这项工作得到了国家重点研发计划（编号：2022YFD1401700）和国家自然科学基金（编号：32090013和32072385）的支持。

1. 昆虫饲养
<ol><li>提高本实验中使用的水稻幼苗（Oryza sativa cv. Japanbonbare）的SBPH。在培养箱（65mm x 200mm）中种植20棵水稻幼苗，并在16小时光照/ 8小时黑暗光周期下在25°C下生长。</li>
</ol>2. 解剖 SBPH 以收集血淋巴
<ol><li>将SBPH放入离心管中，并将它们置于冰浴中10-30分钟。 注意：不要将SBPH放入冰浴中少于10分钟，否则昆虫可能会复活。</li>
	<li>将冷冻的SBPH放在立体显微镜下的载玻片（见材料表）上，其腹部朝上。调整昆虫六条腿的焦点。</li>
	<li>准备两个带有超细尖端的高精度镊子（见 材料表）。用一把镊子压在昆虫的身体上以使其保持在原位，然后用另一把镊子小心地将一条腿从昆虫身上拉下来。 注意：对于硬壳昆虫，可以使用眼科手术刀切断其中一条腿。</li>
	<li>用镊子轻轻按压昆虫的胸部，使血淋巴通过伤口流出。 注意：血淋巴表现为透明飞沫，没有任何可见的白色絮状物。如果有任何白色絮凝物，则丢弃脂质，这代表脂肪体污染。</li>
</ol>3. 使用微量移液器收集血淋巴
<ol><li>准备微量移液器。将体积为1μL的毛细管（见材料表）放入移液器灯泡中（见材料表）。为了进行准确的测量，请确保刻度线可见。 注意：移液器灯泡顶部的一个小孔是必要的。</li>
	<li>收集血淋巴时，握住微量移液器的移液器灯泡，并将毛细管靠近昆虫伤口。通过简单地将毛细血管的尖端接触到血淋巴来收集从伤口渗出的血淋巴。</li>
	<li>当毛细管内的液体达到所需的刻度线时，用手指稍微堵住移液器灯泡顶部的小孔，以停止吸收过程。 注意：连续快速地收集一个 1 μL 血淋巴样本，以避免脂质污染和管堵塞。</li>
	<li>按下移液器灯泡，将收集的血淋巴液排出到 100 μL PBS 缓冲液中（137 毫摩尔/升 NaCI、2.7 毫摩尔/升 KCI、4.3 毫摩尔/升钠 2 HPO 4、1.4 毫摩尔/升KH2 PO 4，pH 7.2-7.4）。 注意：毛细管插入PBS缓冲液中。</li>
	<li>收集另外 1 μL 血淋巴样品时，更换为新的毛细管。</li>
</ol>4. 考马斯蓝染色
<ol><li>根据步骤3中描述的方案从3期幼虫收集3个相同体积（1μL）的血淋巴样品，并将收集的血淋巴排出到PBS缓冲液中，使总体积为100μL。</li>
	<li>将 25 μL 5x 蛋白质上样缓冲液（250 mmol/L Tris-HCI [pH 6.8]、10% W/V SDS、0.05% W/V 溴酚蓝、50% W/V 甘油、5% W/V β-巯基乙醇）加入到 100 μL 稀释的血淋巴样品中，加热使蛋白质变性。</li>
	<li>将 20 μL 煮沸的样品上样到 10% SDS-PAGE 蛋白质凝胶（参见 材料表）上以分离蛋白质。</li>
