这项工作是由国家研究所德拉桑特等德拉RECHERCHEMédicale的支持下，该中心的法国国家科学研究中，圣圣斯特拉斯堡总医院斯特拉斯堡，协会倒拉RECHERCHE河畔勒癌，国立研究所杜癌症，法新社的国立德拉RECHERCHE和地区阿尔萨斯。 彼得拉内韦一直支持基金会倒拉RECHERCHEMédicale。目前，他是一个因诺琴蒂基金会意大利语TECNOLOGIA（IIT）的奖学金的获得者。出版费用由Neurex网络支持（TriNeuron - 计划区域间四上莱茵省）

1，样品采集<ol><li>分离半粘合施耐德2细胞（1-5×10 6个细胞的平均值），通过移液并通过离心（100×g离心1分钟）收获它们。如果在体外培养的贴壁细胞，trypsinize他们在标准条件下，或者直接从板上将它们收集到的RNA提取试剂的便利量，用细胞刮刀的帮助。 </li><li>对于胚胎收集，成长果蝇株苍蝇笼子里，让胚胎积聚在产蛋板。在蒸馏水画笔（DH 2 O） 的胚胎擦拭掉，丢弃杂物用筛子过滤，冲洗和一小瓶48收回胚胎。 </li><li>作为一种替代技术，手动解剖组织/器官。在立体显微镜下（放大20倍）隔离腹果蝇睾丸（在piRNA基因分析的优惠制度）的磷酸盐缓冲液（PBS），用解剖针或镊子作为VISualized沙利文等 49。 </li><li>杵匀化后的0.5容积RNA提取试剂瘾样本。 </li></ol> 2，RNA提取/分析<ol><li>按照厂家的说明书，通过商业化的试剂进行RNA分离。 小心！RNA提取试剂通常毒性和刺激性。可替换地，应用的蛋白酶K的基于协议的RNA分离50，如下所示： <ol><li>样品稀释到400微升停止混合，并在室温下孵育15分钟。 </li><li>回收水溶性组分通过标准的苯酚：氯仿：异戊醇和氯仿提取并用乙醇（EtOH中）沉淀/洗涤。空气干燥样品重悬成焦碳酸二乙酯（DEPC）处理双蒸（DD）H 2 O </li></ol></li><li>评估紫外分光光度法测定RNA浓度。也保证了RNA的纯度高，接近2.0，BAS版上的260/280的阅读。 </li><li>检查RNA制备的变性琼脂糖凝胶如下： <ol><li>在一个干净的烧杯中，溶解1.2克琼脂糖82毫升DEPC-DDH 2 O，在不断搅拌下。煮至完全溶解。 </li><li>允许凝胶溶液冷却下来;当温度达到60℃，加上10×4吗啉酸10毫升（MOPS）缓冲液1X终浓度（FC）和8毫升37甲醛3％至％FC。 小心！甲醛是一种致癌物质。 </li><li>倒胶至水平电泳槽。使凝胶的发动机罩下固化至少1小时。下面凝固，淹没凝胶进入公开资讯观测站1X。 </li><li>分装2微克RNA​​样品和1微克天梯（使用一种RNA标记时，准确的评估需要）。加入3倍体积的琼脂糖样染料（ALD）的RNA和阶梯。热火在70℃下5分钟，立即在冰凉凉的。 加利福尼亚UTION！ALD的内容（溴化乙锭和甲酰胺，见材料）的诱变剂和腐蚀性，分别为。 </li><li>装入样品和运行凝胶为50 V与偶尔的缓冲回收约1小时。检查用UV可视化或凝胶成像RNA的分离模式。识别相应于28S和18S个rRNA和tRNA的到离散的条带（参见图1）。 </li></ol></li><li>继续北检测，或将RNA在-80℃。 </li></ol> 3，变性聚丙烯酰胺凝胶的制备<ol><li>用于该分级步骤一个小型垂直电泳仪（板尺寸长约8厘米×9厘米，梳厚度0.75mm）组合。 </li><li>组装清洁眼镜一起在试镜架。 </li><li>制备将10ml 10％丙烯酰胺/双丙烯酰胺（19:1）在MOPS 1X溶液，7M尿素。立即浇注之前，加入100微升10％过硫酸铵及10微升TEMED，并快速混合的溶液。 小心！丙烯酰胺具有神经毒性和致癌物质。 </li><li>倒入玻璃板之间的凝胶和仔细地插入以及梳，以避免气泡。让凝胶聚合​​在RT使用前至少45分钟。另外，存储凝胶的O / N，包裹在水中浸泡滤纸和保鲜膜密封。 </li></ol> 4，样品制备<ol><li>等分0.5-20总RNA为各样品的微克。如果体积超过3-4微升，真空干燥该样品。 </li><li>对于尺寸确定，并行运行的放射性低的范围梯等分（20-30 CPS）来样订做，作为标记（见4.3和4.4的准备）。在并行处理以样品，如在步骤4.5中描述。 </li><li>排行榜：设置使用0.1微克10个基点，阶梯的磷酸盐正向反应。在37℃下30分钟，使反应停止，加入乙二胺四乙酸（EDTA）以20mM的FC。 </li><li>通过乙醇沉淀/洗衣机，空气干燥和净化悬浮在DDH 2 O（约100微升）。 小心！32 P是一个放射源。 </li><li>向每个样品中添加的丙烯酰胺蓝色染料（ABD）的等体积。