The authors would like to thank all the members of the "Insect Genetics and Genomics" Laboratory, in particular to Lorenzo Ghiringhelli who has worked at developing, adapting and maintaining the rearing of the medfly over the past thirty years. Part of the representative results of this paper have been reprinted from N. Biotechnology, 25(1) by Scolari F. et al., Fluorescent sperm marking to improve the fight against the pest insect Ceratitis capitata (Wiedemann; Diptera: Tephritidae), 76-84, 2008, with permission from Elsevier (License number 3796240759880). This work received support from Cariplo-Regione Lombardia "IMPROVE" (FS).

1.实验装置<ol><li> 养虫要求 <ol><li>维护所有C.实蝇生命阶段在25°C，湿度65％和12/12小时的光/暗光周期。 </li><li>将约1,500-2,000地中海果蝇蛹在6升笼子。使用笼黄铜一侧网有孔小到足以刺激产卵42。插入海绵​​条通过在笼的底部的小开口通过毛细作用的手段，以提供与水蝇。使用酵母和糖（1:10）作为用于成年人（ 图1A）的食物源的混合物。提高酵母的量（高达1:3，酵母:糖）应女性不产卵。 </li><li>将充满水笼下方的板收集通过网格沉积在鸡蛋。随着处女雌可以排卵，等到苍蝇是收集卵子显微注射实验先老，至少6-7天。这将确保女性都会有交配并且该鸡蛋将已受精。 </li><li>通过用细网过滤器过​​滤的水收集鸡蛋。从过滤器转移蛋在塑料盒含有标准幼虫食物（1.5 LH 2 O，加入100ml盐酸1％，5克的广谱抗微生物剂溶解在50毫升乙醇中400克糖，175克软化水啤酒酵母，1使用巴斯德吸管公斤软麦麸）。 </li><li>放置每个方块在用网盖关闭的透明容器，让空气流通，并含有麸皮层有利于化蛹（ 图1B）。 </li><li>经过约10天，检查3 龄幼虫，从幼虫的食物出现并且跳进了化蛹麸皮层。 </li><li> 24小时后，用软刷收集了一小杯蛹并把它们转移到一个新的成人笼子。正常成人化蛹后10天出现。新兴飞使用ptilinum，充气袋位于其头顶次触角电子基，以强制关闭puparium的末尾。出苗后，ptilinum永久性崩溃后脑勺里面。 </li></ol></li><li> 1升食品准备显微注射幼虫 <ol><li>溶解2.5克琼脂在300毫升H 2 O </li><li>温馨500毫升H 2 O于热搅拌盘和化解4克苯甲酸钠，4.5毫升37％的盐酸，41克酵母提取物和115克脱水胡萝卜粉。 </li><li>添加到混合物溶于10毫升无水乙醇2.86克广谱抗微生物剂的溶液中。 </li><li>分发媒体15厘米的圆形培养皿并冷却。如果有必要，应储存于4℃。 </li></ol></li><li> 幻灯片准备 <ol><li>放置双面胶带2厘米长条在显微镜载玻片（75×26 平方毫米）和标记有中国标记的边缘，以防止油扩散和注入胚胎的随后干燥。 </li></ol></li><li><st荣&gt;核酸制品<ol><li>使用市售的试剂盒34分离质粒DNA。用乙醇沉淀质粒，并在注射缓冲液（0.1 mM磷酸盐缓冲液pH 7.4，5mM的KCl）中在所需的浓度重新悬浮。 </li><li>纯化在体外使用苯酚-氯仿原料合成长的dsRNA，沉淀用异丙醇和在注射缓冲液43重新悬浮。 </li></ol></li></ol> 2.胚芽制备<ol><li>将含有6-7日龄成虫笼下一个装满水的托盘。 </li><li>允许雌性产卵30分钟，然后通过使用一个细滤网过滤水收集胚胎。始终保持滤网的水，以避免胚胎干燥。 </li><li>通过浸没式为5秒的过滤器在商业50％漂白溶液Dechorionate胚胎。 </li><li>通过反复佛光普照清洁超纯水过滤（至少仔细清洗胚胎4-5倍）。最后，将干净的超纯水过滤。使用最多2小时内的胚胎。 注:显微注射的定时相关的必要性时细胞化之前核分裂的相位以注入胚前期胚胎，。这允许将要采取行动的注射DNA导入细胞核，具体到原始生殖细胞的细胞核，将发起的配子。在果蝇中 ，细胞化在极细胞开始在约90分钟受精后（在25℃），具有胚形成中发生约30分钟后44，而在地中海果蝇细胞化需要9和12小时45,46之间。 </li><li> 行胚胎方向 <ol><li>使用精细的画笔，收集约50胚胎，并把它们用清水浸泡黑色滤纸的磁盘上。 </li><li>在解剖立体显微镜，安排在一排的胚胎在双面胶带的上表面上用细画笔（000）的显微镜载片（ 图1C）。 注:对齐都在同一个方向的胚胎，作为喷射在后极，其中，种系将发起执行;后极是相反的珠孔区域。 </li><li>盖上三氟了一层油确保油不溢出中国的标记边界（见1.3）的胚胎。 注意:覆盖油胚胎之前，可以执行进一步的步骤:胚胎可以干燥处理几分钟，以减少它们的液体含量，从而促进注入的DNA溶液的入口。这可以帮助避免注射或注射溶液的过量泄漏期间胚胎破裂，因为高的液体压力的结果。 </li></ol></li></ol> 3.胚胎显微注射<ol><li>填充针（1-2微升）用质粒DNA或用微量的dsRNA溶液（1-2微克/微升）。 </li><lI&gt;针连接到微注射装置，其控制喷射时间（0.5秒），压力（800百帕）和背压（150帕斯卡）。 </li><li>在立体显微镜下进行的显微注射，使用显微来控制针（ 图1D）的运动。使用显微镜舞台上移动的幻灯片。 <ol><li>广场上的范围阶段的幻灯片。 </li><li>插入针的尖端进入胚胎（ 图1E）的后极。 </li><li>通过激活显微注射系统与踏板注入该溶液。注入的液体引起的内压增加，从而导致胚胎的轻微运动。 </li><li>松开踏板，马上，而是轻轻，取出胚胎针。 </li><li>观察注射部位稍微细胞质泄漏。 注:由于注射亲DNA /双链RNA / siRNA和死亡率的潜在致命影响区分cedure本身，对照实验必须执行。这些包括缓冲器的注入，或仅在反向遗传学实验的情况下，绿色荧光蛋白（GFP）的衍生双链RNA（dsRNA的-GFP），或siRNA。 </li></ol></li><li>注射后孵育胚胎在25℃。 </li></ol> 4.后喷射过程<ol><li>注射后两天，查体视显微镜下孵幼虫（ 图1F）的幻灯片。幼虫可在油中移动，但需要被尽快转移到幼虫的食物。为此，检查幻灯片，每天数次。 </li><li>使用精细的画笔（000），在孵幼虫轻轻地转移到基于胡萝卜幼虫的食物。传输最高可达每每个培养皿200幼虫。 </li><li>孵育幼虫在65％的湿度（25℃）。确保盖子不沾的菜，影响空气交换。 </li><li>保持培养皿的盖大多关闭，使得仍有空气流的幼虫。为了避免过度干燥，每天检查的菜，如果需要，加水以补偿蒸发。 </li><li>放置培养皿中在由网状盖关闭明确容器和含有麸皮层有利于化蛹。至于日常的养虫室饲养，3 龄幼虫跳出幼虫的食物和化蛹在麸皮。 </li><li>收集蛹，并放置在一个小笼子成人饲养和筛选（这是第0代，G0）。后在标准幼虫食后代。 </li><li>在种系转化实验的情况下，为您在下一代可能改变个人（G1），作为注入个人通常是嵌合体。换位事件，事实上，可能发生在任何胚胎合胞体的核，这将形成任一种系或体细胞的。 <ol><li>出苗后不久，使用二氧化碳麻醉G1个人和检查TRANSF落射荧光立体显微镜下ormation事件。为了筛选红色荧光的存在，可以使用一个DsRedwide滤波器（内线一十二分之五百四十六;电解金属锰七十五分之六百〇五），而到屏幕为绿色荧光使用EYFP滤波器（内线二十○分之五百;电解金属锰三十〇分之五百三十五） 。 </li><li>将转化个人在一个小笼子里与异性的野生型成人和发起新的初始杂，应变。 </li></ol></li></ol>

