Name: nucleofection and in vivo propagation of chicken eimeria parasites
Uploaded: 2020-02-14T12:00:00
Description: Watch this Scientific Journal Video about Nucleofection and In Vivo Propagation of Chicken Eimeria Parasites at JoVE.com

这项工作得到了国家重点研究发展计划（2017YFD0501200）和国家自然科学基金（31572507、31772728和31873007）的支持。

所有动物实验的鸡按照中国农业大学动物护理与使用委员会的指导方针进行饲养和维护，并遵循《涉及动物的生物医学研究国际指导原则》。实验得到了北京市实验室动物管理委员会的批准。
1. 提取和纯化艾梅里亚孢子石（例如，E. tenella）
<ol><li>释放孢子<ol>
<li>在 2，300 x g处将二铬酸钾溶液中的孢子卵母细胞（2.5%，m/v）离心 1 x 107孢子卵母细胞...

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在20世纪90年代，为阿皮兰寄生虫开发了转染系统，并用于研究美洲寄生虫。最近，在E.tenella8，9和E.nieschulzi15进行了稳定的转染。通过转染第二代美罗佐特11，实现了E.necatrix的稳定转染。通过翼静脉接种E.acervulina的转染孢子球，解决了E.acervulina孢子菌无法通过氯气途径接种的药管（张...

Eimeria spp. 引起球菌病，给畜禽业带来巨大的经济损失。虽然抗凝球药物，并在一定程度上，衰减抗凝细胞疫苗，已被广泛用于控制球菌病，但仍存在缺陷，在耐药性，药物残留，和疫苗菌株的潜在扩散，重新获得毒性1。随着分子生物学的发展，转染已成为研究基因功能、研制新型疫苗、筛选Eimeria新药靶点的重要工具。
在过去的几十年中，转染已经成功地应用于疟原虫寄生虫，如疟原虫和弓形虫贡迪2，3，4，5，6。一项使用β-gal作为E.tenella转染报告员的研究在Eimeria7中试行了这种工作。E. tenella8、9、 E. mitis10和E. acervulina（张等人，未公开的数据）在鸡中成功转染。最近，我们通过核化11，利用E.necatrix的美罗佐特体实现了转染。
研究表明，表达异源抗原的Eimeria具有被开发为重组疫苗的潜力，如那些表达弯曲杆菌抗原A（CjaA）或鸡白莱金2（chIL-2）12，13。因此，该协议描述了对鸡中Eimeria spp的成核研究。该程序描述了孢子或美速物的纯化、用质粒DNA核化、克隆接种/静脉注射和体内繁殖，以帮助研究人员开始研究转基因Eimeria寄生虫。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>ATP-disodium</td>
			<td>Sigma</td>
			<td>A26209</td>
			<td></td>
		</tr>
		<tr>
			<td>Cellulose DE-52</td>
			<td>Solarbio</td>
			<td>C8350</td>
			<td></td>
		</tr>
		<tr>
			<td>Constant Flow Pump</td>
			<td>SHANGHAI JINGKE INDUSTRIAL CO., LTD.</td>
			<td>HL-2B</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>MACGENE</td>
			<td>CM15019</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass beads</td>
			<td>Sigma</td>
			<td>Z250473-1PAK</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma</td>
			<td>No. V900116</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Biotopped</td>
			<td>G6200</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS</td>
			<td>MACGENE</td>
			<td>CC016</td>
			<td></td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>Sigma</td>
			<td>No. V900041</td>
			<td></td>
		</tr>
		<tr>
			<td>Low Speed Centrifuge</td>
			<td>BEIJING ERA BEILI CENTRIFUGE CO., LTD.</td>
			<td>DT5-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnetic Mixer</td>
			<td>SCILOGEX</td>
			<td>MS-H280-Pro</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Sigma</td>
			<td>449164</td>
			<td></td>
		</tr>
		<tr>
			<td>MoFlo cell sorter</td>
			<td>BeckMan Coulter, US</td>
			<td>201309995</td>
			<td></td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Sigma</td>
			<td>144-55-8</td>
			<td></td>
		</tr>
		<tr>
			<td>Nucleofection device</td>
			<td>LONZA/amaxa</td>
			<td>90900012 (Nucleofector II)</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Solarbio</td>
			<td>P1010</td>
			<td></td>
		</tr>
		<tr>
			<td>Percoll (DG gradient stock solution)</td>
			<td>GE Healthcare</td>
			<td>17-0891-09</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium taurodeoxycholate hydrate</td>
			<td>Sigma</td>
			<td>T0875</td>
			<td></td>
		</tr>
		<tr>
			<td>Sorvall Legend Micro 17 Microcentrifuge</td>
			<td>ThermoFisher Scientific</td>
			<td>75002430</td>
			<td></td>
		</tr>
		<tr>
			<td>The composition of DMEM: 4.5 g/L glucose with sodium pyruvate, L-glutamine, and 25 mM HEPES.</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Solarbio</td>
			<td>T8150</td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex Mixer</td>
			<td>Beijing North TZ-Biotech Develop.co.</td>
			<td>HQ-60-II</td>
			<td></td>
		</tr>
		<tr>
			<td>Water Bath Thermostat</td>
			<td>Grant Instruments (Cambridge), Ltd.</td>
			<td>GD120,GM0815010</td>
			<td></td>
		</tr>
	</tbody>
</table>