通过加热变性，在80℃下进行5分钟并在冰上冷却直到加载。 小心！甲酰胺（在ABD内容）是腐蚀性的。 </li></ol> 5，电泳分级<ol><li>从铸造支撑取出玻璃板并正确组装它们中的电泳单元。以足够的电泳缓冲液（RB）填充内和外室。 </li><li>轻轻取出梳子和运行前，在200V的凝胶10分钟。装载样品前，洗去从井尿素存款通过注射器喷出一些包。 </li><li>使用平头从步骤4.5在每个孔的底部仔细分层样本。检查阳极和阴极连接，以避免倒跑。后样品进入凝胶在100伏5分钟，我现象越来越多的电压200V。 </li><li>对于约7厘米长的分馏，45分钟，长期来看是足够的。避免溴酚蓝（在ABD跟踪颜料）溢出的胶。 </li><li>可视化的凝胶分级分离的长的RNA通过溴化乙锭（EtBr的）染色，作为一个可选的步骤： <ol><li>切开2厘米的凝胶的顶部，并简要冲洗在DDH 2 O的片</li><li>孵育凝胶切片在室温下进行15分钟，在RB，EtBr的0.5微克/毫升的FC温和正遨游。 </li><li>脱色在RB中的凝胶15分钟，可视化长个rRNA通过UV照射或凝胶成像。检查RNA的装载和质量（ 见图1，面板7）。 小心！溴化乙锭是一种诱变剂。 </li></ol></li><li>同时继续进行印迹含有小RNA种类的凝胶的下部。 </li></ol> 6，凝胶印迹<ol><li>切成6片吸墨纸以适合印迹区域的和等效的一块unchargeð尼龙膜。挫伤张纸张，薄膜和在RB 2印迹垫。请务必反复挤压他们的RB完全浸泡垫。 </li><li>除去从该装置中的凝胶并用抹刀​​的平均开眼镜。用干净的切割，除去孔内。将湿滤纸纸张上胶，轻轻抬不起来。 </li><li>将尼龙膜在凝胶上的自由表面。标志着一个角落里按装样以定向筛选。完成“三明治”（3纸片每边）。卷上的印迹表面的塑料棒，以消除任何可能的气泡。 </li><li>将两个印迹垫，然后之间的杂交，在杂交盒中。组装纸盒的印迹模块中，充满RB室，并检查是否有正确的方向（尼龙膜必须凝胶和正端之间留）。 </li><li>在传输20 V 20分钟。注：为确保均匀的条件下，经过10分钟打开印迹模块和R完成转接之前otate夹心180°。 </li></ol> 7，交联膜<ol><li>制备6毫升新鲜交联溶液（XLS）。饱和一块10厘米×10公分（大于尼龙膜）一张吸水纸，XLS， 小心！盐酸（XLS的一个组成部分）具有很强的腐蚀性。 </li><li>拆下的印迹，并将膜在湿3MM滤纸。注意：RNA必须不与饱和纸直接接触。将两块玻璃板之间的过滤器和纸，敷在萨兰换行。 </li><li>加热膜在60℃2小时，接着是DDH 2 O洗涤。储存在-20°C或进行杂交。 </li></ol> 8，膜预杂交<ol><li>如果装载的RNA已检查（可选步骤5.5），直接进行杂交。否则，切有1厘米的尼龙过滤器的顶部，以避免探针跨hybridizations至分馏长的RNA。 </li><li>预热在37℃10毫升杂交液（HS）的。变性1毫克鲑鱼精子在95℃下5分钟，在冰上冷却并添加到HS。 </li><li>在杂交炉中孵育协过滤器在37℃至少1小时，下旋转。检查过滤器的RNA的侧面爆头。 </li></ol> 9探针合成<ol><li>设立磷酸正向反应在10皮摩尔的反义寡核苷酸，如4.3节。 </li><li>加入5倍体积的Tris-EDTA-NaCl的（TEN）的缓冲液来进行反应和纯化来自未掺入的核苷酸的G-25葡聚糖凝胶柱的标记的引物通过凝胶排阻色谱法（或也通过乙醇沉淀）。探头活性可以通过液体闪烁计数器进行评估。 </li></ol> 10膜杂交<ol><li>更换用尽HS采用了新的10毫升一份（准备和补充如上所述）。热火在探头95℃下进行10分钟，在冰上冷却并添加到新的HS。孵育过滤器在37℃开。 </li></ol> 11膜的清洗<ol><li>恢复的杂交溶液中，它在-20℃下储存在放射性屏蔽盒。 </li><li>在冲洗洗涤缓冲液（WB）过滤器彻底清洗它在室温下10分钟。 </li><li>用盖革 - 米勒探测器为在滤波器的角落检查剩余放射性。当背景辐射大约是5厘，进入膜的论述。 </li></ol> 12，信号检测<ol><li>暴露在放射自显影胶片或在分子成像仪屏幕上的过滤器。揭示了照相处理或数字转换信号。 </li><li>分析由专门的量化软件（ImageJ的或Optiquant）的谱带强度。 </li></ol> 13，剥膜<ol><li>要删除基色信号，淹没膜在500毫升煮沸剥离液，然后在室温下孵育1分钟。前进到新的（预）杂交或存储滤波器在-20℃。 </li></ol>

