我们感谢：萨科Gompel和本雅明本议定书的发展所作出的贡献，以Prud&#39;homme梅利莎威廉姆斯和四个匿名评审专家对稿件的意见;研究奖学金战争的代顿大学研究生院和代顿大学生物学部和研究所（UDRI）研究TMW的支持。这项工作是由美国心脏协会授予11BGIA7280000 TMW的支持。

概述： 该视频演示了一个协议，能够定量测量果蝇（D.）果蝇的顺式调控元件（CRE）序列的基因调控活动。这个协议可以用来比较附体的监管活动：野生型和突变型的综合招聘考试形式，自然发生在一个物种，或分歧物种之间的同源CRES发现华润创业等位基因。 1。记者转基因定点整合到果蝇基因组答记者转基因建设<ol><li>华润创业作为第一步，任何评估必须单独成一个记者载体，包含（1）attB位细菌附着部位顺序为站点特定的基因整合8-11使用，（2）多克隆位点上游（3）克隆（4）编码序列的异源启动子荧光蛋白质（如EGFP或DsRed的）。 </li><li>虽然任何矢量可以兼容phiC31介导的定点整合引入attB位序列的载体骨架，载体pS3aG12 - 14可从addgene质粒库<a href="http://www.addgene.org/pgvec1">（ http://www.addgene.org/pgvec1）</a> pS3aG是一个定制版本的P -型转变vector15两个吉普赛绝缘体之一，其中一个SfIb绝缘体取代，它包含一个增强型的多克隆位点，并拥有一个250碱基对的序列，包含attB位的附着部位。利益CRES插入到多克隆位点上游HSP70的最小启动子和EGFP基因编码绿色荧光蛋白定位于细胞核的增强版本。 </li></ol> B.记者转基因定点整合<ol><li>对于CRES的活动是比较d为记者转基因载体的一部分，创建载体分别集成到相同的基因组的着陆点。这里phiC31噬菌体整合酶蛋白的催化（attB位）在转基因载体的细菌附着部位和基因组位于噬菌体（attP）附着部位之间的单向重组。 8-11几组有多种，包括attP网站（所谓的着陆地点），并包含一个基因组的源 phiC31 8表示在生殖细胞中这种蛋白质的转基因株系。这些飞股票可以很容易地获得布卢明顿果蝇库存中心<a href="http://flystocks.bio.indiana.edu/">（http://flystocks.bio.indiana.edu/） 。</a> </li><li>发布协议的存在，详细说明如何建立转基因果蝇网站专门使用phiC31 整合 16 。设备和技术专长，使转基因果蝇李，未列名不再需要几家厂商的存在（见表1），这项服务可以外包。 </li><li>转基因的行为，可以相差很大，从一个组织在接下来的11。因此，一个单一的 attP登陆网站，是不是所有CRES，发育阶段，和/或组织的研究分析的最佳。这是可取的，以评估在几个不同的着陆地点的利益的综合招聘考试活动，以便找到一个网站，华润创业活动为代表的内源性靶基因（S）的表达模式。 </li><li>所获得的转基因株系，最好是让他们对转基因纯合子。华润创业活动通常是在转基因的两个副本和定量测量，当务之急是比较个人之间的相同数量的转基因拷贝的人更可靠。可通过使用平衡器染色体的纯合个体。特点登陆网站虽然，它往往是小号impler收集处女男性和女性，和交叉与更加激烈的眼睛的颜色赋予迷你白色基因（图1），提供综合的转基因载体的白色突变救援是在个人更戏剧性的表型与两个向量副本。 </li></ol> 2。获取标本或组织适当的发展阶段开发时间获取标本进行分析，当利益（S）的基因表达模式（S）内源性，因此正在调查创建的时候要激活报告基因的表达发生。对于果蝇 ，这可无论是胚胎，幼虫，蛹或成人阶段。下面我们描述一个阶段成人和变质苍蝇的方法，其中后者内puparium，硬幼虫皮肤包含不动的标本，直到它作为一个成年人ecloses。 <ol class="ualpha"><li>展开转基因株系，以确保specimENS的适当阶段时，可以使用共聚焦显微镜分析报告基因的表达。设置两个小瓶，每个雄性和雌性果蝇与几个（约5 - 10）。在交替的日子里，凹凸的苍蝇从一个小瓶未使用的小瓶。重复这一个星期。