The authors declare that they have no competing financial interests.

核酸在昆虫胚胎显微注射是一个普遍的技术，该技术有利于基因的功能和生物技术应用中的两个分析。 最近从越来越多的昆虫物种的基因组序列的出版物导致为未知功能的基因的功能表征工具的迫切需要。 RNA干扰已被证明是最有价值的方法之一来推断分子功能49和胚胎显微注射利于这些研究。 质粒DNA注射可用于修改使用转座介导的基因插入昆虫?...

地中海果蝇（地中海果蝇） 地中海实蝇是一个世界性的物种，广泛破坏水果栽培作物。它属于实蝇科家族，包括若干害虫种类，如那些属于属实蝇和实蝇 。在地中海果蝇是这个家庭的研究最多的物种，它不仅已成为昆虫入侵1的研究，同时也为优化虫害治理策略2的模式。 在地中海果蝇是一种多化性品种可以攻击300余种野生和栽培植物3,4。该损害是由成年人和幼体阶段既造成:交配雌刺穿水果的表面产卵，从而使微生物影响他们的商业品质，而在果肉幼虫饲料。经过三年幼虫阶段，幼虫从主机出现，化蛹成土壤。 实蝇结球甘蓝显示一个几乎全世界发行，包括非洲，中东，澳大利亚西部，中美洲和南美洲，欧洲，和美国5的领域。 最常见的策略，以限制地中海果蝇虫害包括使用杀虫剂（ 如马拉硫磷，多杀菌素）和环境友好型昆虫不育技术（SIT）6。后一种方法涉及释放到几十万暴露进行消毒，以电离辐射的男性的野性。这样灭菌的男性野生雌性交配导致没有后代，导致种群大小的减少，最终导致消除。虽然坐在多个广告系列已被证明有效的全球，它的主要缺点包括饲养和灭菌数以百万计的昆虫被释放的高成本。发布的个人标记是必要区分期间在现场捕获的野生昆虫无菌监测活动，它是使用荧光粉末目前实现的。这些过程是昂贵的，并有不希望的副作用7。 为了优化和/或制定这种害虫的控制更加有效的办法，地中海果蝇生物学和遗传学已广泛被全球众多研究人员探讨。在地中海果蝇基因组序列8,9的可用性，将有利于对基因功能的新的调查。 RNA干扰是这类研究的强有力的工具，它可以通过双链RNA（双链RNA）或siRNA（小干扰RNA）的显微注射来实现。这一技术已被使用，例如，以证明在C中的性别决定分子级联实蝇仅部分相对于该果蝇 10的保守的。 协议的发展microinject允许C.地中海果蝇胚胎实蝇是第一非-Drosophilid蝇种进行遗传修饰。作为它的卵是相似的果蝇的，无论是在形态和性方面对干燥11中 ，协议递送质粒DNA到第一对D.开发胚前期胚胎黑 12,13 最初适合用于C.实蝇 。这些第一实验允许基于转座子米诺斯11地中海果蝇种系改造。接着，将原来的系统是使用其他基于转座子的方法改性14。这是piggyBac转从鳞翅目夜蛾 15的情况。该协议已经被进一步优化，这已经允许的其他品种tephritid 16-21的转型也有许多其他双翅目22-31的。所有这些系统依赖于使用一个典型的二元矢量/辅助质粒转化体系:人为的，有缺陷的TRANSPO含有目的基因的儿子被组装到质粒DNA并通过提供转座酶32集成到昆虫的基因组中。已经产生了许多转基因地中海果蝇线，具有多种功能，包括携带诱导杀伤力有条件的显性致死基因的菌株，株产仅限男性后代，因此不需要额外的性别鉴定的策略，并用荧光精子的菌株，这可以提高精度的SIT监测阶段33-37。虽然在转基因生物的野生释放在导频测试已发生针对蚊子仅38,39，至少一个公司正在评估一些转基因地中海果蝇菌株对它们在字段40使用。 胚胎显微注射也可以偏向的新的基因组编辑工具，例如转录活化剂 - 样效应核酸（TALENS），群集定期相互间隔短回文重复序列的发展（CRISPR）/ CRISPR相关蛋白9核酸酶（Cas9）和归巢核酸内切酶基因（HEGs），这将使新的进化和发展的研究，以及扩大现有的生物技术工具箱。基因组编辑的办法已经允许在蚊子的基因41驱动系统的产生，和他们转移到地中海果蝇是迫在眉睫。在这里，我们描述了在显微注射胚胎地中海果蝇核酸可以为上述所有应用程序非常有用的通用协议。