The authors declare that they have no competing financial interests.

虽然我们的阅历相互交织，通过它的RNA调节神经的升值​​不断增加，基于RNA的设计电路影响神经干细胞生物学，神经细胞的分化/功能集成，神经病变和癌症的发展机理的影响尚未被探索。这样的网络通过王国的维护使得遗传系统的潜力作为果蝇有助于揭开未开发的细胞途径，其中短的非编码RNA和转录因子被认为是主要的分子球员。在此视图中，候选小分子RNA表达水平的概要是propaedeutic到...

RNA生物化学经历了惊人的进步，在过去的1年。我们的基因表达的对照RNA的潜在的理解已经爆裂通过强大的非编码核糖调节2的识别，通过新颖的基于RNA的调节机制3,4的发现和利用已公知的转录后事件更深的表征5。总之这些研究使RNA的生物学大大使场景，成为了目前的科学景观的一个主要研究课题。特别是近年来，我们越来越的“RNA世界”的分子神经生物学6，在现代生命科学中最具活力的研究领域之一的普遍冲击感。在过去的一个世纪的最后十年，整体科学的情景已经被彻底改变了小调控RNA 9,10的RNA干扰7,8的发现和特别是关于微小RNA 11，内源性表达牵连的几乎所有细胞功能基因表达的多效性和组合调节控制的小非编码RNA。 近10年后的miRNA最初发现在秀丽隐杆线虫由安布罗斯和Ruvkun的实验室，重新关注被拒绝到外地时，在果蝇和人类细胞以及12-15确定了高数量的miRNA。从那时起，这要归功于灵活的转基因方法， 果蝇已经站在了一个宝贵的生物背景的深入研究miRNA的生物合成和活性。 果蝇的miRNA揭示了不同的功能的昆虫特异性或进化上保守的过程，老化代谢，信号转导通路跨越，行为，当然，神经发生。沿着这个方向，我们最近推出了有趣的合作一个新的链接16rrelation主基因GCM /滑翔和RNA的通路之间发生。飞转录因子GCM /滑翔17-19构成的细胞命运的决定因素，这决定了胶质细胞与神经元命运的选择多能飞的神经前体20一个独特的例子。二十年对这个话题很长的研究已经清楚地强调了基因表达调控多和重叠的投入发生了融合GCM /滑行 21-28建立所需的神经发育过程中神经的平衡和神经胶质同行之间的微妙比例的阈值水平。 我们发现，通过DMEL-miR279目标，监管是进一步控制水平有助于转录后微调的GCM /滑动式16。从全球来看，这些研究都行需要具体的方法改进：沿几年，一些技术最初开发分析TRADitional的RNA被转化为定量小的非编码RNA，等作为RNA酶保护测定，cDNA芯片29-31，实时PCR的方法32-35和测序36,37。在另一边，技术方法校准助长了持续进展的领域。 Northern印迹法（NB，或RNA凝胶印迹法）构成了一个有代表性的实例：它主要用于型材RNA的积累，因为它确保了表达水平的定量和尺寸确定。然而，该方法的特性差的灵敏度被限制时，它是被应用到低丰度的基因表达细调谐器，像短的RNA。甲不利后果是大量的总RNA，这使得难以应用到特定的生物样品的要求。对于这样的理由，小分子RNA检测特定NB变种已经发展38-40：我们采取了一种改进的NB PROC的优势edure 41（ENB，增强Northern印迹），同时阐明DMEL-miR279和GCM /滑翔之间的上述相互作用。 此方法依赖于基于碳二亚胺衍生物的活性的化学交联步骤[1 - 乙基 - 3（3-二甲基氨基丙基）碳化二亚胺，EDC]固定核酸到固体支持物。碳二亚胺是已知催化的酰胺键的活化羧基或磷酸基团和胺基团42之间形成一个多功能的交联剂。此属性可通过其单磷酸5&#39;-羟基基团被利用共价耦合的小分子RNA，以氨基基团在尼龙膜的表面上。所得的附接结构增加了固定化的核酸的可访问性，反过来，探针-靶杂交，这导致显着的检测增强43的效率。 该技术假设特定relevancE在果蝇的分子生物学研究，通过这些新颖独特的类小非编码RNA的产生是新兴的44。在这些中，rasiRNAs 45,46构成PIWI相互作用RNA（了piRNA 47），参与序列特异性基因沉默的特异性亚型。此方法的执行细节中充分描述和可视化在下文中，相对于微小RNA DMEL-miR279和DMEL-miR286的分析，并在第一时间，有rasiRNA，rasi4的。我们推走极端此方法，该方法允许我们从RNA（小于1微克）的最小量的揭示表达很差目标。