两个星期结束，将转基因果蝇可用范围从幼虫到成年的发育阶段。 </li><li> 举办成蝇的期限内puparium花的时间是98小时为女性，男性为102小时，饲养在25 ° C 。成蝇，可以很容易地收集，当他们从puparium（称为羽化）出现。这个时间点，可以考虑0小时后羽化。成蝇，可以饲养，直到所需的时间点进行分析。例如，一个综合招聘考试称为MEL - OE1控制D.成人oenocytes desatF基因的表达果蝇女性不活跃后立即羽化但CRE活动SWItches后，有一天14。当获得正确的阶段，麻醉苍蝇和位于在幻灯片上的卤烃油的下降，并进行共聚焦评价。 </li><li> 分期在变态的标本 ：在puparium形成的第三龄幼虫阶段结束。虽然在这个阶段的动物在技术上仍然是一个三龄幼虫，这一发展阶段容易辨认标本作为非移动，puparium是软的，白色的，和气孔有外翻（图2A）。 Puparium形成（HAPF）0小时后，这个时间点，可以考虑。在这个阶段，男性可以从女性的杰出出现半透明各界的身体的中点附近存在双边位于性腺（盒装图2A和2A黑色箭头表示“）。 </li></ol> 1。变态的长度后，分期的基础： 在0 HAPF，标本可以被转移到一个新的小瓶或培养皿中，用湿的油漆刷。要保持干燥标本，添加了一块Kimwipe和水滋润。传输瓶或培养皿至25 ° C培养箱，直到所需的HAPF。 2。分期由可见的形态学特征： 它往往是不方便0 HAPF标本收集后，华润创业在一个特定的时间点活动的标本进行分析。另外，可以识别正确上演标本进行分析，在方便的时候，根据形态标记（详见下文），通过puparium或puparium去除后（图2）可见的存在和位置。 2A。逼近变质阶段的形态标准： <ul><li> 0 HAPF：白prepupa阶段幼虫停止不动的;前气孔外翻（图2A中的箭头。）;气管外侧可见;白嫩puparium（图2A ）。 </li><li> 〜14 HAPF：Puparium鞣制和硬化，头SAC外翻;横向气管和性腺鲜明，充分延长沿腹部的腿和翅膀，马氏管尚未突出和绿色（虚线盒装区域图2B）。眼睛是unpigmented（图2B） 。 </li><li> 〜25 HAPF：两条平行马氏管突出的颜色和绿色（盒装地区。虚线图2C）。 </li><li> 〜40 HAPF：深绿色的黄体定位之间的马氏管（图2D的红色箭头。）前结束。 </li><li> 〜50 HAPF：黄体定位在马氏管（盒装区域图2E和扩大2E“）和眼睛的中点琥珀的颜色，如果白色基因的野生型（红眼睛的颜色所需） 。眼睛颜色是缺乏在这个阶段， 当白色的突变表型是由小型的白色基因是综合记者转基因载体（图2E“）的一部分救出。 </li><li><strONG&gt;〜70 HAPF：（图盒装地区2F盒装和扩大2F“）背胸microchaetae macrochaetae可见（图2F，箭头）定位在马氏管后的黄体;眼睛的颜色是苍白无力的的粉红色的迷你白色基因（图2F“）救出时。 </li><li> 〜80 HAPF：翼尖灰色;位于前腹部的中点（图2G和扩大在2G盒装地区“）之间的马氏管;腹节上腹部tergites刷毛之间的折叠可见（图2G 2G“）;并救出眼睛的颜色是鲜艳的红色（图2G）。 </li><li> 〜90 HAPF：翼黑暗为黑色;马氏管和黄体tergites鞣遮蔽;胸（箭头）和腹部刷毛成熟和黑暗（图2H）;和绿色胎便出现在腹部背后提示（图。2H“）。 </li></ul>注： <ol class="lalpha"><li>在LaTE阶段的蜕变，标本的性别可以由生殖器官的形态（图2I和2J箭头）或黑暗的性别梳子上的第一套男性双腿的存在。 </li><li>可以实现更加精确的分期考虑额外的描述以前17的形态标记。 </li></ol> D.步骤，消除标本从puparium： <ol><li>使用实验室磁带坚持了一块到夹层板包装胶带粘表面朝上。 </li><li>湿画笔，并适用于水分pupariated标本。使用油漆刷，转移标本到Kimwipe。 </li><li>使用产钳或画笔，转移标本包装胶带，并坚持与背表面朝上（puparium弯曲侧）。 </li><li>允许标本干〜15分钟。