Name: an electroporation method to transform rickettsia spp. with a fluorescent protein-expressing shuttle vector in tick cell lines
Uploaded: 2022-10-11T14:20:00
Description: Watch this Scientific Journal Video about An Electroporation Method to Transform Rickettsia spp. with a Fluorescent Protein-Expressing Shuttle Vector in Tick Cell Lines at JoVE.com

我们感谢Timothy J. Kurtti和Benjamin Cull富有洞察力的讨论和建议。这项研究得到了美国国立卫生研究院（2R01AI049424）对U.G.M.的资助和明尼苏达州农业实验站（MIN-17-078）对U.G.M.的资助。

1. 从蜱细胞培养物中繁殖和纯化帕氏螨
注意：所有细胞培养程序均应在II类生物安全柜中进行。
<ol><li>准备 R. parkeri-受感染的蜱细胞<ol><li>在 34 °C 的 5 mL L15C300 培养基17 中的 25 cm2 细胞培养瓶中培养 ISE6 细胞，并补充有 5% 胎牛血清 （FBS）、5% 色糖磷酸盐肉汤 （TPB） 和 0.1% 脂蛋白浓缩物 （LPC）。 注意：ISE6（肩突硬?...

<ol>
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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNAi+reveals+proteins+for+metabolism+and+protein+processing+associated+with+Langat+virus+infection+in+Ixodes+scapularis+(black-legged+tick)+ISE6+cells.">RNAi reveals proteins for metabolism and protein processing associated with Langat virus infection in Ixodes scapularis (black-legged tick) ISE6 cells.</a> Parasites &amp; Vectors. 10 (1), 1-4 (2017).</li><li>Al-Rofaai, A., Bell-Sakyi, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tick+cell+Lines+in+research+on+tick+control.">Tick cell Lines in research on tick control.</a> Frontiers in Physiology. 11, 152 (2020).</li><li>Wang, X. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrion-dependent+apoptosis+is+essential+for+Rickettsia+parkeri+infection+and+replication+in+vector+cells.">Mitochondrion-dependent apoptosis is essential for Rickettsia parkeri infection and replication in vector cells.</a> mSystems. 6 (2), 01209-01220 (2021).</li><li>Berney, M., Hammes, F., Bosshard, F., Weilenmann, H. U., Egli, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+and+interpretation+of+bacterial+viability+by+using+the+LIVE/DEAD+BacLight+Kit+in+combination+with+flow+cytometry.">Assessment and interpretation of bacterial viability by using the LIVE/DEAD BacLight Kit in combination with flow cytometry.</a> Applied and Environmental Microbiology. 73 (10), 3283-3290 (2007).</li><li>Riley, S. P., Macaluso, K. 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在这里，我们展示了一种使用电穿孔将穿梭质粒pRAM18dSFA上编码的外源性DNA引入立克次体的方法。在此过程中，从宿主细胞中纯化游离立克次体，用立克次体穿梭载体转化，并释放到蜱细胞上进行感染。还描述了一种共聚焦免疫荧光程序，用于检测蜱细胞中表达红色荧光蛋白的 R. parkeri 。类似的方法适用于其他 立克次体 物种，并且通过进一步的修改可能适用于能够维持质粒的其他专?...