	<li>将凝胶染色在考马斯蓝溶液中（将1g考马斯亮蓝R-250与250mL异丙醇，100mL冰醋酸和650mLddH2O混合，并使用滤纸过滤掉任何颗粒）在室温下1小时，然后用脱色溶液（100mL乙酸， 400 mL 甲醇，500 mLddH 2O）。</li>
</ol>5. 蛋白质浓度测定
<ol><li>使用两倍稀释法将 10 mg/mL 牛血清白蛋白 （BSA） 稀释至 5 mg/mL、2.5 mg/mL、1.25 mg/mL、0.625 mg/mL 和 0.3125 mg/mL。</li>
	<li>在步骤5.1中吸出5μLPBS缓冲液和每种浓度的BSA溶液，加入1mL的1x布拉德福德染料试剂（参见 材料表），并在室温下孵育5分钟。使用布拉德福德染料试剂中的PBS缓冲液作为对照，OD = 595nm时为零。测量每种剩余蛋白质浓度的吸光度值，并制作标准曲线。</li>
	<li>将来自幼虫、雌性或雄性 SBPH 的 1 μL 血淋巴液加入 100 μL PBS 缓冲液中，然后在 4 °C 下以 1，000 x g 离心样品 10 分钟以除去血细胞。</li>
	<li>将90μL上清液吸入新的离心管中，并根据步骤5.2中的说明测量上清液的吸光度值。</li>
	<li>根据标准曲线的回归方程计算血淋巴样品的蛋白质浓度。在实验中包括三个生物学重复和三个技术重复。</li>
</ol>6. 显微检测
<ol><li>使用微量移液器收集不同发育阶段的10-15 SBPH的血淋巴。将收集的血淋巴液加入含有20μL4%多聚甲醛溶液的管中（参见 材料表）。将混合物在室温下孵育30分钟以固定血淋巴。</li>
	<li>将20μL固定的血细胞移液到涂有硅烷的载玻片的中心（参见 材料表）。</li>
	<li>在载玻片晾衣架中干燥液滴。 注意：避免过度干燥。</li>
	<li>用金抗淬灭试剂4'，6-二脒基-2-苯基吲哚（DAPI）覆盖样品（见材料表），并在倒置显微镜下检查载玻片（见材料表）。</li>
</ol>7. 细胞定量
<ol><li>用微量移液器从SBPH中收集1μL血淋巴，并将其溶解到20μLPBS缓冲液中。</li>
	<li>用PBS缓冲液将血淋巴样品稀释五倍。</li>
	<li>将20μL血淋巴溶液移液到细胞计数室的中心（参见 材料表）。用盖玻片盖住液滴（见 材料表）。</li>
	<li>在倒置显微镜下对细胞计数室（补充图1）上的五个方块中的细胞进行计数（参见 材料表）。在每个实验中包括三个生物学重复和三个技术重复。 细胞/μL = 5 个中间平方的总计数 × 5 × 稀释因子 × 104</li>
</ol>8. 统计分析
<ol><li>使用相应的软件进行统计分析（见 材料表）。创建一个新文件，选择列，导入数据，然后生成带有条形的散点图。</li>
	<li>使用列统计量计算每组数据的平均值和 SD，并使用单因子方差分析工具评估组间差异的显著性。通过三个独立实验从三个生物学重复中确定平均值和SD。</li>
</ol>