<table border="0" cellpadding="0" cellspacing="0" width="791">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">1 x injection Buffer&#160;</td>
			<td style="width:107px;">Buffer</td>
			<td style="width:93px;"></td>
			<td style="width:119px;"></td>
			<td style="width:257px;">0.1 mM phosphate buffer pH 7.4, 5mM KCl</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Construct Plasmid</td>
			<td style="width:107px;">DNA</td>
			<td style="width:93px;"></td>
			<td style="width:119px;"></td>
			<td style="width:257px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Helper Plasmid</td>
			<td style="width:107px;">DNA</td>
			<td style="width:93px;"></td>
			<td style="width:119px;"></td>
			<td style="width:257px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">dsRNA</td>
			<td style="width:107px;">RNA</td>
			<td style="width:93px;"></td>
			<td style="width:119px;"></td>
			<td style="width:257px;">Phenol-Chloroform purified</td>
		</tr>
		<tr height="126">
			<td height="126" style="height:126px;width:216px;">Standard Larval food</td>
			<td style="width:107px;">Rearing Food&#160;</td>
			<td style="width:93px;"></td>
			<td style="width:119px;"></td>
			<td style="width:257px;">1.5 L H2O, 100 ml HCl 1%, 5 g broad-spectrum antimicrobial agent used in pharmaceutical products&#160; dissolved in 50 ml of ethanol, 400 g sugar, 175 g demineralized brewer&#8217;s yeast, 1 kg soft wheat bran</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Carrot Larval Food</td>
			<td style="width:107px;">Rearing food</td>
			<td style="width:93px;"></td>
			<td style="width:119px;"></td>
			<td style="width:257px;">2.5 g Agar, 4 g Sodium Benzoate, 4.5 ml 37% HCl, 42 g yeast extract, 115 g carrot powder, 2.86 g broad-spectrum antimicrobial agent , water to 1L</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Adult Food</td>
			<td style="width:107px;">Rearing food</td>
			<td style="width:93px;"></td>
			<td style="width:119px;"></td>
			<td>yeast extract and sugar (1:10)&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Microscope slides</td>
			<td style="width:107px;"></td>
			<td style="width:93px;">Sigma-Aldrich</td>
			<td>Z692247</td>
			<td style="width:257px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Injection needles&#160;</td>
			<td style="width:107px;"></td>
			<td style="width:93px;">Eppendorf</td>
			<td align="right">5242956000</td>
			<td style="width:257px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Microloaders</td>
			<td style="width:107px;"></td>
			<td style="width:93px;">Eppendorf</td>
			<td align="right">5242956003</td>
			<td style="width:257px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Double slided tape</td>
			<td style="width:107px;"></td>
			<td style="width:93px;"></td>
			<td style="width:119px;"></td>
			<td style="width:257px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Whatman Black circle paper</td>
			<td style="width:107px;"></td>
			<td style="width:93px;"></td>
			<td></td>
			<td style="width:257px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Bleach</td>
			<td style="width:107px;">Generic reagent</td>
			<td style="width:93px;"></td>
			<td style="width:119px;"></td>
			<td style="width:257px;">Diluite 1:2 before use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Paintbrush (000)</td>
			<td style="width:107px;">Generic tool</td>
			<td style="width:93px;"></td>
			<td style="width:119px;"></td>
			<td style="width:257px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Micromanipulator</td>
			<td style="width:107px;">Instrument</td>
			<td style="width:93px;">Narishige</td>
			<td style="width:119px;">MN-153</td>
			<td style="width:257px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Microinjector</td>
			<td style="width:107px;">Instrument</td>
			<td style="width:93px;">Eppendorf</td>
			<td style="width:119px;">Femtojet</td>
			<td style="width:257px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Adult cages</td>
			<td style="width:107px;">Generic tool</td>
			<td style="width:93px;"></td>
			<td style="width:119px;"></td>
			<td style="width:257px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Halocarbon oil 700</td>
			<td style="width:107px;">Reagent</td>
			<td style="width:93px;">Sigma-Aldrich</td>
			<td style="width:119px;">H8898</td>
			<td style="width:257px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ceratitis capitata</td>
			<td style="width:107px;">Animal</td>
			<td style="width:93px;"></td>
			<td style="width:119px;"></td>
			<td style="width:257px;">The strain used is ISPRA</td>
		</tr>
	</tbody>
</table>

delivery of nucleic acids through embryo microinjection in the worldwide agricultural pest insect, ceratitis capitata

地中海果蝇（地中海果蝇） 地中海实蝇 （威德曼）（双翅目:实蝇科）是一种害虫具有极高的相关性农业。这是由于其繁殖行为:女性损坏的水果和蔬菜的外表面时，他们产卵和他们的纸浆孵仔鱼饲料。野生C.实蝇种群是通过传统的杀虫剂喷洒控制和/或环保的方法，最成功的是昆虫不育技术（SIT）。到SIT依赖于大规模饲养，基于辐射灭菌，并且保持它们交配能力，但不能够生成可育后代的男性字段释放。的出现和生物技术工具在随后的快速发展，与地中海果蝇基因组序列的可用性在一起，极大地增强了我们这一物种的生物学的理解。这种青睐的新战略增殖基因组操纵，其中CAN为适用于控制人口。 在这种情况下，胚胎显微注射对扩大工具箱为地中海果蝇控制的双重角色。与该调节键的生物过程的基因的功能，以干扰的能力，事实上，扩展了我们的分子机械地中海果蝇侵袭下层的理解。另外，为了达到种系转化的能力有利于生产，可以在新的SIT设置将来的现场应用进行测试的多个转基因品系。事实上，基因操作可以用来赋予能，例如，可以使用在该领域并监控育雄性能理想性状，或可能导致生命早期阶段致死。这里，我们描述的方法来microinject核酸导入地中海果蝇胚胎来实现这两个主要目标。

在这里，我们分别报告指示在感兴趣的基因的功能表征胚胎显微注射的两个应用程序（情况1）中，在（情况2）的代转基因品系的。 dsRNA的交付成胚胎解开基因的功能。 所述innexin-5基因编码，在昆虫，具体在雄性和雌性性腺43,47,48表示的间隙连接。根据可用于密切相...

Watch this Scientific Journal Video about Delivery of Nucleic Acids through Embryo Microinjection in the Worldwide Agricultural Pest Insect, Ceratitis capitata at JoVE.com

Delivery of Nucleic Acids through Embryo Microinjection in the Worldwide Agricultural Pest Insect, Ceratitis capitata

The Mediterranean fruit fly (medfly) Ceratitis capitata (Diptera: Tephritidae) is a worldwide pest of agriculture. A deeper understanding of its biology is key to control medfly populations and thus reduce economic impact. Embryo microinjection is a fundamental tool allowing both germ-line transformation and reverse genetics studies in this species.

