Name: using the gal4-uas system for functional genetics in anopheles gambiae
Uploaded: 2021-04-15T13:00:00
Description: Watch this Scientific Journal Video about Using the GAL4-UAS System for Functional Genetics in Anopheles gambiae at JoVE.com

我们非常感谢LSTM和IVCC（Adriana Adolfi），BBSRC（新研究员奖（AL），MRC（BCP：MR / P016197 / 1博士生奖学金），Wellcome（Henry Wellcome爵士博士后奖学金LG：215894 / Z / 19 / Z）的资助，这些资金已将Gal4UAS分析纳入提案中。

1. 无人机系统结构的设计和建造
<ol><li>候选基因表达载体的设计和组装
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<li>确定用于候选基因上调的序列。<ol>
<li>从感兴趣的菌株中对cDNA / gDNA进行测序，并将其与已发表的序列进行比较，以验证其身份并确定潜在的SNP和限制性位点，以进行诊断消化。</li>
			<li>确保用于基因扩增的前向引物覆盖天然Kozak序列，并在适当的情况下启动密码子。起始密码子上游约10 bp结?...

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了解蚊子的基因功能对于开发控制按蚊和影响疟疾传播的新方法至关重要。所描述的GAL4-UAS系统是一种多功能且功能强大的系统，用于候选基因的功能分析，迄今为止，我们已使用该系统来检查杀虫剂抗性17和角质层碳氢化合物生产的遗传基础15，23，以及荧光标记不同的蚊子细胞群8.到目前为止，已经通过限制?...