立克次体病是由属于立克次体属（立克次体科，立 克次 体目）的各种专性细胞内细菌引起的。 立克次 体属根据系统发育特征1，2 分为四大类：斑疹热组 （SFG），其中包含导致最严重和致命的蜱传立克次体病的立克次体（例如，立克次氏体，落基山斑疹热的病原体 ）、 斑疹伤寒组（TG，例如， 普罗瓦泽基立克次体，流行性斑疹伤寒的病原体）， 过渡组（TRG，例如费利立 克次体，跳蚤传播斑疹热的病原体）和祖先组（AG，例如 贝氏立克次体）。
在已知最古老的媒介传播疾病中，立克次体病主要是在病原体通过受感染的节肢动物媒介（包括蜱、跳蚤、虱子和螨虫）叮咬传播病原体后获得的3，4。尽管有效抗生素的发现改善了治疗结果，但新出现和重新出现的流行立克次体病继续挑战传统的预防和控制策略。因此，全面了解立克次体/宿主/媒介相互作用最终将为开发预防和治疗这些古老疾病的新方法奠定坚实的基础。
在自然界中，细菌中的水平基因转移（HGT）通过偶联，转导和转化发生5。体外细菌转化利用这些HGT概念，尽管立克次体的细胞内性质提出了一些挑战。不同种类立克次体中受限的生长条件和对共轭和转导系统知之甚少，阻碍了立克次体6，7，8中结合和转导方法的应用。与其他专性细胞内细菌属（例如衣原体、柯克斯体、无形体和埃立克体）相比，立克次体属在细胞质内的生长和复制策略方面有所不同，由于其独特的生活方式特征，这对立克次体的遗传修饰提出了特定的挑战9。
在尝试对立克次体进行基因改造时，要克服的最初障碍是实现成功的转化。因此，设计一种具有高转化效率的可行方法对于开发立克次体遗传工具具有极其有价值的价值。在这里，我们专注于电穿孔，这是一种被广泛认可的转化方法，已被用于将外源性DNA成功引入几种立克次体，包括普罗瓦泽氏立克次体，伤寒立克次氏体，圆锥立克次氏体，帕氏立克次氏体，蒙坦立克次氏体，贝氏立克次体，孔雀立克次体和布氏立克次体10，11，12，13，14，15，16.
本文描述了一种电穿孔帕 克利 菌株泰特地狱（加入：GCA_000965145.1）的程序，其穿梭载体pRAM18dSFA源自弱 细胞立克次氏立克次氏 菌株AaR/SC质粒pRAM18，该梭载体被工程化以编码mKATE（一种远红荧光蛋白）和 aadA，赋予壮观霉素和链霉素抗性13，15，20。转化的 R. parkeri 在蜱细胞系的抗生素选择下是可行的，并且稳定地维持。此外，我们表明，通过共聚焦显微镜在活蜱细胞中转化的 R. parkeri 的定位可用于评估载体细胞系中转化率的质量。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.1 cm gap gene pulser electroporation cuvette</td>
			<td>Bio-Rad</td>
			<td>1652083</td>
			<td></td>
		</tr>
		<tr>
			<td>2 &#956;m pore size filter&#160;</td>
			<td>GE Healthcare Life Sciences Whatman</td>
			<td>6783-2520</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL Luer-lock syringe&#160;</td>
			<td>BD</td>
			<td>309646</td>
			<td></td>
		</tr>
		<tr>
			<td>60-90 silicon carbide grit&#160;</td>
			<td>LORTONE, inc</td>
			<td>&#160;591-056</td>
			<td></td>
		</tr>
		<tr>
			<td>absolute methanol&#160;</td>
			<td>Fisher Scientific</td>
			<td>A457-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacto tryptose phosphate broth&#160;</td>
			<td>BD</td>
			<td>260300</td>
			<td></td>
		</tr>
		<tr>
			<td>Cytospin centrifuge Cytospin4</td>
			<td>Thermo Fisher Scientific</td>
			<td>A78300003</td>
			<td>The rotor is detatchable so the whole rotor can be put into the hood to load infectious samples</td>
		</tr>
		<tr>
			<td>EndoFree Plasmid Maxi Kit (10)</td>
			<td>QIAGEN</td>
			<td>12362</td>
			<td>used to obtain endotoxin-free pRAM18dSFA plasmid</td>
		</tr>
		<tr>
			<td>extended fine tip transfer pipet&#160;</td>
			<td>Perfector Scientific</td>
			<td>TP03-5301</td>
			<td></td>
		</tr>
		<tr>
			<td>fetal bovine serum&#160;</td>
			<td>Gemini Bio</td>
			<td>900-108</td>
			<td>The FBS batch has to be tested to make sure ISE6 cells will grow well in it.</td>
		</tr>
		<tr>
			<td>Gene Pulser II electroporator with Pulse Controller PLUS</td>
			<td>Bio-Rad</td>
			<td>165-2105 &#38; 165-2110</td>
			<td></td>
		</tr>
		<tr>
			<td>hemocytometer</td>
			<td>Thermo Fisher Scientific</td>
			<td>267110</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Millipore-Sigma</td>
			<td>H4034</td>
			<td></td>
		</tr>
		<tr>
			<td>ImageJ Fiji</td>
			<td>National Institute of Health</td>
			<td></td>
			<td>raw image editing</td>
		</tr>
		<tr>
			<td>KaryoMAX Giemsa stain&#160;</td>
			<td>Gibco</td>
			<td>&#160;2021-10-30</td>
			<td></td>
		</tr>
		<tr>
			<td>Leibovitz&#39;s L-15 medium</td>
			<td>Gibco</td>
			<td>41300039</td>
			<td></td>
		</tr>
		<tr>
			<td>lipoprotein concentrate&#160;</td>
			<td>MP Biomedicals</td>
			<td>191476</td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon Diaphot</td>
			<td>Nikon</td>
			<td></td>
			<td>epifluorescence microscope</td>
		</tr>
		<tr>
			<td>NucBlue Live ReadyProbes Reagent&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>R37605</td>
			<td></td>
		</tr>
		<tr>
			<td>Olympus Disc Scanning Unit (DSU) confocal microscope&#160;</td>
			<td>Olympus</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Petroff-Hausser Counting Chamber</td>
			<td>Hausser Scientific</td>
			<td>&#160;Chamber 3900</td>
			<td></td>
		</tr>
		<tr>
			<td>sodium bicarbonate</td>
			<td>Millipore-Sigma</td>
			<td>S5761</td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex</td>
			<td>Fisher Vortex Genie 2</td>
			<td>12-812</td>
			<td></td>
		</tr>
	</tbody>
</table>

an electroporation method to transform rickettsia spp. with a fluorescent protein-expressing shuttle vector in tick cell lines