核酸的传递通过显微注射胚胎在世界范围内的农业害虫，<em&gt;地中海实蝇</em

The Mediterranean fruit fly (medfly) Ceratitis capitata (Wiedemann) (Diptera: Tephritidae) is a pest species with extremely high agricultural relevance. ...

delivery-nucleic-acids-through-embryo-microinjection-worldwide

Research

JoVE Journal

Genetics

Delivery of Nucleic Acids through Embryo Microinjection in the Worldwide Agricultural Pest Insect, Ceratitis capitata

8.9K 次观看.  University of Pavia. The Mediterranean fruit fly (medfly) Ceratitis capitata (Diptera: Tephritidae) is a worldwide pest of agriculture. A deeper understanding of its biology is key to control medfly populations and thus reduce economic impact. Embryo microinjection is a fundamental tool allowing both germ-line transformation and reverse genetics studies in this species.

视频: Delivery of Nucleic Acids through Embryo Microinjection in the Worldwide Agricultural Pest Insect, Ceratitis capitata

The Terroir Concept Interpreted through Grape Berry Metabolomics and Transcriptomics

Terroir refers to the combination of environmental factors that affect the characteristics of crops such as grapevine (Vitis vinifera) according to particular habitats and management practices. This article shows how certain terroir signatures can be detected in the berry metabolome and transcriptome of the grapevine cultivar Corvina using multivariate statistical analysis. The method first requires an appropriate sampling plan. In this case study, a specific clone of the Corvina cultivar was selected to minimize genetic differences, and samples were collected from seven vineyards representing three different macro-zones during three different growing seasons. An untargeted LC-MS metabolomics approach is recommended due to its high sensitivity, accompanied by efficient data processing using MZmine software and a metabolite identification strategy based on fragmentation tree analysis. Comprehensive transcriptome analysis can be achieved using microarrays containing probes covering ~99% of all predicted grapevine genes, allowing the simultaneous analysis of all differentially expressed genes in the context of different terroirs. Finally, multivariate data analysis based on projection methods can be used to overcome the strong vintage-specific effect, allowing the metabolomics and transcriptomics data to be integrated and analyzed in detail to identify informative correlations.

This article describes the application of untargeted metabolomics, transcriptomics and multivariate statistical analysis to grape berry transcripts and metabolites in order to gain insight into the terroir concept, i.e., the impact of the environment on berry quality traits.

风土通过概念葡萄果实代谢组学和转录组解释的

Terroir refers to the combination of environmental factors that affect the characteristics of crops such as grapevine (Vitis vinifera) according to ...

科研

遗传学

Laser-assisted Cytoplasmic Microinjection in Livestock Zygotes

Cytoplasmic microinjection into one-cell embryos is a very powerful technique. As an example, it enables the delivery of genome editing tools that can create genetic modifications that will be present in every cell of an adult organism. It can also be used to deliver siRNA, mRNAs or blocking antibodies to study gene function in preimplantation embryos. The conventional technique for microinjecting embryos used in rodents consists of a very thin micropipette that directly penetrates the plasma membrane when advanced into the embryo. When this technique is applied to livestock animals it usually results in low efficiency. This is mainly because in contrast to mice and rats, bovine, ovine, and porcine zygotes have a very dark cytoplasm and a highly elastic plasma membrane that makes visualization during injection and penetration of the plasma membrane hard to achieve. In this protocol, we describe a suitable microinjection method for the delivery of solutions into the cytoplasm of cattle zygotes that has proved to be successful for sheep and pig embryos as well. First, a laser is used to create a hole in the zona pellucida. Then a blunt-end glass micropipette is introduced through the hole and advanced until the tip of the needle reaches about 3/4 into the embryo. Then, the plasma membrane is broken by aspiration of cytoplasmic content inside the needle. Finally, the aspirated cytoplasmic content followed by the solution of interest is injected back into the embryonic cytoplasm. This protocol has been successfully used for the delivery of different solutions into bovine and ovine zygotes with 100% efficiency, minimal lysis, and normal blastocysts development rates.

This protocol shows how to perform cytoplasmic microinjection in farm animal zygotes. This technique can be used to deliver any solution into the one-cell embryo such as genome editing tools to generate knockout animals.

激光辅助畜禽受精卵细胞质显微注射

Cytoplasmic microinjection into one-cell embryos is a very powerful technique. As an example, it enables the delivery of genome editing tools that can ...