二分GAL4-UAS系统是昆虫模式生物果蝇中基因功能表征的主力1，2，3。为了使用GAL4-UAS系统，在调节序列的控制下表达酵母转录因子GAL4的转基因驱动系与携带目的基因或RNA干扰（RNAi）构建体的响应系交叉，该响应系由GAL4识别的上游激活序列（UAS）控制。该杂交的后代以由控制GAL4表达的启动子决定的时空模式表达感兴趣的转基因（图1）。可以评估驾驶员 - 响应者杂交后代显示的表型，以阐明候选基因的功能。虽然D. melanogaster已被用于检查来自其他生物的基因4，5，6，7，但GAL4-UAS系统现已适应用于具有医学和农业重要性的昆虫，以提供对感兴趣的物种的直接分析8，9，10，11，12，13，14。
在非洲疟疾蚊子冈比亚按蚊中，GAL4-UAS系统首先通过细胞系共转染9进行测试。对多种结构在不同的成对组合中测定了效率，发现当与GAL4驱动器面板一起使用时，14个串联重复的UAS补充了一个小的人造内含子（UAS-14i），显示出最宽的活化电位。为了证明体内功能，然后使用这些构建体通过PiggyBac转化8创建两个单独的转基因冈比亚锥系：一个由中肠特异性启动子驱动的携带GAL4的驱动系，以及一个在UAS序列调节下含有荧光素酶和增强型黄色荧光蛋白（eYFP）基因的响应系。肠道特异性荧光素酶活性和后代荧光表明该系统在按蚊中是有效的。从那时起，已经创建了驱动系，以接近无处不在的模式10表达对载体能力和杀虫剂耐药性很重要的其他组织中表达转基因。还产生了许多UAS系，以测定被认为参与代谢和封存介导的杀虫剂耐药性，角质层碳氢化合物合成以及荧光标记不同细胞和组织类型的基因（表1）。对于响应者系，现在通过ΦC31催化的重组盒交换17，18进行转基因的位点导向整合，以固定UAS调节基因的基因组上下文。通过这种方式，转基因表达在基因组插入位置方面被归一化，从而可以更准确地比较不同候选基因的表型效应。
迄今为止创造的响应细胞系被设计为在升高的水平上表达转基因或通过RNA干扰（RNAi）减少基因表达。 通常，cDNA克隆被融合到UAS序列中以产生合适的表达质粒，但是假设它们对于克隆来说不太大，完整的基因组序列也是可行的。为了产生沉默构建体，我们使用三种不同的方法来获得合适的串联倒置序列，这些序列形成刺激RNAi的发夹dsRNA。这些包括融合PCR，不对称PCR和发夹构建体的商业合成。每种方法的共同点是在倒置序列之间包含一个内含子序列，以提供克隆稳定性。已经开发出可插入目的基因/RNAi构建体的应答质粒15。这些质粒还携带RMCE所需的ΦC31 attB 位点（在Adolfi随附的JoVE论文中详细描述了RCME技术）。本文包含了在选择插入这些质粒之一进行过表达时所需的重要步骤的实验方案。此外，还描述了并说明了用于RNAi发夹构建体创建的两种方案。
在创建新品系时，鉴定稀有转基因个体对于繁殖以建立和维持转基因菌落至关重要。最重要的是，对于GAL4-UAS系统，有必要区分响应者和驾驶员线，以建立交叉并识别携带两种转基因的个体后代。这是通过使用与驱动和应答盒相连的不同显性可选择标记基因来实现的。最常见的是荧光标记基因，使用滤光片（例如，eYFP，eCFP，dsRed）可以清楚地区分。重要的是，标记物以已知且可靠的时空模式表达，因为这可以更容易地识别异常和污染。荧光标记基因表达通常由合成的 3xP3 启动子调节，其在 冈比亚锥发育 的所有阶段引起眼睛和腹侧神经节特异性表达19。由 3xP3 控制的荧光标记物包含在本文描述的所有转化质粒中。本文详细介绍了用于筛选荧光 冈比亚蛹 GAL4-UAS线的常用方法的方案。
GAL4-UAS系统的关键要素之一是必须跨越差分标记的驾驶员和响应者线。为此，每条线的雄性和雌性必须在交配前分开。成虫很容易通过视觉来区分，然而，为了建立遗传杂交，在成虫出现之前将性别分开是明智的，以确保没有发生交配。雄性和雌性 冈比亚蛹 之间的一般大小差异变化太大，无法成为有效和可靠的性别确定方法20。相反，外生殖器的明显形态差异为 冈比亚羚的性行为提供了可靠的基础。在本文中，我们描述了一种可靠的方法，用于对 冈比亚蛹 进行，以建立适当的杂交。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62131/62131fig1v3.jpg"> 图1 -在冈比亚按蚊中使用二分GAL4-UAS系统的过程示意图。 （A）描述了示例载体（pSL-attB-UAS14-gyp[3xp3-eYFP]）的主要成分，详细说明了多个克隆位点中可用于插入目标基因的发夹构建体或编码序列的可用限制位点（EcoRI，NheI，XhoI和NcoI）。还描绘了对接线的结构。（B）交叉步骤图示了来自驱动线的雄性（由感兴趣的启动子携带GAL4驱动并由3xP3启动子驱动的eCFP）和来自响应线的雌性（携带由UAS启动子控制的兴趣基因或发夹构建体和由3xP3启动子控制的eYFP标记）。（C）GAL4驱动B中杂交后代中目的基因表达的图表表示，以及评估的一些典型表型的列表。缩写：多克隆位点（MCS），重组酶介导的盒交换（RMCE），上游激活序列（UAS），增强型黄色荧光蛋白（eYFP），增强型青色荧光蛋白（eCFP）。 <a href="https://www.jove.com/files/ftp_upload/62131/62131fig1v2large.jpg" target="_blank">请点击此处查看此图的放大版本。</a>
正是交叉的使用提供了GAL4-UAS系统的二分性质，与更线性的方法相比，它具有明显的优势。例如，可以评估的驱动和响应系组合比必须为每个启动子/基因组合生成和维护新的转基因系时可行得多。更重要的是，它允许分析当其表达受到干扰时产生致命或无菌表型的基因，这些基因在线性系统中难以创建/维持。这种致命的表型可以在所有发育阶段表现出来，这取决于基因功能和时空表达，但最常在胚胎发育过程中观察到。可视化蚊子胚胎发育需要清除覆盖卵子的不透明绒毛膜。按照Trpiš（1970）21 和Kaiser等人（2014）22中描述的方法，我们描述了我们用于固定胚胎的方案，同时保持结构完整性，并漂白以清除允许微观可视化和成像的内脏。

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using the gal4-uas system for functional genetics in anopheles gambiae

二分GAL4-UAS系统是功能性遗传分析的多功能且功能强大的工具。该系统的本质是穿越以组织特异性方式表达酵母转录因子GAL4的转基因"驱动"系，转基因"响应者"系携带候选基因/RNA干扰构建体，其表达由结合GAL4的上游激活序列（UAS）控制。在随后的后代中，基因或沉默构建体因此以规定的时空方式表达，从而能够测定所得的表型并推断基因功能。二元系统使实验方法能够灵活地筛选由多种组织特异性模式的转基因表达产生的表型，即使引起严重的适应性成本。我们针对非洲主要疟疾病媒冈 比亚按蚊调整了这一系统。
在本文中，我们提供了GAL4-UAS分析期间使用的一些常用过程。我们描述了已经生成的 An. gambiae GAL4-UAS谱系，以及用于上调和RNAi敲低的新响应器结构的克隆。我们指定了蚊子蛹的分步指南，以建立遗传杂交，其中还包括筛选后代以跟踪标记驾驶员和响应者插入的荧光基因标记的遗传。我们还提出了一个清除 冈比亚锥胚胎 以研究胚胎发育的协议。最后，我们介绍了该方法的潜在适应性，通过CRISPR / Cas9插入靶基因下游的GAL4来产生驱动系。