干puparium更容易比那些潮湿的开拓。在这一点上的标本可以粗略地上演由形态通过的puparium可见的标记。 </li><li>使用镊子，逐步开放puparium开始前鳃盖，并继续朝后结束。如果需要一个更精细的大规模清扫隔离特定的组织，这是可以做到在一个手表玻璃用PBS液。否则，转让蛹载玻片上的粘性HC700卤烃油（Sigma - Aldrich公司）下降。 </li><li>位于标本幻灯片上，使用立体显微镜，并使用上面描述的标准（第2A）标本的阶段才能确定.. </li></ol> 3。共聚焦显微镜评价报告基因的表达在整个装入样品<ol><li>对于整装标本，使用4X或10X的目标。使用刺激荧光汞灯曝光，或者在明亮的领域观看，调整显微镜阶段，在光学领域的标本位置，然后将标本成为关注的焦点。 </li><li>使用共焦microscoPE软件，调整为EGFP的激发波长（或一个合适的波长，如果使用其他荧光蛋白）。 </li><li>为了防止漂白标本，开始与激光强度的5-10％。如果信号是给人留下深刻印象，则需要加大力度。 </li><li>为了提高共聚焦图像的信噪比，使软件的设置，平均像素复制扫描测量，如卡尔曼平均线和堆栈平均。根据我们的经验卡尔曼三次扫描平均提高信噪比而不进行图像采集时间过长。 </li><li>华润创业活动的定量比较，它是必不可少的，标本的荧光强度不饱和多少像素。使用的Z -荧光出现最激烈的部分，调整使几个像素饱和设置。这可以切换到一个饱和的预警查找表和调整通道的电压，增益，偏移设置几个像素饱和。最后，以收集足够的信号从一个标本，它往往是必要的调整焦光圈。 </li><li>一旦确定了最佳设置，运行Z -扫描。 </li><li>当扫描完成后，转换成一个投影图像堆栈，保存在标签图像文件格式或“TIFF”的形象。 </li></ol> 注意： <ol type="lalpha"><li>重要的是使用相同的共聚焦设置所有复制的标本和记者转基因线，将在华​​润创业活动的定量比较。 </li></ol> 4。量化顺式调控元件活动虽然有些焦收购软件允许投影图像进行进一步的处理，我们更愿意使用免费提供的Image J软件程序来评价共聚焦图像 18 。使用图像J 18记录GFP的表达模式可以量化为一个指定的区域内的像素值统计。虽然不需要灰度图像，我们喜欢这样做。 <ol><li>与图像J计划启动后，打开TIFF图像进行评估。 </li><li>点击写意的选择按钮和轮廓面积的定量所需的标本;例如在图3中虚线的黄色边框内的区域。 （见注意以下方面选择一个区域进行定量。） </li><li>要获得一个像素值统计，单击“分析”选项卡上，然后选择措施。一个结果“框中将显示面积，像素值平均，最低和最高分数。记录像素值平均得分和重复复制标本来产生像素值统计（见注b复制数量）。 </li><li>我们建议收集第二意味着从控制地区是不活跃的综合招聘考试的像素值，例如在图3中虚线的红色边框内的区域。这后者VALUE可以从前者的价值减去去除背景效果的测量，得到调整后的平均像素值。在我们的经验，这进一步降低了复制标本（见注c控制区的大小选择）之间的变化。 </li><li>使用调整后的平均像素值复制标本，计算出一个综合招聘考试及平均值的标准误差平均的监管活动。这种监管活动的价值和测量误差可以比其他的记者外源基因在综合招聘考试的顺序不同（例如图3）。 </li></ol>注： <ol type="lalpha"><li>图像J允许选择一个定义的形状和面积，平均像素值评分将取决于用户。然而，作为试样尺寸往往不同，我们更喜欢用写意的选择工具来指定要对每个标本进行评估的面积。 </li><li>一个较大的复制数（N）的结果在一个更好的估计意味着调控latory活动对于一个给定的综合招聘考试。我们发现，4和8之间的N是一般足够。 </li><li>根据我们的经验，控制选定区域的大小影响很小测得的平均控制区域的像素值。在一般情况下，我们选择感兴趣的区域面积大小成正比。 </li></ol> 5。代表性的成果： 野生型的一个四版果蝇综合招聘考试，被称为二态元素13，驱动的EGFP记者表达女性蛹（图3A）在A6腹段的高水平。 