Controllable Ion Channel Expression through Inducible Transient Transfection

Transfection, the delivery of foreign nucleic acids into a cell, is a powerful tool in protein research. Through this method, ion channels can be investigated through electrophysiological analysis, biochemical characterization, mutational studies, and their effects on cellular processes. Transient transfections offer a simple protocol in which the protein becomes available for analysis within a few hours to days. Although this method presents a relatively straightforward and time efficient protocol, one of the critical components is calibrating the expression of the gene of interest to physiological relevant levels or levels that are suitable for analysis. To this end, many different approaches that offer the ability to control the expression of the gene of interest have emerged. Several stable cell transfection protocols provide a way to permanently introduce a gene of interest into the cellular genome under the regulation of a tetracycline-controlled transcriptional activation. While this technique produces reliable expression levels, each gene of interest requires a few weeks of skilled work including calibration of a killing curve, selection of cell colonies, and overall more resources. Here we present a protocol that uses transient transfection of the Transient Receptor Potential cation channel subfamily V member 1 (TRPV1) gene in an inducible system as an efficient way to express a protein in a controlled manner which is essential in ion channel analysis. We demonstrate that using this technique, we are able to perform calcium imaging, whole cell, and single channel analysis with controlled channel levels required for each type of data collection with a single transfection. Overall, this provides a replicable technique that can be used to study ion channels structure and function.

Studying ion channels through a heterologously expressing system has become a core technique in biomedical research. In this manuscript, we present a time efficient method to achieve tightly controlled ion channel expression by performing transient transfection under the control of an inducible promoter.

通过诱导瞬时转染可控离子通道表达

Transfection, the delivery of foreign nucleic acids into a cell, is a powerful tool in protein research. Through this method, ion channels can be ...

Sequence-specific and Selective Recognition of Double-stranded RNAs over Single-stranded RNAs by Chemically Modified Peptide Nucleic Acids

RNAs are emerging as important biomarkers and therapeutic targets. Thus, there is great potential in developing chemical probes and therapeutic ligands for the recognition of RNA sequence and structure. Chemically modified Peptide Nucleic Acid (PNA) oligomers have been recently developed that can recognize RNA duplexes in a sequence-specific manner. PNAs are chemically stable with a neutral peptide-like backbone. PNAs can be synthesized relatively easily by the manual Boc-chemistry solid-phase peptide synthesis method. PNAs are purified by reverse-phase HPLC, followed by molecular weight characterization by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF). Non-denaturing polyacrylamide gel electrophoresis (PAGE) technique facilitates the imaging of the triplex formation, because carefully designed free RNA duplex constructs and PNA bound triplexes often show different migration rates. Non-denaturing PAGE with ethidium bromide post staining is often an easy and informative technique for characterizing the binding affinities and specificities of PNA oligomers. Typically, multiple RNA hairpins or duplexes with single base pair mutations can be used to characterize PNA binding properties, such as binding affinities and specificities. 2-Aminopurine is an isomer of adenine (6-aminopurine); the 2-aminopurine fluorescence intensity is sensitive to local structural environment changes, and is suitable for the monitoring of triplex formation with the 2-aminopurine residue incorporated near the PNA binding site. 2-Aminopurine fluorescence titration can also be used to confirm the binding selectivity of modified PNAs towards targeted double-stranded RNAs (dsRNAs) over single-stranded RNAs (ssRNAs). UV-absorbance-detected thermal melting experiments allow the measurement of the thermal stability of PNA-RNA duplexes and PNA&#183;RNA2 triplexes. Here, we describe the synthesis and purification of PNA oligomers incorporating modified residues, and describe biochemical and biophysical methods for characterization of the recognition of RNA duplexes by the modified PNAs.

We report the protocols for the synthesis and purification of Peptide Nucleic Acid (PNA) oligomers incorporating modified residues. The biochemical and biophysical methods for the characterization of the recognition of RNA duplexes by the modified PNAs are described.

化学修饰肽核酸对单链 rna 双链 rna 的序列特异和选择性识别

RNAs are emerging as important biomarkers and therapeutic targets. Thus, there is great potential in developing chemical probes and therapeutic ligands ...

Transient Expression of Foreign Genes in Insect Cells (sf9) for Protein Functional Assay

The transient gene expression system is one of the most important technologies for performing protein functional analysis in the baculovirus in vitro cell culture system. This system was developed to express foreign genes under the control of the baculoviral promoter in transient expression plasmids. Furthermore, this system can be applied to a functional assay of either the baculovirus itself or foreign proteins. The most widely and commercially available transient gene expression system is developed based on the immediate-early gene (IE) promoter of Orgyia pseudotsugata multicapsid nucleopolyhedrovirus (OpMNPV). However, a low expression level of foreign genes in insect cells was observed. Therefore, a transient gene expression system was constructed for improving protein expression. In this system, recombinant plasmids were constructed to contain the target sequence under the control of the Drosophila heat shock 70 (Dhsp70) promoter. This protocol presents the application of this heat shock-based pDHsp/V5-His (V5 epitope with 6 histidine)/Spodoptera frugiperda cell (sf9 cell) system; this system is available not only for gene expression but also for evaluating the anti-apoptotic activity of candidate proteins in insect cells. Furthermore, this system can be either transfected with one recombinant plasmid or co-transfected two potentially functionally antagonistic recombinant plasmids in insect cells. The protocol demonstrates the efficiency of this system and provides a practical case of this technique.