nucleofection and in vivo propagation of chicken eimeria parasites

转染是一个技术过程，通过它遗传物质，如DNA和双链RNA，被输送到细胞中，以修改感兴趣的基因。目前，转基因技术正在成为研究Eimeria（家禽和牲畜球菌病的病原体）不可缺少的工具。该协议提供了对美洲寄生虫稳定转染的详细描述：孢子石或第二代美罗索石的纯化和核化，以及转染寄生虫在体内传播。利用这个协议，我们实现了几种艾梅里亚的转染。总之，核素是一个有用的工具，以促进基因操作的人类寄生虫。

该协议已用于转染美洲寄生虫。在这项研究中，图2A和图2B显示了E.necatrix的第二代梅龙特和美罗佐石，而图2C和图2D显示了使用密度梯度溶液后E.tenella的孢子和孢子石。还显示了在核化了麦罗索特或孢子石之后的...

Watch this Scientific Journal Video about Nucleofection and In Vivo Propagation of Chicken Eimeria Parasites at JoVE.com

Nucleofection and In Vivo Propagation of Chicken Eimeria Parasites

在这里，我们提供了一种通过核化孢子菌或第二代美罗索特物实现鸡艾梅里亚寄生虫稳定转染的方法。表达异源抗原基因的转基因生物寄生虫可用作疫苗输送工具。

鸡寄生虫的成核和活体繁殖

Transfection is a technical process through which genetic material, such as DNA and double-stranded RNA, are delivered into cells to modify the gene of ...

建立Eimeria物种的转染方案将有助于推进基因功能的研究，开发新型疫苗，并筛选新的Eimeria药物靶点。利用这项技术，我们实现了几种Eimeria的转染，核分体是促进基因操作在艾默利寄生虫中应用的有用工具。Eimeria的核分为体外无法培养的其他寄生虫的转染提供了参考，并将加速其基因操纵的研究。如果您第一次尝试这种技术，您应该在转染开始前做好所有试剂及其仪器的准备，以避免在实验过程中争先恐后。这种方法的实现有助于关注Eimeria转染的人看到这个实验的复杂细节。首先释放孢子囊，将1倍10离二氯酸钾溶液中的7个孢子卵母体，5分钟。用 PBS 洗三次。用一毫升PBS将颗粒重新暂停，然后将混合物转移到15毫升管中。添加一毫米直径的玻璃珠的同等体积，并使用涡流混合器振荡卵母质悬浮液以释放孢子。每分钟，通过显微镜监测孢子细胞的释放。当超过 90% 的卵母细胞破裂时，停止涡旋。将孢子悬浮液转移到新的 1.5 毫升管中，以 1，600 次 g 离心，5 分钟。用一毫升50%密度梯度溶液重新沉淀，以10，000倍g离心1分钟。要释放孢子虫，请用排泄物缓冲液重新沉淀，并在42摄氏度的水浴中孵育40至60分钟，以释放孢子虫。在出站期间每五分钟摇动一次管子。当超过90%的孢子虫被释放时，停止孵育。在 600 次 g 下离心 10 分钟。用一毫升55%密度梯度溶液重新沉淀，以10000倍g离心管一分钟。用一毫升 PBS 重新暂停沉淀，并使用血细胞计计算孢子。为了收集Eimeria necatrix的第二代美生动物，将鸡肠从蛋黄茎纵向切到蛋黄孔，并在培养皿中用PBS或HBSS轻轻洗三次。将肠切成碎片，并把它们放在一个带消化缓冲液的圆锥烧瓶中。将烧瓶放在37摄氏度的磁力搅拌器上，用搅拌棒放置30至60分钟，以释放红石。孵育30分钟后，每5分钟通过显微检查监测一次美罗佐石的释放。为了净化美洛佐石，将含有消化的美罗佐石的悬浮液倒入四层纱布中，过滤成50毫升管，以600倍g离心管10分钟。离心后，丢弃含有肠碎屑的上经剂。将沉淀物与纯化的美罗佐石转移到1.5毫升管中。用一毫升PBS重新暂停沉淀，并使用血细胞计计算美罗佐特。首先，将孢子虫或梅罗佐特悬浮液在600倍g下离心10分钟。然后丢弃上一液。按照以下顺序，在含有孢子虫或异质的1.5毫升管中加入85微升的核转染缓冲液、10微克质粒和25个限制性酶单位。将暂停转移到核转染杯上。将杯子放入核转移槽中。使用电源按钮打开核分子设备，然后选择转染过程 U-033。程序完成后，按核分子设备的 X 按钮，屏幕显示正常，表示分核成功。将 0.5 添加到 Dulbecco 的修改鹰的介质中 0.5 到核分杯中，以停止反应。轻轻混合后，将悬浮液转移到1.5毫升管中。将核感染寄生虫接种到七天大的鸡体内。通过丁香途径接种Eimeria necatrix的雌激素或Eimeria tenella的孢子虫，但通过静脉注射接种Eimeria acervulina孢子虫。从粪便中收集卵母细胞后，使用荧光素活性细胞分类和每公斤150毫克的苯丙胺来增加转基因人群比例。该协议已用于转染艾默利寄生虫。在这项研究中，这里显示了Eimeria necatrix的第二代梅龙和梅罗佐石，以及使用密度梯度溶液后，Eimeria tenella的孢子和孢子虫。Eimeria necatrix 感染核感染的雌性动物后，与细胞核感染的雌性细胞的卵母体图像和感染核感染的孢子体后，Eimeria tenella 的卵母体图像表明细胞分切成功。尝试此过程时，最重要的是确保孢子虫和紫杉石的纯化成功，并使用 50% 密度梯度溶液去除 OS2，同时确保孢子虫和孢子虫更纯化。按照此过程，您可以优化此协议，如转染卵母细胞或孢子，以简化 Eimeria 寄生虫的转染程序。随着Eimeria转染技术的发展，Eimeria的CRISPR-Cas9技术将显著提高对Eimeria的谨慎基因处理，并作为合适的疫苗输送工具应用。