13碱基对序列，发现在这个元素是一个DSX转录因子的结合位点， 并在体内的这种序列的重要性，可以通过量化此CRE的监管活动，这个结合位点发生突变时证明。考虑DSX的网站，这个突变的野生型二态要100元的监管活动的± 5％，减少的第ulatory活动至28 ± 3％。类似的比较，可以做的种内华润创业等位基因或从不同的物种之间的同源CRES的监管活动。例如，考虑了D.驱动EGFP的女段A6水平果蝇广小号应变调节活性为100 ± 4％，为物种的同源CRES部simulans和D 。 willistoni发现分别拥有监管活动等于75 ± 4％和0 ± 0％（图3B）。 <img alt="图1" src="/files/ftp_upload/3395/3395fig1.jpg" /> 图1：使用眼表型的白救援的力度，以使转基因株系的纯合子。为了定量评估记者转基因重要的是，每个标本具有相同的记者基因的基因型。 （一）一个白色基因突变与attP着陆点序列的遗传背景是利用网站- S<html> pecific转基因整合。 （二）转基因个体的半合子（三）为一体的综合载体的纯合子通常区分获救的眼睛集成的迷你白色基因的拷贝数的颜色表型的强度。可以建立纯合线交叉（三）男性和女性最黑暗的眼睛颜色的表型的苍蝇。 <img alt="图2" src="/files/ftp_upload/3395/3395fig2.jpg" /> 图2：使用形态标记，以确定果蝇变质阶段。从幼虫到成年果蝇的变态期间，通过一系列的刻板印象，可以用来确定性别和近似的发展阶段的形态标本过渡。他们puparium在波黑的标本已被删除。面板中的A，E，F，G和H框表示的地区分别为面板放大的“E”，F“地下”，和H“。 <img alt="&lt;/ HTML">“图3”SRC =“/ files/ftp_upload/3395/3395fig3.jpg”/&gt; 图3。定量比较顺式调控元件的活动。所有样品由一个特别的双态元素介导的EGFP报告基因的表达进行了评估，女性在80小时后puparium形成。在每个图像的顶部是A6腹段的平均像素值（左上角虚线的黄色边框内的区域形象表示）减去平均计算的样本的EGFP的表达水平为A4段的像素值（背景校正;左上角虚线的红色边框内的区域形象表示）。对于每个记者的转基因四个复制被评定为计算平均分部A6像素值。监管活动报道％的野生型（一）或（B）广小号应变女性平均像素值± SEM。 （一）绿色荧光蛋白表达对女性拥有的野生型等位基因二形元素和一个单一的DSX转录因子结合位点的突变版本烧蚀。这种突变体的结合位点减少至28 ± 3％的野生型序列二态元素活动。 （b）比较了D.序列同源监管活动果蝇 （广东小号应变）二态元素。考虑到GFP的表达介导了D 果蝇为100％的双态元素等位基因，从相关物种的同源序列部simulans和D 。 willistoni分别拥有75个活动的± 4％和0 ± 0％。 6。材料<table border="1"><tbody><tr><td> 材料名称 </td><td> 类型 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td>网站特定的基因整合</td><td>服务</td><td>最佳基因</td><td>计划H </td><td>选择一个降落地点，接收转化线</td></tr><tr><td>卤烃油</td><td>试剂</td><td> Sigma - Aldrich公司</td><td> H8898 </td><td>用于安装标本共焦成像</td></tr><tr><td>杜蒙＃5镊子</td><td>工具</td><td>精细科学工具</td><td> 11252-40 </td><td>精细指出耐用和清扫</td></tr><tr><td> SZ61变焦体视显微镜</td><td>工具</td><td>奥林巴斯</td><td>可定制</td><td>水果工作的优秀光学苍蝇</td></tr><tr><td> FluoView共聚焦显微镜</td><td>工具</td><td>奥林巴斯</td><td>可定制</td><td>提供高分辨率的共聚焦图像</td></tr></tbody></table>