enhanced northern blot detection of small rna species in drosophila melanogaster

在过去的几十年目睹身边的基因表达调控机制的科学兴趣在RNA水平的爆炸。分子生物学的这个分支已经大大助长了非编码RNA作为主要参与者的转录后调控的发现。这样一种革命性透视一直伴随着与由强大的技术开发的仿形短的RNA表达触发，无论是在高通量水平（全基因组鉴定）或单候补分析（特定物种的稳态积累）。虽然国家的最先进的几种策略是目前可用于定量或可视化等逃离分子，RNA印迹分析仍然是分子生物学的RNA表达的即时和准确的评估资格的办法。它代表了对更复杂的，昂贵的技术，在许多情况下，应用程序的第一步，仍然是一个优先的方法来轻松地深入了解成RNA生物学。在这里，我们概述一个有效的协议（增强Northern杂交），用于检测果蝇弱表达的microRNA（或其他小调控RNA种类）全果蝇胚胎，人工解剖幼虫/成人组织或体外培养的细胞。是必需的RNA的一个非常有限的量，并利用材料从流式细胞仪，分离的细胞可以是也可以设想。

通过下面的文本中， 如图1中所描述和示意性地表示的整体过程中，我们评估不同来源的复杂的RNA样品的小RNA的表达。在图1中给出的实验中，RNA从果蝇施耐德2细胞通过蛋白酶K提取，检查其完整性分离（可选步骤：变性琼脂糖凝胶分离），并在双加载。凝胶的一半印迹在中性膜和化学交联的EDC;第二半被转移上带正电的尼龙过滤器，然后UV交联的（1.2×10 5个μ...

Watch this Scientific Journal Video about Enhanced Northern Blot Detection of Small RNA Species in Drosophila Melanogaster at JoVE.com

Enhanced Northern Blot Detection of Small RNA Species in Drosophila Melanogaster

本刊物的目的是可视化和探讨手术步骤，从果蝇的RNA提取的增强Northern杂交协议的果蝇胚胎，细胞和组织。这个协议是用于有效地检测小RNA种类是特别有用的。

增强Northern印迹检测小RNA种类的<em&gt;果蝇</em

The last decades have witnessed the explosion of scientific interest around gene expression control mechanisms at the RNA level. This branch of molecular ...

enhanced-northern-blot-detection-small-rna-species-drosophila

Research

JoVE Journal

Biology

Enhanced Northern Blot Detection of Small RNA Species in Drosophila Melanogaster

23.8K 次观看.  Institut de Génétique et de Biologie Moléculaire et Cellulaire. 本刊物的目的是可视化和探讨手术步骤，从果蝇的RNA提取的增强Northern杂交协议的果蝇胚胎，细胞和组织。这个协议是用于有效地检测小RNA种类是特别有用的。 