从小型节肢动物中收集血淋巴

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血淋巴是节肢动物循环系统的介质，虫媒病毒只有在恶劣的血淋巴环境中生存下来，才能侵入其他节肢动物组织。收集高质量的血淋巴样本是研究血淋巴中发生的载体-病毒相互作用的第一步。据报道，昆虫血淋巴可以从昆虫身体的几个部位获得，包括前腿上的伤口、头部区域的小切口或腹部的撕裂伤口26、27、28、<sup class="xref"...

动植物病毒都可以通过节肢动物传播，这些病毒对人类健康构成严重威胁，给农业造成巨大的经济损失1，2，3。重要的是，节肢动物血淋巴作为节肢动物的循环系统和免疫系统的重要组成部分，在调节虫媒病毒传播方面起着重要作用。通过节肢动物肠道获得的病毒只有在成功逃脱不良血淋巴环境后才会转运到其他组织4，5，6，7。节肢动物血淋巴中病毒的生命周期涉及病毒在血浆中存活、进入血细胞并转运到其他组织，血淋巴中发生各种病毒-载体相互作用机制8、9、10、11、12。例如，SBPH 对 RSV 的垂直传播取决于 SBPH 卵黄素蛋白和 RSV（米条纹病毒）衣壳蛋白13，14 之间的分子相互作用。一些病毒可能通过结合特定的载体因子15，16，17，18来逃避血淋巴的免疫反应。因此，研究节肢动物血淋巴中的载体-病毒相互作用对于更好地了解虫媒病毒传播非常重要。
一些小昆虫的血淋巴，如飞虱、叶蝉和一些蚊子，由于其体型的原因，很难收集。为了解决这个问题，已经开发了几种收集血淋巴的方法，包括将注射器针头直接插入昆虫体内以提取微量的血淋巴，用细尖镊子从伤口部位收集渗出物，以及直接离心。这些方法能够测量血淋巴内的相对基因表达水平和病毒滴度19，20，21。然而，对于这些小昆虫，目前还没有一种有效的血淋巴体积定量方法，这是血细胞计数、蛋白质定量和酶活性分析所必需的。
SBPH（小棕色飞虱）是一种体长约2-4毫米的小型昆虫媒介。SBPH能够传播多种植物病毒，包括RSV，玉米粗矮病毒和水稻黑条纹矮病毒22，23，24。在过去十年中，对SBPH和RSV之间的相互作用进行了深入研究。为了便于使用SBPH，我们开发了一种新颖而简单的血淋巴收集方法。这种方法基于毛细管力原理，使用带有刻度标记的毛细管以精确和可量化的方式获取昆虫的血淋巴。这使我们能够有效地从小昆虫中收集特定体积的血淋巴，并更详细地研究小载体的血淋巴环境。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>10% SDS-PAGE protein gel</td>
			<td>Bio-rad</td>
			<td>4561035</td>
			<td>Protein separation and detection</td>
		</tr>
		<tr>
			<td>4% paraformaldehyde</td>
			<td>Solarbio</td>
			<td>P1110</td>
			<td>For fixation of the cells or tissues&#160;</td>
		</tr>
		<tr>
			<td>Bradford dye reagent</td>
			<td>Bio-rad</td>
			<td>5000205</td>
			<td>Protein concentration detection</td>
		</tr>
		<tr>
			<td>Capillary</td>
			<td>Hirschmann</td>
			<td>9000101</td>
			<td>For collecting hemolymph</td>
		</tr>
		<tr>
			<td>Cell counting chamber</td>
			<td>ACMEC</td>
			<td>AYA0810</td>
			<td>Hemocytes counting</td>
		</tr>
		<tr>
			<td>Glass slide</td>
			<td>Gitoglas</td>
			<td>10127105A</td>
			<td>For holding insects</td>
		</tr>
		<tr>
			<td>Glass slide coated with silane</td>
			<td>Sigma</td>
			<td>S4651-72EA</td>
			<td>For holding microscope samples</td>
		</tr>
		<tr>
			<td>Gold antifade reagent with DAPI</td>
			<td>Invitrogen</td>
			<td>P36935</td>
			<td>Nucleus staining</td>
		</tr>
		<tr>
			<td>Microscope cover glass</td>
			<td>Gitoglas</td>
			<td>10212424C</td>
			<td>For microscopic observation</td>
		</tr>
		<tr>
			<td>Pipette bulb</td>
			<td>Hirschmann</td>
			<td>9000101</td>
			<td>For collecting hemolymph</td>
		</tr>
		<tr>
			<td>Prism 8.0 software</td>
			<td>GraphPad Software</td>
			<td>/</td>
			<td>Statistical analyses</td>
		</tr>
		<tr>
			<td>Stereomicroscope&#160;</td>
			<td>Motic</td>
			<td>SMZ-168</td>
			<td>For insect dissection</td>
		</tr>
		<tr>
			<td>Tweezers</td>
			<td>Tianld</td>
			<td>P5622</td>
			<td>For insect dissection</td>
		</tr>
		<tr>
			<td>Zeiss inverted microscope</td>
			<td>Zeiss</td>
			<td>Observer Z1</td>
			<td>Hemocytes observation</td>
		</tr>
	</tbody>
</table>