<ol>
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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evo-devo+and+an+expanding+evolutionary+synthesis:+a+genetic+theory+of+morphological+evolution.">Evo-devo and an expanding evolutionary synthesis: a genetic theory of morphological evolution.</a> Cell. 134, 25-36 (2008).</li><li>Sethupathy, P., Collins, F. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNA+target+site+polymorphisms+and+human+disease.">MicroRNA target site polymorphisms and human disease.</a> Trends Genet. 24, 489-497 (2008).</li><li>Visel, A., Rubin, E. M., Pennacchio, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomic+views+of+distant-acting+enhancers.">Genomic views of distant-acting enhancers.</a> Nature. 461, 199-205 (2009).</li><li>Spradling, A. C., Rubin, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transposition+of+cloned+P+elements+into+Drosophila+germ+line+chromosomes.">Transposition of cloned P elements into Drosophila germ line chromosomes.</a> Science. 218, 341-347 (1982).</li><li>Bischof, J., Maeda, R. K., Hediger, M., Karch, F., Basler, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+optimized+transgenesis+system+for+Drosophila+using+germ-line-specific+phiC31+integrases.">An optimized transgenesis system for Drosophila using germ-line-specific phiC31 integrases.</a> Proc. Natl. Acad. Sci. U. S. A. 104, 3312-3317 (2007).</li><li>Groth, A. C., Fish, M., Nusse, R., Calos, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Construction+of+transgenic+Drosophila+by+using+the+site-specific+integrase+from+phage+phiC31.">Construction of transgenic Drosophila by using the site-specific integrase from phage phiC31.</a> Genetics. 166, 1775-1782 (2004).</li><li>Venken, K. J., He, Y., Hoskins, R. A., Bellen, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=P[acman]:+a+BAC+transgenic+platform+for+targeted+insertion+of+large+DNA+fragments+in+in+D.+melanogaster.">P[acman]: a BAC transgenic platform for targeted insertion of large DNA fragments in in D. melanogaster.</a> Science. 314, 1747-1751 (2006).</li><li>Markstein, M., Pitsouli, C., Villalta, C., Celniker, S. E., Perrimon, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Exploiting position effects and the gypsy retrovirus insulator to engineer precisely expressed transgenes. , (2008).</li><li>Rebeiz, M., Pool, J. E., Kassner, V. A., Aquadro, C. F., Carroll, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stepwise+modification+of+a+modular+enhancer+underlies+adaptation+in+a+Drosophila+population.">Stepwise modification of a modular enhancer underlies adaptation in a Drosophila population.</a> Science. 326, 1663-1667 (2009).</li><li>Williams, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+regulation+and+evolution+of+a+genetic+switch+controlling+sexually+dimorphic+traits+in+Drosophila.">The regulation and evolution of a genetic switch controlling sexually dimorphic traits in Drosophila.</a> Cell. 134, 610-623 (2008).</li><li>Shirangi, T. R., Dufour, H. D., Williams, T. M., Carroll, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+evolution+of+sex+pheromone-producing+enzyme+expression+in+Drosophila.">Rapid evolution of sex pheromone-producing enzyme expression in Drosophila.</a> PLoS biology. 7, e1000168-e1000168 (2009).</li><li>Barolo, S., Carver, L. A., Posakony, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GFP+and+beta-galactosidase+transformation+vectors+for+promoter/enhancer+analysis+in+Drosophila.">GFP and beta-galactosidase transformation vectors for promoter/enhancer analysis in Drosophila.</a> BioTechniques. 29, 726-732 (2000).</li><li>Fish, M. P., Groth, A. C., Calos, M. P., Nusse, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Creating transgenic Drosophila by microinjecting the site-specific phiC31 integrase mRNA and a transgene-containing donor plasmid. , (2007).</li><li>Ashburner, M., Golic, K. G., Hawley, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Drosophila: A laboratory handbook. , (2005).</li><li>Abramoff, M. D., Magelhaes, P. S., Ram, S. 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我们什么都没有透露。