视频: Enhanced Northern Blot Detection of Small RNA Species in Drosophila Melanogaster

Dissection of Larval CNS in Drosophila Melanogaster

The central nervous system (CNS) of Drosophila larvae is complex and poorly understood.  One way to investigate the CNS is to use immunohistochemistry to examine the expression of various novel and marker proteins.  Staining of whole larvae is impractical because the tough cuticle prevents antibodies from penetrating inside the body cavity.  In order to stain these tissues it is necessary to dissect the animal prior to fixing and staining.  In this article we demonstrate how to dissect Drosophila larvae without damaging the CNS.  Begin by tearing the larva in half with a pair of fine forceps, and then turn the cuticle "inside-out" to expose the CNS.  If the dissection is performed carefully the CNS will remain attached to the cuticle.  We usually keep the CNS attached to the cuticle throughout the fixation and staining steps, and only completely remove the CNS from the cuticle just prior to mounting the samples on glass slides.  We also show some representative images of a larval CNS stained with Eve, a transcription factor expressed in a subset of neurons in the CNS.  The article concludes with a discussion of some of the practical uses of this technique and the potential difficulties that may arise.

In this article we demonstrate how to dissect the central nervous system from third instar Drosophila larvae.

在果蝇幼虫中枢神经系统的剖析

The central nervous system (CNS) of Drosophila larvae is complex and poorly understood.  One way to investigate the CNS is to use immunohistochemistry to ...

科研

生物学

High-Resolution Video Tracking of Locomotion in Adult Drosophila Melanogaster

Flies provide an important model for studying complex behavior due to the plethora of genetic tools available to researchers in this field. Studying locomotor behavior in Drosophila melanogaster relies on the ability to be able to quantify changes in motion during or in response to a given task. For this reason, a high-resolution video tracking system, such as the one we describe in this paper, is a valuable tool for measuring locomotion in real-time. Our protocol involves the use of an initial air pulse to break the flies momentum, followed by a thirty second filming period in a square chamber. A tracking program is then used to calculate the instantaneous speed of each fly within the chamber in 10 msec increments. Analysis software then compiles this data, and outputs a variety of parameters such as average speed, max speed, time spent in motion, acceleration, etc. This protocol will discuss proper feeding and management of flies for behavioral tasks, handling flies without anesthetization or immobilization, setting up a controlled environment, and running the assay from start to finish.

The study of complex locomotor behavior in Drosophila melanogaster is dependent upon the ability to quantify changes in a given fly's movement. This article demonstrates how to do this using a high-resolution tracking system.

高分辨率视频跟踪移动的，在成年果蝇。

Flies provide an important model for studying complex behavior due to the plethora of genetic tools available to researchers in this field.  Studying ...

Live Imaging Of Drosophila melanogaster Embryonic Hemocyte Migrations

Many studies address cell migration using in vitro methods, whereas the physiologically relevant environment is that of the organism itself. Here we present a protocol for the mounting of Drosophila melanogaster embryos and subsequent live imaging of fluorescently labeled hemocytes, the embryonic macrophages of this organism. Using the Gal4-uas system1 we drive the expression of a variety of genetically encoded, fluorescently tagged markers in hemocytes to follow their developmental dispersal throughout the embryo. Following collection of embryos at the desired stage of development, the outer chorion is removed and the embryos are then mounted in halocarbon oil between a hydrophobic, gas-permeable membrane and a glass coverslip for live imaging. In addition to gross migratory parameters such as speed and directionality, higher resolution imaging coupled with the use of fluorescent reporters of F-actin and microtubules can provide more detailed information concerning the dynamics of these cytoskeletal components.

Live Imaging Of Drosophila melanogaster Embryonic Hemocyte Migrations

Drosophila hemocytes disperse over the entirety of the developing embryo. This protocol demonstrates how to mount and image these migrations using embryos with fluorescently labelled hemocytes.

实时成像果蝇胚胎血细胞迁移

Many studies address cell migration using in vitro methods, whereas the physiologically relevant environment is that of the organism itself. Here we ...