a precise and quantifiable method for collecting hemolymph from small arthropods

众所周知，节肢动物通过其血淋巴传播各种具有医学和农业重要性的病毒，这对于病毒传播至关重要。血淋巴采集是研究病毒-载体相互作用的基本技术。在这里，我们描述了一种新颖而简单的方法，用于定量收集小型节肢动物的血淋巴，使用 Laodelphax striatellus （小棕色飞虱，SBPH）作为研究模型，因为这种节肢动物是水稻条纹病毒（RSV）的主要载体。在该协议中，该过程首先用细尖镊子轻轻捏掉冷冻节肢动物的一条腿，然后将血淋巴从伤口中压出。然后，根据毛细管力原理，使用由毛细管和移液器灯泡组成的简单微量移液器从伤口收集漏出性血淋巴。最后，可以将收集的血淋巴溶解到特定的缓冲液中以供进一步研究。这种从小型节肢动物收集血淋巴的新方法是进一步研究虫媒病毒和载体-病毒相互作用的有用且有效的工具。

微量移液器模型和血淋巴收集 我们开发了一种简单的微量移液器，其作用基于毛细管的毛细管力。微量移液器由毛细管和移液器灯泡组成（图1A）。毛细管有不同的体积尺寸，范围从 1 μL 到 20 μL，毛细管体积可根据要求选择。不建议使用体积较小的毛细管，因为较小体积的毛细管的额外细孔会使吸收血淋巴等液体变得困难。移液器灯泡顶部有一个孔，在血淋...

Watch this Scientific Journal Video about A Precise and Quantifiable Method for Collecting Hemolymph from Small Arthropods at JoVE.com

作者聚焦：从小型节肢动物中收集血淋巴液的精确且可量化的方法  

我们描述了一种从小型节肢动物中有效收集可量化血淋巴以进行后续分析的方法。

一种精确且可量化的小型节肢动物血淋巴收集方法

Arthropods are known to transmit a variety of viruses of medical and agricultural importance through their hemolymph, which is essential for virus ...

我们的研究重点是虫媒病毒、昆虫和植物相互作用。从小型节肢动物中获取血淋巴样本对昆虫传播疾病的研究提出了技术挑战。该协议显示了解决此问题的精确且可量化的技术。虫媒病毒传播的最新研究进展发现，病毒可以操纵几个重要的媒介因素来突破宿主屏障。该方案是一种简单，具有成本效益且有价值的技术，可以精确定量血淋巴。这种方法还为启动病毒和载体之间的相互作用提供了有价值的技术。我们即将进行的研究将侧重于了解媒介昆虫传播病毒的分子机制，同时探索和增强新的操作技术。

a-precise-quantifiable-method-for-collecting-hemolymph-from-small

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

A Precise and Quantifiable Method for Collecting Hemolymph from Small Arthropods

1.5K 次观看.  Chinese Academy of Sciences. 我们的研究重点是虫媒病毒、昆虫和植物相互作用。从小型节肢动物中获取血淋巴样本对昆虫传播疾病的研究提出了技术挑战。该协议显示了解决此问题的精确且可量化的技术。虫媒病毒传播的最新研究进展发现，病毒可以操纵几个重要的媒介因素来突破宿主屏障。该方案是一种简单，具有成本效益且有价值的技术，可以精确定量血淋巴。这种方法还为启动病毒和载体?...

视频: 作者聚焦：从小型节肢动物中收集血淋巴液的精确且可量化的方法  

Method for Culture of Early Chick Embryos ex vivo (New Culture)

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying  the formation and patterning of brain, neural tube, somite and heart primordia. Applications of chick embryo culture include electroporation of DNA or RNA constructs in order to analyze gene function, grafts of growth factor coated beads such as FGFs and BMPs , as well as whole mount in situ hybridization and immunohistochemistry. This video demonstrates the different steps in chick embryo culture; First, the embryo is explanted in saline. Then, the embryo is centered on a glass ring. The membranes surrounding the embryo are lifted along the walls of the ring. The ring is then placed in a culture dish containing a pool of albumine. The culture dish is sealed and placed in a humid chamber, where the embryo is cultured for up to 24 hrs. Finally, the embryo is removed from the ring, fixed and processed for further applications. A troubleshooting guide is also presented.

Method for Culture of Early Chick Embryos ex vivo New Culture

This video demonstrates New culture, a method by which chick embryos are cultured outside the egg for up to 24 hr. This method enables one to study early development (primitive streak to 14 som.), a period corresponding to E7-9 in mouse. Applications of this technique include electroporation, in situ hybridization and immunohistochemistry.

