identification of genetic variants that are statistically associated with a clinical disease or phenotype. For many diseases and traits, most variants are non-coding, and are thus likely to act by impacting subtle, comparatively hard to predict mechanisms controlling gene expression. Here, we describe a general strategic approach to prioritize non-coding variants, and screen them for their function. This approach involves computational prioritization using functional genomic databases followed by experimental analysis of differential binding of transcription factors (TFs) to risk and non-risk alleles. For both electrophoretic mobility shift assay (EMSA) and DNA affinity precipitation assay (DAPA) analysis of genetic variants, a synthetic DNA oligonucleotide (oligo) is used to identify factors in the nuclear lysate of disease or phenotype-relevant cells. For EMSA, the oligonucleotides with or without bound nuclear factors (often TFs) are analyzed by non-denaturing electrophoresis on a tris-borate-EDTA (TBE) polyacrylamide gel. For DAPA, the oligonucleotides are bound to a magnetic column and the nuclear factors that specifically bind the DNA sequence are eluted and analyzed through mass spectrometry or with a reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by Western blot analysis. This general approach can be widely used to study the function of non-coding genetic variants associated with any disease, trait, or phenotype.

Screening for Functional Non-coding Genetic Variants Using Electrophoretic Mobility Shift Assay EMSA and DNA-affinity Precipitation Assay DAPA

We present a strategic plan and protocol for identifying non-coding genetic variants affecting transcription factor (TF) DNA binding. A detailed experimental protocol is provided for electrophoretic mobility shift assay (EMSA) and DNA affinity precipitation assay (DAPA) analysis of genotype-dependent TF DNA binding.

利用电泳迁移分析（EMSA）和筛选功能的非编码基因变异DNA的亲和沉淀试验（DAPA）

Population and family-based genetic studies typically result in the identification of genetic variants that are statistically associated with a clinical ...