Dissection of Oenocytes from Adult Drosophila melanogaster

In Drosophila melanogaster, as in other insects, a waxy layer on the outer surface of the cuticle, composed primarily of hydrocarbon compounds, provides protection against desiccation and other environmental challenges. Several of these cuticular hydrocarbon (CHC) compounds also function as semiochemical signals, and as such mediate pheromonal communications between members of the same species, or in some instances between different species, and influence behavior. Specialized cells referred to as oenocytes are regarded as the primary site for CHC synthesis. However, relatively little is known regarding the involvement of the oenocytes in the regulation of the biosynthetic, transport, and deposition pathways contributing to CHC output. Given the significant role that CHCs play in several aspects of insect biology, including chemical communication, desiccation resistance, and immunity, it is important to gain a greater understanding of the molecular and genetic regulation of CHC production within these specialized cells. The adult oenocytes of D. melanogaster are located within the abdominal integument, and are metamerically arrayed in ribbon-like clusters radiating along the inner cuticular surface of each abdominal segment. In this video article we demonstrate a dissection technique used for the preparation of oenocytes from adult D. melanogaster. Specifically, we provide a detailed step-by-step demonstration of (1) how to fillet prepare an adult Drosophila abdomen, (2) how to identify the oenocytes and discern them from other tissues, and (3) how to remove intact oenocyte clusters from the abdominal integument. A brief experimental illustration of how this preparation can be used to examine the expression of genes involved in hydrocarbon synthesis is included. The dissected preparation demonstrated herein will allow for the detailed molecular and genetic analysis of oenocyte function in the adult fruit fly.

Dissection of Oenocytes from Adult Drosophila melanogaster

In insects, the oenocytes produce cuticular hydrocarbon compounds. These compounds protect against desiccation and facilitate chemical communication. Here we demonstrate a dissection technique used to isolate the oenocytes from adult Drosophila melanogaster, and illustrate how this preparation can be utilized to study genes involved in hydrocarbon synthesis.

从成人Oenocytes剖析果蝇</em

In Drosophila melanogaster, as in other insects, a waxy layer on the outer surface of the cuticle, composed primarily of hydrocarbon compounds, provides ...

Isolation of Drosophila melanogaster Testes

The testes of Drosophila melanogaster provide an important model for the study of stem cell maintenance and differentiation, meiosis, and soma-germline interactions. Testes are typically isolated from adult males 0-3 days after eclosion from the pupal case. The testes of wild-type flies are easily distinguished from other tissues because they are yellow, but the testes of white mutant flies, a common genetic background for laboratory experiments are similar in both shape and color to the fly gut. Performing dissection on a glass microscope slide with a black background makes identifying the testes considerably easier. Testes are removed from the flies using dissecting needles. Compared to protocols that use forceps for testes dissection, our method is far quicker, allowing a well-practiced individual to dissect testes from 200-300 wild-type flies per hour, yielding 400-600 testes. Testes from white flies or from mutants that reduce testes size are harder to dissect and typically yield 200-400 testes per hour.

Isolation of Drosophila melanogaster Testes

Drosophila melanogaster testes can be rapidly and efficiently isolated from adult males using dissecting needles. With practice, one can readily isolate in one or two days an amount of testes sufficient for the analysis of DNA or RNA by high throughput sequencing or more traditional molecular biology methods or of protein for antibody- or enzyme-based assays.

分离果蝇睾丸

The testes of Drosophila melanogaster provide an important model for the study of stem cell maintenance and differentiation, meiosis, and soma-germline ...

A Noninvasive Hair Sampling Technique to Obtain High Quality DNA from Elusive Small Mammals

Noninvasive genetic sampling approaches are becoming increasingly important to study wildlife populations. A number of studies have reported using noninvasive sampling techniques to investigate population genetics and demography of wild populations1. This approach has proven to be especially useful when dealing with rare or elusive species2. While a number of these methods have been developed to sample hair, feces and other biological material from carnivores and medium-sized mammals, they have largely remained untested in elusive small mammals. In this video, we present a novel, inexpensive and noninvasive hair snare targeted at an elusive small mammal, the American pika (Ochotona princeps). We describe the general set-up of the hair snare, which consists of strips of packing tape arranged in a web-like fashion and placed along travelling routes in the pikas&#x2019; habitat. We illustrate the efficiency of the snare at collecting a large quantity of hair that can then be collected and brought back to the lab. We then demonstrate the use of the DNA IQ system (Promega) to isolate DNA and showcase the utility of this method to amplify commonly used molecular markers including nuclear microsatellites, amplified fragment length polymorphisms (AFLPs), mitochondrial sequences (800bp) as well as a molecular sexing marker. Overall, we demonstrate the utility of this novel noninvasive hair snare as a sampling technique for wildlife population biologists. We anticipate that this approach will be applicable to a variety of small mammals, opening up areas of investigation within natural populations, while minimizing impact to study organisms.