Research

JoVE Journal

Biology

Nucleofection and In Vivo Propagation of Chicken Eimeria Parasites

Propagation of Human Embryonic Stem (ES) Cells

Propagation of Human Embryonic Stem ES Cells

人类胚胎干细胞（ES细胞）的繁殖

科研

生物学

Windowing Chicken Eggs for Developmental Studies

发育研究的窗口鸡蛋

The study of development has been greatly aided by the use of the chick embryo as an experimental model.  The ease of accessibility of the embryo has ...

Placing Growth Factor-Coated Beads on Early Stage Chicken Embryos

配售早期鸡胚生长因子包被的磁珠

The neural tube expresses many proteins in specific spatiotemporal patterns during development.   These proteins have been shown to be critical for cell ...

In Ovo Electroporations of HH Stage 10 Chicken Embryos

在OVO的HH阶段10个鸡胚Electroporations

Large size and external development of the chicken embryo have long made it a valuable tool in the study of developmental biology.  With the advent of ...

In vivo and in vitro Studies of Adaptor-clathrin Interaction

In vivo and in vitro Studies of Adaptor-clathrin Interaction

适配器，网格蛋白相互作用在体内和体外研究

A major endocytic pathway initiates with the formation of clathrin-coated vesicles (CCVs) that transport cargo from the cell surface to endosomes1-6. CCVs ...

Mechanical Stimulation-induced Calcium Wave Propagation in Cell Monolayers: The Example of Bovine Corneal Endothelial Cells

机械刺激引起的钙波传播的细胞单层：牛角膜内皮细胞的例子

Intercellular communication is essential for the coordination of physiological processes between cells in a variety of organs and tissues, including the ...

Stimulation of Cytoplasmic DNA Sensing Pathways In Vitro and In Vivo

Stimulation of Cytoplasmic DNA Sensing Pathways In Vitro and In Vivo

细胞质DNA的检测途径的刺激<em&gt;在体外</em&gt;和<em&gt;体内</em

In order to efficiently stimulate an innate immune response, DNA must be of sufficient length and purity. We present a method where double stranded DNA ...

In Vivo Microinjection and Electroporation of Mouse Testis

In Vivo Microinjection and Electroporation of Mouse Testis

在小鼠睾丸活体显微注射和电穿孔

This video and article contribution gives a comprehensive description of microinjection and electroporation of mouse testis in vivo. This particular ...

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

测量的刚度<em&gt;离体</em&gt;鼠标主动脉用原子力显微镜

Arterial stiffening is a significant risk factor and biomarker for cardiovascular disease and a hallmark of aging. Atomic force microscopy (AFM) is a ...

Preparation and Observation of Thick Biological Samples by Scanning Transmission Electron Tomography

准备厚生物样品的观察扫描透射电子断层扫描

This report describes a protocol for preparing thick biological specimens for further observation using a scanning transmission electron microscope. It ...