文化早期鸡胚体外（新文化）的方法

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

科研

生物学

Method for Whole Mount Antibody Staining in Chick

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying antibody expression in developing brain, neural tube and somite. This video demonstrates the different steps in whole-mount antibody staining using HRP conjugated secondary antibodies; First, the embryo is dissected from the egg and fixed in paraformaldehyde. Second, endogenous peroxidase is inactivated; The embryo is then exposed to primary antibody. After several washes, the embryo is incubated with secondary antibody conjugated to HRP. Peroxidase activity is revealed using reaction with diaminobenzidine substrate. Finally, the embryo is fixed and processed for photography and sectioning. The advantage of this method over the use of fluorescent antibodies is that embryos can be processed for wax sectioning, thus enabling the study of antigen sites in cross section. This method was originally introduced by Jane Dodd and Tom Jessell 1.

This video demonstrates whole mount immunohistochemistry, a method by which the spatial and temporal expression pattern of an antigen can be visualized in young chick embryos. This method was originally introduced by Jane Dodd and Tom Jessell.

整装在鸡的抗体染色方法

Collecting and Measuring Nociceptive and Inflammatory Mediators in Surgical Wounds 

We describe a methodology by which we are able to collect and measure inflammatory and nociceptive biochemical mediators at the surgical wound site. Collecting site-specific biochemical markers allows us to evaluate the relationship between surgical wound and serum levels; determine any associations between mediator release, pain and analgesic consumption; and evaluate the effect of systemic and peripheral drug administration on surgical wound biochemistry.
This methodology has been applied to healthy women undergoing elective cesarean delivery with spinal anesthesia. Wound exudate and serum mediators, in conjunction with pain scores and analgesics consumption were measured at 1, 6, 24, and 48 hours post-cesarean delivery. Biochemical mediators that were detected included IL-1&beta;, IL-2, IL-4, IL-6, IL-7, IL-8, IL-10, IL-12, IL-13, IL-17, TNF&alpha;, INF&gamma;, G-CSF, GM-CSF, MCP-1 and MIP-1&beta;, nerve growth factor (NGF), prostaglandin E2 (PG-E2) and substance P. We found no correlations between wound and serum cytokines concentrations or time-release profiles (J Pain. 2008 Jul 9(7):650-7). This article describes and demonstrates the feasibility of collecting and assaying nociceptive and inflammatory mediators in surgical wounds at specific time points. The lack of significant correlations between serum and wound levels shows the importance of determining site-specific release if surgical wounds and localized pathologies are to be studied.

Collecting and Measuring Nociceptive and Inflammatory Mediators in Surgical Wounds

A technique to collect and measure surgical wound biochemical mediators at specific time points.

在手术伤口的痛觉及炎症介质的收集和测量

We describe a methodology by which we are able to collect and measure inflammatory and nociceptive biochemical mediators at the surgical wound site. ...

Collecting Variable-concentration Isothermal Titration Calorimetry Datasets in Order to Determine Binding Mechanisms

Isothermal titration calorimetry (ITC) is commonly used to determine the thermodynamic parameters associated with the binding of a ligand to a host macromolecule. ITC has some advantages over common spectroscopic approaches for studying host/ligand interactions. For example, the heat released or absorbed when the two components interact is directly measured and does not require any exogenous reporters. Thus the binding enthalpy and the association constant (Ka) are directly obtained from ITC data, and can be used to compute the entropic contribution. Moreover, the shape of the isotherm is dependent on the c-value and the mechanistic model involved. The c-value is defined as c = n[P]tKa, where [P]t is the protein concentration, and n is the number of ligand binding sites within the host. In many cases, multiple binding sites for a given ligand are non-equivalent and ITC allows the characterization of the thermodynamic binding parameters for each individual binding site. This however requires that the correct binding model be used. This choice can be problematic if different models can fit the same experimental data. We have previously shown that this problem can be circumvented by performing experiments at several c-values. The multiple isotherms obtained at different c-values are fit simultaneously to separate models. The correct model is next identified based on the goodness of fit across the entire variable-c dataset. This process is applied here to the aminoglycoside resistance-causing enzyme aminoglycoside N-6'-acetyltransferase-Ii (AAC(6')-Ii). Although our methodology is applicable to any system, the necessity of this strategy is better demonstrated with a macromolecule-ligand system showing allostery or cooperativity, and when different binding models provide essentially identical fits to the same data. To our knowledge, there are no such systems commercially available. AAC(6')-Ii, is a homo-dimer containing two active sites, showing cooperativity between the two subunits. However ITC data obtained at a single c-value can be fit equally well to at least two different models a two-sets-of-sites independent model and a two-site sequential (cooperative) model. Through varying the c-value as explained above, it was established that the correct binding model for AAC(6')-Ii is a two-site sequential binding model. Herein, we describe the steps that must be taken when performing ITC experiments in order to obtain datasets suitable for variable-c analyses.