We present a noninvasive sampling approach to efficiently collect hair samples from elusive small mammals, as shown for the American pika. We demonstrate the utility of this method by extracting DNA from sampled hair and amplifying several types of molecular markers commonly used in studies of wildlife ecology and conservation.

一种无创性的发采样技术，以获得难以捉摸的小型兽类高质量的DNA

Noninvasive genetic sampling approaches are becoming increasingly important to study wildlife populations. A number of studies have reported using ...

Substrate Generation for Endonucleases of CRISPR/Cas Systems

The interaction of viruses and their prokaryotic hosts shaped the evolution of bacterial and archaeal life. Prokaryotes developed several strategies to evade viral attacks that include restriction modification, abortive infection and CRISPR/Cas systems. These adaptive immune systems found in many Bacteria and most Archaea consist of clustered regularly interspaced short palindromic repeat (CRISPR) sequences and a number of CRISPR associated (Cas) genes (Fig. 1) 1-3. Different sets of Cas proteins and repeats define at least three major divergent types of CRISPR/Cas systems 4. The universal proteins Cas1 and Cas2 are proposed to be involved in the uptake of viral DNA that will generate a new spacer element between two repeats at the 5' terminus of an extending CRISPR cluster 5. The entire cluster is transcribed into a precursor-crRNA containing all spacer and repeat sequences and is subsequently processed by an enzyme of the diverse Cas6 family into smaller crRNAs 6-8. These crRNAs consist of the spacer sequence flanked by a 5' terminal (8 nucleotides) and a 3' terminal tag derived from the repeat sequence 9. A repeated infection of the virus can now be blocked as the new crRNA will be directed by a Cas protein complex (Cascade) to the viral DNA and identify it as such via base complementarity10. Finally, for CRISPR/Cas type 1 systems, the nuclease Cas3 will destroy the detected invader DNA 11,12 .
These processes define CRISPR/Cas as an adaptive immune system of prokaryotes and opened a fascinating research field for the study of the involved Cas proteins. The function of many Cas proteins is still elusive and the causes for the apparent diversity of the CRISPR/Cas systems remain to be illuminated. Potential activities of most Cas proteins were predicted via detailed computational analyses. A major fraction of Cas proteins are either shown or proposed to function as endonucleases 4.
Here, we present methods to generate crRNAs and precursor-cRNAs for the study of Cas endoribonucleases. Different endonuclease assays require either short repeat sequences that can directly be synthesized as RNA oligonucleotides or longer crRNA and pre-crRNA sequences that are generated via in vitro T7 RNA polymerase run-off transcription. This methodology allows the incorporation of radioactive nucleotides for the generation of internally labeled endonuclease substrates and the creation of synthetic or mutant crRNAs. Cas6 endonuclease activity is utilized to mature pre-crRNAs into crRNAs with 5'-hydroxyl and a 2',3'-cyclic phosphate termini.

CRISPR/Cas systems mediate adaptive immunity in Bacteria and Archaea. Many Cas proteins are proposed to act as endoribonucleases acting on crRNA precursors of varying length. Here we illustrate three different approaches to generate pre-crRNA substrates for the biochemical analysis of Cas endonuclease activity.

CRISPR / CAS系统内切酶的底物生成

The  interaction of viruses and their prokaryotic hosts shaped the evolution of  bacterial and archaeal life. Prokaryotes developed several strategies to ...