eYFP，dsRed和eCFP的3xP3表达提供了可靠，易于区分的个体，这些个体具有在冈比亚蛹的眼睛和腹侧神经节中产生表达的标记基因（图3）。 图 4 突出显示了在用于的男性和女性外生殖器中观察到的差异形态以及无法识别的蛹的示例。从蛹中去除所有水分会增加性交难度，因为肛门桨模糊了生殖器的可视化（图4D，E<...

Watch this Scientific Journal Video about Using the GAL4-UAS System for Functional Genetics in Anopheles gambiae at JoVE.com

Using the GAL4-UAS System for Functional Genetics in Anopheles gambiae

二分GAL4-UAS系统是一种多功能工具，用于以受控的时空方式修饰基因表达，从而允许在 冈比亚按蚊中进行功能性遗传分析。使用该系统所描述的程序是半标准化的克隆策略，性别化和蛹荧光蛋白标志物的筛选以及胚胎固定。

使用GAL4-UAS系统在冈比亚按蚊的功能遗传学中

The bipartite GAL4-UAS system is a versatile and powerful tool for functional genetic analysis. The essence of the system is to cross transgenic 'driver' ...

二分GAL4-UAS系统是通过时空控制方式对基因表达进行遗传修饰，对冈比亚按蚊进行功能遗传分析的多功能工具。二元系统使转基因表达表型表征的实验方法具有灵活性，即使引起严重的适应性成本。该系统可用于可能涉及杀虫剂耐药性，病原体传播或用于遗传控制方法的基因的表型表征。研究技术人员Fraser Coleman将帮助演示这一程序，首先使用三毫升塑料巴斯德移液器收集蛹，末端被切断到适合与体视显微镜一起使用的透明平盘上。小心地去除蛹周围的大部分水。打开荧光灯泡，让它变暖。考虑在筛选荧光标记物时预期的表达的颜色时空模式和不同表型的比例。在荧光立体镜上选择所需的滤光片，并检查是否有一束可见的彩色光束指向载物台板的中心。在白光下，将蛹居中置于视野中，并将它们聚焦。打开白光并使用精细焦点将携带具有可见荧光图案的感兴趣表型的蛹区域聚焦。使用精细的细节画笔轻轻转动蛹并移动肛门桨，以便识别外生殖器。根据独特的外生殖器将蛹分开。雄性有一根长管，从最后的背节挤出，大约是肛门桨长度的一半。雌性蛹的外生殖器相当短且分叉。将有性化的蛹分成10个或更少的组，在透明的20毫升管中用水，并用棉絮球密封管子。将所需数量的雄性和雌性成虫从管中吸入笼子或小桶中，注意在转移过程中不要损坏成虫。在喂血前保持十字架五到七天。第二天，可以收集卵子来研究胚胎发育。组装产卵室，并小心地将10至15名血液喂养的雌性引入其中。盖上产卵室，静置 20 分钟。小心地将50毫升聚丙烯管从产卵罐中取出，确保不会释放蚊子。盖上锅盖，让鸡蛋成熟到感兴趣的发育阶段。使用精细细节画笔从锅中捡起鸡蛋，并将它们放在40平方毫米的挖掘玻璃块中的水中。用微量移液器小心地从玻璃块中取出水，并将鸡蛋盖在500微升FAA中。在室温下在轨道振荡器上轻轻振荡 30 分钟。用蒸馏水彻底冲洗鸡蛋15次，以去除FAA溶液的所有痕迹。使用1000微升微量移液管添加，然后一次除去一毫升蒸馏水，注意不要损坏鸡蛋。用一毫升Trpis溶液覆盖固定的鸡蛋，并在室温下孵育30分钟。卵在孵化五分钟后会开始形成苍白的斑块，最终在清除后达到乳白色。用蒸馏水彻底冲洗鸡蛋15次，以去除Trpis溶液的所有痕迹。在冈比亚按蚊蛹的眼睛和腹侧神经节中观察到由3xP3启动子驱动的荧光标记物的表达。使用不同颜色的荧光标记物可以选择和筛选携带不同GAL4和UAS组分的改良个体。当使用二分GAL4-UAS系统时，有必要交叉来自不同菌株的雄性和雌性，以获得携带GAL4和UAS组分的后代。在蛹的男性和女性外生殖器中观察到的差异形态用于。这里突出显示了一个无法识别的蛹的例子。如右图所示，从蛹中去除所有水分会增加性交难度，因为肛门桨模糊了生殖器的可视化。二分GAL4-UAS系统允许以受控的时空方式修饰基因表达。这可以通过将卵子细胞和无处不在的GAL4四个表达的驱动线与响应线UAS-mCherry交叉来可视化。使用二分GAL4-UAS系统的一个关键好处是即使在胚胎阶段也易于研究致命的表型。为此，有必要可视化胚胎的内部形态。在胚胎清除方案完成后，可以在产后12，24和36小时观察到胚胎发育的不同阶段的正常形态特征。在整个过程中，不允许蛹和卵干燥至关重要。因此，在去除液体时快速工作非常重要。GAL4-UAS方法使我们能够在功能上表征参与杀虫剂抗性的基因以及在基因驱动控制方法中具有潜在用途的基因。