Transient Expression of Foreign Genes in Insect Cells sf9 for Protein Functional Assay

This protocol describes a heat shock-induced protein expression system (pDHsp/V5-His/sf9 cell system), which can be used for either expressing foreign proteins or evaluating the anti-apoptotic activity of potential foreign proteins and their truncated amino acids in insect cells.

昆虫细胞 (sf9) 外源基因在蛋白质功能检测中的瞬态表达

The transient gene expression system is one of the most important technologies for performing protein functional analysis in the baculovirus in vitro cell ...

Embryo Microinjection and Transplantation Technique for Nasonia vitripennis Genome Manipulation

The jewel wasp Nasonia vitripennis has emerged as an effective model system for the study of processes including sex determination, haplo-diploid sex determination, venom synthesis, and host-symbiont interactions, among others. A major limitation of working with this organism is the lack of effective protocols to perform directed genome modifications. An important part of genome modification is delivery of editing reagents, including CRISPR/Cas9 molecules, into embryos through microinjection. While microinjection is well established in many model organisms, this technique is particularly challenging to perform in N. vitripennis primarily due to its small embryo size, and the fact that embryonic development occurs entirely within a parasitized blowfly pupa. The following procedure overcomes these significant challenges while demonstrating a streamlined, visual procedure for effectively removing wasp embryos from parasitized host pupae, microinjecting them, and carefully transplanting them back into the host for continuation and completion of development. This protocol will strongly enhance the capability of research groups to perform advanced genome modifications in this organism.

Embryo Microinjection and Transplantation Technique for Nasonia vitripennis Genome Manipulation

Microinjection of Nasonia vitripennis embryos is an essential method for generating heritable genome modifications. Described here is a detailed procedure for microinjection and transplantation of Nasonia vitripennis embryos, which will greatly facilitate future genome manipulation in this organism.

金小蜂金小蜂基因组操作的胚胎显微注射和移植技术

The jewel wasp Nasonia vitripennis has emerged as an effective model system for the study of processes including sex determination, haplo-diploid sex ...

Determining the Egg Fertilization Rate of Bemisia tabaci Using a Cytogenetic Technique

A few species of sap-sucking whiteflies are some of the most damaging terrestrial pests worldwide because of the crop damage they inflict and plant viruses they vector. Despite numerous studies of the biology of these species in different environments, a key life history parameter, offspring sex ratios, has received little attention, yet is important for predicting population dynamics. The primary sex ratio (sex ratio at oviposition) of Bemisia tabaci has never been reported but can be found by determining the egg fertilization rate of this haplodiploid insect. The technique involves the dechorionation of eggs with bleach, a series of fixation steps, and the application of the general DNA fluorescent stain, DAPI (4&#8242;,6-diamidino-2-phenylindole, a DNA-binding fluorescent dye), to bind to female and male pronuclei. Here, we present the technique, and an example of its application, to test whether an endosymbiotic bacterium, Rickettsia sp. nr. bellii, influenced the primary sex ratio of B. tabaci. This method may assist in population studies of whiteflies, or in determining if sex allocation exists with certain environmental stimuli.

Determining the Egg Fertilization Rate of Bemisia tabaci Using a Cytogenetic Technique

We present a simple cytogenetic technique using 4&#8242;,6-diamidino-2-phenylindole (DAPI) to determine the fertilization rate and primary sex ratio of the haplodiploid invasive pest Bemisia tabaci.

用细胞遗传学技术测定芽孢杆菌的受精率

A few species of sap-sucking whiteflies are some of the most damaging terrestrial pests worldwide because of the crop damage they inflict and plant ...

In Vitro Biochemical Assays using Biotin Labels to Study Protein-Nucleic Acid Interactions

Protein-nucleic acid interactions play important roles in biological processes such as transcription, recombination, and RNA metabolism. Experimental methods to study protein-nucleic acid interactions require the use of fluorescent tags, radioactive isotopes, or other labels to detect and analyze specific target molecules. Biotin, a non-radioactive nucleic acid label, is commonly used in electrophoretic mobility shift assays (EMSA) but has not been regularly employed to monitor protein activity during nucleic acid processes. This protocol illustrates the utility of biotin labeling during in vitro enzymatic reactions, demonstrating that this label works well with a range of different biochemical assays. Specifically, in alignment with previous findings using radioisotope 32P-labeled substrates, it is confirmed via biotin-labeled EMSA that MEIOB (a protein specifically involved in the meiotic recombination) is a DNA-binding protein, that MOV10 (an RNA helicase) resolves biotin-labeled RNA duplex structures, and that MEIOB cleaves biotin-labeled single-stranded DNA. This study demonstrates that biotin is capable of substituting 32P in various nucleic acid-related biochemical assays in vitro.&#160;

Presented here are protocols for in vitro biochemical assays using biotin labels that may be widely&#160;applicable for studying protein-nucleic acid interactions.