ITC is a powerful tool for studying the binding of a ligand to its host. In complex systems however, several models may fit the data equally well. The method described here provides a means to elucidate the appropriate binding model for complex systems and extract the corresponding thermodynamic parameters.

收集可变浓度等温滴定量热数据集，以确定约束机制

Isothermal titration calorimetry (ITC) is commonly used to determine the thermodynamic parameters associated with the binding of a ligand to a host ...

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 ...

Culturing Primary Rat Inner Medullary Collecting Duct Cells

Arginine-vasopressin (AVP) facilitates water reabsorption by renal collecting duct principal cells and thereby fine-tunes body water homeostasis. AVP binds to vasopressin V2 receptors (V2R) on the surface of the cells and thereby induces synthesis of cAMP. This stimulates cellular signaling processes leading to changes in the phosphorylation of the water channel aquaporin-2 (AQP2). Protein kinase A phoshorylates AQP2 and thereby triggers the translocation of AQP2 from intracellular vesicles into the plasma membrane facilitating water reabsorption from primary urine. Aberrations of AVP release from the pituitary or AVP-activated signaling in principal cells can cause central or nephrogenic diabetes insipidus, respectively; an elevated blood plasma AVP level is associated with cardiovascular diseases such as chronic heart failure and the syndrome of inappropriate antidiuretic hormone secretion.
Here, we present a protocol for cultivation of primary rat inner medullary collecting duct (IMCD) cells, which express V2R and AQP2 endogenously. The cells are suitable for elucidating molecular mechanisms underlying the control of AQP2 and thus to discover novel drug targets for the treatment of diseases associated with dysregulation of AVP-mediated water reabsorption. IMCD cells are obtained from rat renal inner medullae and are used for experiments six to eight days after seeding. IMCD cells can be cultured in regular cell culture dishes, flasks and micro-titer plates of different formats, the procedure only requires a few hours, and is appropriate for standard cell culture laboratories.

Arginine-vasopressin (AVP) controls fine-tuning of body water homeostasis through facilitating water reabsorption by renal principal cells. Here, we present a protocol for the cultivation of primary rat inner medullary collecting duct cells suitable for the elucidation of molecular mechanisms underlying AVP-mediated water reabsorption.

原代培养大鼠内髓集合管细胞

Arginine-vasopressin (AVP) facilitates water reabsorption by renal collecting duct principal cells and thereby fine-tunes body water homeostasis. AVP ...

Isolation of Small Noncoding RNAs from Human Serum

The analysis of RNA and its expression is a common feature in many laboratories. Of significance is the emergence of small RNAs like microRNAs, which are found in mammalian cells. These small RNAs are potent gene regulators controlling vital pathways such as growth, development and death and much interest has been directed at their expression in bodily fluids. This is due to their dysregulation in human diseases such as cancer and their potential application as serum biomarkers. However, the analysis of miRNA expression in serum may be problematic. In most cases the amount of serum is limiting and serum contains low amounts of total RNA, of which small RNAs only constitute 0.4-0.5%1. Thus the isolation of sufficient amounts of quality RNA from serum is a major challenge to researchers today. In this technical paper, we demonstrate a method which uses only 400 &#181;l of human serum to obtain sufficient RNA for either DNA arrays or qPCR analysis. The advantages of this method are its simplicity and ability to yield high quality RNA. It requires no specialized columns for purification of small RNAs and utilizes general reagents and hardware found in common laboratories. Our method utilizes a Phase Lock Gel to eliminate phenol contamination while at the same time yielding high quality RNA. We also introduce an additional step to further remove all contaminants during the isolation step. This protocol is very effective in isolating yields of total RNA of up to 100 ng/&#181;l from serum but can also be adapted for other biological tissues.