Research

JoVE Journal

Genetics

Using the GAL4-UAS System for Functional Genetics in Anopheles gambiae

Analysis of the Ambient Particulate Matter-induced Chromosomal Aberrations Using an In Vitro System

Analysis of the Ambient Particulate Matter-induced Chromosomal Aberrations Using an In Vitro System

使用了大气颗粒物引起的染色体畸变分析<em&gt;体外</em&gt;系统

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科研

遗传学

Detection of Copy Number Alterations Using Single Cell Sequencing

单细胞测序拷贝数改变的检测

Detection of genomic changes at single cell resolution is important for characterizing genetic heterogeneity and evolution in normal tissues, cancers, and ...

Systemic Delivery of MicroRNA Using Recombinant Adeno-associated Virus Serotype 9 to Treat Neuromuscular Diseases in Rodents

重组腺相关病毒9型 MicroRNA 系统分娩治疗啮齿动物神经肌肉疾病

RNA interference via the endogenous miRNA pathway regulates gene expression by controlling protein synthesis through post-transcriptional gene silencing. ...

Determination of the Optimal Chromosomal Location(s) for a DNA Element in Escherichia coli Using a Novel Transposon-mediated Approach

Determination of the Optimal Chromosomal Locations for a DNA Element in Escherichia coli Using a Novel Transposon-mediated Approach

用新的转介导法测定大肠杆菌中 DNA 元素的最佳染色体位置 (s)

The optimal chromosomal position(s) of a given DNA element was/were determined by transposon-mediated random insertion followed by fitness selection. In ...

High Resolution Fluorescent In Situ Hybridization in Drosophila Embryos and Tissues Using Tyramide Signal Amplification

High Resolution Fluorescent In Situ Hybridization in Drosophila Embryos and Tissues Using Tyramide Signal Amplification

高分辨率荧光原位杂交在果蝇胚胎和组织中使用 Tyramide 信号放大

In our efforts to determine the patterns of expression and subcellular localization of Drosophila RNAs on a genome-wide basis, and in a variety of ...

CRISPR-Mediated Reorganization of Chromatin Loop Structure

CRISPR 的染色质环结构重组

Recent studies have clearly shown that long-range, three-dimensional chromatin looping interactions play a significant role in the regulation of gene ...

Measuring Exercise Levels in Drosophila melanogaster Using the Rotating Exercise Quantification System (REQS)

Measuring Exercise Levels in Drosophila melanogaster Using the Rotating Exercise Quantification System REQS

使用旋转运动量化系统测量果蝇中的运动水平 (满足要求)

Drosophila melanogaster is a new model organism for studies in exercise biology. To date, two main exercise systems, the Power Tower and the Treadwheel ...

Characterizing Histone Post-translational Modification Alterations in Yeast Neurodegenerative Proteinopathy Models

酵母神经退行性蛋白病模型中组蛋白后转化修饰改变的表征

Neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) and Parkinson&#39;s disease (PD), cause the loss of hundreds of thousands of lives ...

In vivo Application of the REMOTE-control System for the Manipulation of Endogenous Gene Expression

remtoe 控制系统在内源基因表达调控中的应用

Here we describe a protocol for implementing the REMOTE-control system (Reversible Manipulation of Transcription at Endogenous loci), which allows for ...

In Vivo Functional Study of Disease-associated Rare Human Variants Using Drosophila

In Vivo Functional Study of Disease-associated Rare Human Variants Using Drosophila

在Vivo功能研究的疾病相关的稀有人类变异使用果蝇

Advances in sequencing technology have made whole-genome and whole-exome datasets more accessible for both clinical diagnosis and cutting-edge human ...