使用生物素标签研究蛋白质-核酸相互作用的体外生化分析

Protein-nucleic acid interactions play important roles in biological processes such as transcription, recombination, and RNA metabolism. Experimental ...

Discrimintion and Mapping of the Primary and Processed Transcripts in Maize Mitochondrion Using a Circular RT-PCR-based Strategy

In plant mitochondria, some steady-state transcripts have 5&#39; triphosphate derived from transcription initiation (primary transcripts), while the others contain 5&#39; monophosphate generated post-transcriptionally (processed transcripts). To discriminate between the two types of transcripts, several strategies have been developed, and most of them depend on presence/absence of 5&#39; triphosphate. However, the triphosphate at primary 5&#39; termini is unstable, and it hinders a clear discrimination of the two types of transcripts. To systematically differentiate and map the primary and processed transcripts stably accumulated in maize mitochondrion, we have developed a circular RT-PCR (cRT-PCR)-based strategy by combining cRT-PCR, RNA 5&#39; polyphoshpatase treatment, quantitative RT-PCR (RT-qPCR), and Northern blot. As an improvement, this strategy includes an RNA normalization step to minimize the influence of unstable 5&#39; triphosphate.
In this protocol, the enriched mitochondrial RNA is pre-treated by RNA 5&#39; polyphosphatase, which converts 5&#39; triphsophate to monophosphate. After circularization and reverse transcription, the two cDNAs derived from 5&#39; polyphosphatase-treated and non-treated RNAs are normalized by maize 26S mature rRNA, which has a processed 5&#39; end and is insensitive to 5&#39; polyphosphatase. After normalization, the primary and processed transcripts are discriminated by comparing cRT-PCR and RT-qPCR products obtained from the treated and non-treated RNAs. The transcript termini are determined by cloning and sequencing of the cRT-PCR products, and then verified by Northern blot.
By using this strategy, most steady-state transcripts in maize mitochondrion have been determined. Due to the complicated transcript pattern of some mitochondrial genes, a few steady-state transcripts were not differentiated and/or mapped, though they were detected in a Northern blot. We are not sure whether this strategy is suitable to discriminate and map the steady-state transcripts in other plant mitochondria or in plastids.

We present a circular RT-PCR-based strategy by combining circular RT-PCR, quantitative RT-PCR, RNA 5&#39; polyphosphatase-treatment, and Northern blot. This protocol includes a normalization step to minimize the influence of unstable 5&#39; triphosphate, and it is suitable for discriminating and mapping the primary and processed transcripts stably accumulated in maize mitochondrion.

使用基于循环 RT-PCR 的策略对玉米 Mitochondrion 中初级和已处理转录的区分和映射

In plant mitochondria, some steady-state transcripts have 5&#39; triphosphate derived from transcription initiation (primary transcripts), while the ...

CRISPR-Cas9-Mediated Genome Editing in the Filamentous Ascomycete Huntiella omanensis

The CRISPR-Cas9 genome editing system is a molecular tool that can be used to introduce precise changes into the genomes of model and non-model species alike. This technology can be used for a variety of genome editing approaches, from gene knockouts and knockins to more specific changes like the introduction of a few nucleotides at a targeted location. Genome editing can be used for a multitude of applications, including the partial functional characterization of genes, the production of transgenic organisms and the development of diagnostic tools. Compared to previously available gene editing strategies, the CRISPR-Cas9 system has been shown to be easy to establish in new species and boasts high efficiency and specificity. The primary reason for this is that the editing tool uses an RNA molecule to target the gene or sequence of interest, making target molecule design straightforward, given that standard base pairing rules can be exploited. Similar to other genome editing systems, CRISPR-Cas9-based methods also require efficient and effective transformation protocols as well as access to good quality sequence data for the design of the targeting RNA and DNA molecules. Since the introduction of this system in 2013, it has been used to genetically engineer a variety of model species, including Saccharomyces cerevisiae, Arabidopsis thaliana, Drosophila melanogaster and Mus musculus. Subsequently, researchers working on non-model species have taken advantage of the system and used it for the study of genes involved in processes as diverse as secondary metabolism in fungi, nematode growth and disease resistance in plants, among many others. This protocol detailed below describes the use of the CRISPR-Cas9 genome editing protocol for the truncation of a gene involved in the sexual cycle of Huntiella omanensis, a filamentous ascomycete fungus belonging to the Ceratocystidaceae family.

CRISPR-Cas9-Mediated Genome Editing in the Filamentous Ascomycete Huntiella omanensis

The CRISPR-Cas9 genome editing system is an easy-to-use genome editor that has been used in model and non-model species. Here we present a protein-based version of this system that was used to introduce a premature stop codon into a mating gene of a non-model filamentous ascomycete fungus.

CRISPR-Cas9-中切基因组编辑在纤维阿莫西特 亨蒂埃拉阿曼尼西斯

The CRISPR-Cas9 genome editing system is a molecular tool that can be used to introduce precise changes into the genomes of model and non-model species ...