This protocol describes a method for extracting small RNAs from human serum. We have used this method to isolate microRNAs from cancer serum for use in DNA arrays and also singleplex quantitative PCR. The protocol utilizes phenol and guanidinium thiocyanate reagents with modifications to yield high quality RNA.

小非编码RNA从人血清中分离

The analysis of RNA and its expression is a common feature in many laboratories. Of significance is the emergence of small RNAs like microRNAs, which are ...

A Method of Permeabilization of Drosophila Embryos for Assays of Small Molecule Activity

The Drosophila embryo has long been a powerful laboratory model for elucidating molecular and genetic mechanisms that control development. The ease of genetic manipulations with this model has supplanted pharmacological approaches that are commonplace in other animal models and cell-based assays. Here we describe recent advances in a protocol that enables application of small molecules to the developing fruit fly embryo. The method details steps to overcome the impermeability of the eggshell while maintaining embryo viability. Eggshell permeabilization across a broad range of developmental stages is achieved by application of a previously described d-limonene embryo permeabilization solvent (EPS1) and by aging embryos at reduced temperature (18 &#176;C) prior to treatments. In addition, use of a far-red dye (CY5) as a permeabilization indicator is described, which is compatible with downstream applications involving standard red and green fluorescent dyes in live and fixed preparations. This protocol is applicable to studies using bioactive compounds to probe developmental mechanisms as well as for studies aimed at evaluating teratogenic or pharmacologic activity of uncharacterized small molecules.

A Method of Permeabilization of Drosophila Embryos for Assays of Small Molecule Activity

Introduction of small molecules to the developing Drosophila embryo offers great potential for characterizing biological activity of novel compounds, drugs, and toxins as well as for probing fundamental developmental pathways. Methods described herein outline steps that overcome natural barriers to this approach, expanding the utility of the Drosophila embryo model.

的透的一种方法<em&gt;果蝇</em&gt;胚胎用于小分子活性的测定

The Drosophila embryo has long been a powerful laboratory model for elucidating molecular and genetic mechanisms that control development. The ease of ...

A Method for Extracting Pigments from Squid Doryteuthis pealeii

Cephalopods can undergo rapid and adaptive changes in dermal coloration for sensing, communication, defense, and reproduction purposes. These capabilities are supported in part by the areal expansion and retraction of pigmented organs known as chromatophores. While it is known that the chromatophores contain a tethered network of pigmented granules, their structure-function properties have not been fully detailed. We describe a method for isolating the nanostructured granules in squid Doryteuthis pealeii chromatophores and demonstrate how their associated pigments can be extracted in acidic solvents. To accomplish this, the chromatophore containing dermal layer is first manually isolated using a superficial dissection, and the pigment granules are removed using sonication, centrifugation, and washing cycles. Pigments confined within the purified granules are then extracted via acidic methanol solutions, leaving nanostructures with smaller diameters that are void of visible color. This extraction procedure produces a 58% yield of soluble pigments isolated from granules. Using this method, the composition of the chromatophore pigments can be determined and used to provide insight into the mechanism of adaptive coloration in cephalopods.

A Method for Extracting Pigments from Squid Doryteuthis pealeii

A protocol for the extraction of pigments from the nanostructured granules in squid Doryteuthis pealeii chromatophores is presented.

一种从鱿鱼提取色素的方法<em&gt; Doryteuthis pealeii</em

Cephalopods can undergo rapid and adaptive changes in dermal coloration for sensing, communication, defense, and reproduction purposes. These capabilities ...

一种精确且可量化的小型节肢动物血淋巴收集方法

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