顺式调控元件编码基因组计划，指定基因表达模式，从而发展的过程中，是两种基本形态演变2-4和5,6,19人类性状的表型变异的突变的显着位置。尽管在这方面的重要性，CRES监管逻辑仍然知之甚少。一个突出的原因，这种认识赤字一直缺乏合适的方法来定量比较CRES功能序列。在这里，我们提出了一个协议， 资本对果蝇添加一种定量方面的研究CRES改良的转基因方法。 ...

quantitative comparison of cis-regulatory element (cre) activities in transgenic drosophila melanogaster

基因表达模式指定的顺式调控元件（CRE）序列，也称为增强剂或顺式调控模块。一个典型的综合招聘考试拥有为几个转录因子的蛋白质，赋予监管的逻辑指定时，何地，在什么水平调控基因（S）表示结合位点的安排。一种动物基因组内的CRES的全套编码生物体的计划发展1和经验以及理论研究表明，在CRES突变发挥了突出的作用， 在形态进化2-4。此外，人类基因组范围关联研究表明，遗传变异，在CRES作出重大贡献的表型变异 5,6 。因此，了解监管逻辑和突变如何影响这样的逻辑，是遗传学的一个中心目标。 记者转基因提供了强大的的方法， 研究在体内功能TION的CRES。在这里，已知或可疑的综合招聘考试序列加上记者容易观察到的蛋白产物基因编码的异源启动子和编码序列。当记者转基因插入到宿主生物体，华润创业活动形式编码的记者蛋白质变成可见的。几十年来一直使用的P -元素介导转基因果蝇种果蝇（D.）果蝇 7引入这种模式生物的记者转基因，虽然转基因的基因组的位置是随机的。因此，报告基因的活性是由当地的染色质和基因环境的强烈影响，综合招聘考试比较限制被定性。近年来，phiC31集成系统被改编为在果蝇中使用转基因插入到特定的基因组的着陆点 8-10 。这种能力的基因的定量测量，以及与此有关，华润创业活动 11-13 FEasible。生产的转基因果蝇，可以外包，包括phiC31为基础的一体化，无需购买昂贵的设备和/或有能力在专门的转基因注入协议。 在这里，我们目前一般协议定量评价一个综合招聘考试的活动，并表明这种方法可以用来衡量一个综合招聘考试的活动引入突变的影响，并比较同源CRES活动。虽然给出的例子是为华润创业积极，在果蝇的变态，这种方法可以应用到其他发育阶段，果蝇物种，或模式生物。最终，一个更广泛地使用这种方法研究CRES应该提前了解监管逻辑和逻辑可以如何变化和演变。

Watch this Scientific Journal Video about Quantitative Comparison of cis-Regulatory Element CRE Activities in Transgenic Drosophila melanogaster at JoVE.com