Measurement of Lifespan in Drosophila melanogaster

Aging is a phenomenon that results in steady physiological deterioration in nearly all organisms in which it has been examined, leading to reduced physical performance and increased risk of disease. Individual aging is manifest at the population level as an increase in age-dependent mortality, which is often measured in the laboratory by observing lifespan in large cohorts of age-matched individuals. Experiments that seek to quantify the extent to which genetic or environmental manipulations impact lifespan in simple model organisms have been remarkably successful for understanding the aspects of aging that are conserved across taxa and for inspiring new strategies for extending lifespan and preventing age-associated disease in mammals.
The vinegar fly, Drosophila melanogaster, is an attractive model organism for studying the mechanisms of aging due to its relatively short lifespan, convenient husbandry, and facile genetics. However, demographic measures of aging, including age-specific survival and mortality, are extraordinarily susceptible to even minor variations in experimental design and environment, and the maintenance of strict laboratory practices for the duration of aging experiments is required. These considerations, together with the need to practice careful control of genetic background, are essential for generating robust measurements. Indeed, there are many notable controversies surrounding inference from longevity experiments in yeast, worms, flies and mice that have been traced to environmental or genetic artifacts1-4. In this protocol, we describe a set of procedures that have been optimized over many years of measuring longevity in Drosophila using laboratory vials. We also describe the use of the dLife software, which was developed by our laboratory and is available for download (<a href="http://sitemaker.umich.edu/pletcherlab/software">http://sitemaker.umich.edu/pletcherlab/software</a>). dLife accelerates throughput and promotes good practices by incorporating optimal experimental design, simplifying fly handling and data collection, and standardizing data analysis. We will also discuss the many potential pitfalls in the design, collection, and interpretation of lifespan data, and we provide steps to avoid these dangers.

Measurement of Lifespan in Drosophila melanogaster

Drosophila melanogaster is a powerful model organism for exploring the molecular basis of longevity regulation. This protocol will discuss the steps involved in generating a reproducible, population-based measurement of longevity as well as potential pitfalls and how to avoid them.

寿命测量<em&gt;果蝇</em

Aging is a phenomenon that results in steady physiological deterioration in nearly all organisms in which it has been examined, leading to reduced ...

Imaging Through the Pupal Case of Drosophila melanogaster

The longstanding use of Drosophila as a model for cell and developmental biology has yielded an array of tools. Together, these techniques have enabled analysis of cell and developmental biology from a variety of methodological angles. Live imaging is an emerging method for observing dynamic cell processes, such as cell division or cell motility. Having isolated mutations in uncharacterized putative cell cycle proteins it became essential to observe mitosis in situ using live imaging. Most live imaging studies in Drosophila have focused on the embryonic stages that are accessible to manipulation and observation because of their small size and optical clarity. However, in these stages the cell cycle is unusual in that it lacks one or both of the gap phases. By contrast, cells of the pupal wing of Drosophila have a typical cell cycle and undergo a period of rapid mitosis spanning about 20 hr of pupal development. It is easy to identify and isolate pupae of the appropriate stage to catch mitosis in situ. Mounting intact pupae provided the best combination of tractability and durability during imaging, allowing experiments to run for several hours with minimal impact on cell and animal viability. The method allows observation of features as small as, or smaller than, fly chromosomes. Adjustment of microscope settings and the details of mounting, allowed extension of the preparation to visualize membrane dynamics of adjacent cells and fluorescently labeled proteins such as tubulin. This method works for all tested fluorescent proteins and can capture submicron scale features over a variety of time scales. While limited to the outer 20 &#181;m of the pupa with a conventional confocal microscope, this approach to observing protein and cellular dynamics in pupal tissues in vivo may be generally useful in the study of cell and developmental biology in these tissues.

Imaging Through the Pupal Case of Drosophila melanogaster

This paper demonstrates the use of a fast scanning confocal microscope to image cell behavior directly through the puparium. By leaving the pupal case intact, this method allows observation and measurement of dynamic cell processes at a stage of Drosophila development that is difficult to study directly.

通过影像学的蛹壳<em&gt;果蝇</em

The longstanding use of Drosophila as a model for cell and developmental biology has yielded an array of tools. Together, these techniques have enabled ...

Maintenance of a Drosophila melanogaster Population Cage

Large quantities of DNA, RNA, proteins and other cellular components are often required for biochemistry and molecular biology experiments. The short life cycle of Drosophila enables collection of large quantities of material from embryos, larvae, pupae and adult flies, in a synchronized way, at a low economic cost. A major strategy for propagating large numbers of flies is the use of a fly population cage. This useful and common tool in the Drososphila community is an efficient way to regularly produce milligrams to tens of grams of embryos, depending on uniformity of developmental stage desired. While a population cage can be time consuming to set up, maintaining a cage over months takes much less time and enables rapid collection of biological material in a short period. This paper describes a detailed and flexible protocol for the maintenance of a Drosophila melanogaster population cage, starting with 1.5 g of harvested material from the previous cycle.

Maintenance of a Drosophila melanogaster Population Cage

This manuscript reports a detailed protocol for culturing, on a regular basis, a population of Drosophila melanogaster using a fly population cage.

的维持<em&gt;果蝇</em&gt;人口笼

Large quantities of DNA, RNA, proteins and other cellular components are often required for biochemistry and molecular biology experiments. The short life ...