Quantitative Comparison of cis-Regulatory Element CRE Activities in Transgenic Drosophila melanogaster

性状的表型变异可能导致的顺式调控元件（CRE）序列控制的基因表达模式的突变。在果蝇中使用派生的方法可以定量比较的空间和时间修改或自然发生的综合招聘考试的变体介导的基因表达模式的水平。

定量比较顺调节元件（CRE）的活动，在转基因果蝇</em

Gene expression patterns are specified by cis-regulatory element (CRE) sequences, which are also called enhancers or cis-regulatory modules. A typical CRE ...

quantitative-comparison-cis-regulatory-element-cre-activities

Research

JoVE Journal

Biology

Quantitative Comparison of cis-Regulatory Element (CRE) Activities in Transgenic Drosophila melanogaster

11.7K 次观看.  University of Dayton. 性状的表型变异可能导致的顺式调控元件（CRE）序列控制的基因表达模式的突变。在果蝇中使用派生的方法可以定量比较的空间和时间修改或自然发生的综合招聘考试的变体介导的基因表达模式的水平。

视频: Quantitative Comparison of cis-Regulatory Element CRE Activities in Transgenic 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 ...

Monitoring Heart Function in Larval Drosophila melanogaster for Physiological Studies

We present various methods to record cardiac function in the larval Drosophila. The approaches allow heart rate to be measured in unrestrained and restrained whole larvae. For direct control of the environment around the heart another approach utilizes the dissected larvae and removal of the internal organs in order to bathe the heart in desired compounds. The exposed heart also allows membrane potentials to be monitored which can give insight of the ionic currents generated by the myocytes and for electrical conduction along the heart tube. These approaches have various advantages and disadvantages for future experiments that are discussed. The larval heart preparation provides an additional model besides the Drosophila skeletal NMJ to investigate the role of intracellular calcium regulation on cellular function. Learning more about the underlying ionic currents that shape the action potentials in myocytes in various species, one can hope to get a handle on the known ionic dysfunctions associated to specific genes responsible for various diseases in mammals.

Monitoring Heart Function in Larval Drosophila melanogaster for Physiological Studies

We present various ways to monitor heart function in the larva of Drosophila for assessing questions dealing with the function of gap junctions, ion channel mutations, modulation of pacemaker activity and pharmacological studies.

在幼虫的监测心脏功能果蝇生理研究

We present various methods to record cardiac function in the larval Drosophila. The approaches allow heart rate to be measured in unrestrained  and  ...

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 ...

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 ...

Enhanced Northern Blot Detection of Small RNA Species in Drosophila Melanogaster

The last decades have witnessed the explosion of scientific interest around gene expression control mechanisms at the RNA level. This branch of molecular biology has been greatly fueled by the discovery of noncoding RNAs as major players in post-transcriptional regulation. Such a revolutionary perspective has been accompanied and triggered by the development of powerful technologies for profiling short RNAs expression, both at the high-throughput level (genome-wide identification) or as single-candidate analysis (steady state accumulation of specific species). Although several state-of-art strategies are currently available for dosing or visualizing such fleeing molecules, Northern Blot assay remains the eligible approach in molecular biology for immediate and accurate evaluation of RNA expression. It represents a first step toward the application of more sophisticated, costly technologies and, in many cases, remains a preferential method to easily gain insights into RNA biology. Here we overview an efficient protocol (Enhanced Northern Blot) for detecting weakly expressed microRNAs (or other small regulatory RNA species) from Drosophila melanogaster whole embryos, manually dissected larval/adult tissues or in vitro cultured cells. A very limited amount of RNA is required and the use of material from flow cytometry-isolated cells can be also envisaged.

Enhanced Northern Blot Detection of Small RNA Species in Drosophila Melanogaster

The aim of this publication is to visualize and discuss the operative steps of an Enhanced Northern Blot protocol on RNA extracted from Drosophila melanogaster embryos, cells, and tissues. This protocol is particularly useful for the efficient detection of small RNA species.

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 ...

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 ...