立克次体病是由属于 立克次体 属的各种专性细胞内细菌引起的，这些细菌可以通过受感染的节肢动物媒介的叮咬传播给脊椎动物宿主。迄今为止，由于诊断困难，新出现或重新出现的流行性立克次体病仍然是一个公共卫生风险，因为诊断方法有限，没有标准化或普遍可及。由于缺乏对体征和症状的认识而导致的误诊可能导致抗生素治疗延迟和不良的健康结局。全面了解 立克次 体的特征最终将改善临床诊断、评估和治疗，改善疾病的控制和预防。 
立克次体基因的功能研究对于理解其在发病机制中的作用至关重要。本文描述了使用穿梭载体pRAM18dSFA电穿孔帕氏立克次氏立 克次 氏菌株泰特地狱的程序，以及用抗生素（大观霉素和链霉素）在蜱细胞培养中选择转化的 帕氏立 克次体菌落的程序。还描述了一种使用共聚焦免疫荧光显微镜定位蜱细胞中转化的 R. parkeri 的方法，这是一种检查载体细胞系转化的有用技术。类似的方法也适用于其他立克次体的转化。

吉姆萨染色后在光学显微镜下ISE6细胞中帕克里R. parkeri的形态如图1所示。在图2中，使用共聚焦显微镜显示了在ISE6细胞中表达红色荧光蛋白的转化R. parkeri。从孵育的第7天到（B）第10天，ISE6细胞（蓝色，对应于细胞核）中转化的R. parkeri（红色）的感染率显着增加。
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Watch this Scientific Journal Video about An Electroporation Method to Transform Rickettsia spp. with a Fluorescent Protein-Expressing Shuttle Vector in Tick Cell Lines at JoVE.com

An Electroporation Method to Transform Rickettsia spp. with a Fluorescent Protein-Expressing Shuttle Vector in Tick Cell Lines

电穿孔是一种快速、广泛采用的方法，用于将外源性 DNA 引入 立克次体属。该协议为立 克次体属中专性细胞内细菌的转化提供了一种有用的电穿孔方法。

一种在蜱细胞系中用荧光蛋白表达穿梭载体转化立 克次 体属的电穿孔方法

Rickettsioses are caused by a broad range of obligate intracellular bacteria belonging to the genus Rickettsia that can be transmitted to vertebrate hosts ...

立克次体基因的功能研究对于理解它们在发病机制中的作用至关重要，因此我们需要可靠的方法来以受控方式改变基因。我们的协议使用电穿孔将外源性DNA引入立克次体属的专性细胞内细菌中，从而使我们能够研究引入的DNA的影响。今天和我一起演示该程序的是Lisa Price，来自Munderloh博士和Kurtti博士实验室的研究员。首先，如文中所述制备90%至100%帕氏立克次氏体感染的ISE6细胞培养物。然后，为了通过吉姆萨染色确定感染率，将细胞悬液与完全培养基稀释至1：5的比例，并通过以113G离心将100微升细胞悬液沉积到玻璃显微镜载玻片上五分钟。让载玻片风干后，在室温下将其固定在无水甲醇中五分钟。然后，将载玻片和吉姆萨染色在 30 摄氏度下孵育 37 分钟。接下来，用水冲洗载玻片，干燥后，在带有油物镜的光学显微镜下观察。通过计数感染和未感染细胞来确定感染细胞的百分比。为了制备无细胞立克次氏体，将约0.2毫升无菌60至90碳化硅砂砾倒入两个两毫升无菌微量离心管中，并将管放在一边。从先前接种的含有五毫升培养基的帕克次氏立克次氏体感染烧瓶中，除去两毫升培养基，注意不要干扰细胞层。然后，用剩余的三毫升培养基重悬细胞。将该悬浮液平均分配给两个准备好的碳化硅砂砾管，并高速涡旋管30秒。然后，将它们放在冰上。接下来，通过伸出柱塞来准备无菌的 5 毫升鲁尔锁注射器，并将柱塞端插入用于固定 15 毫升锥形管的聚苯乙烯基座中。使用带有橡胶球操作的屏障两毫升巴斯德移液器，小心地去除涡旋细胞的上清液，不要吸入任何砂砾。然后，将移液器吸头插入注射器集线器开口，并以轻压将内容物弹出注射器中。接下来，通过安装在注射器集线器上的无菌两微米孔径过滤器将帕氏立克次氏体培养物过滤到无菌的 1.5 毫升管中，并在 13， 600 G 下以 4 摄氏度离心管五分钟。除去上清液后，用1.2毫升300毫摩尔冰冷蔗糖溶液洗涤细胞沉淀两次。离心样品并在两次洗涤之间除去上清液。在离心结束时，分别用100至150微升冰冷的蔗糖溶液重悬混合的细胞沉淀，用于两到三个转化样品。然后，将样品在预冷、无菌、1.5 微升微量离心管中分成 50 微升等分试样。为了将3微克无内毒素pRAM18sSFA质粒DNA转化为含有50微升帕氏立克次氏体的每管冰上，并用移液器吸头轻轻搅拌。将混合物转移到间隙尺寸为0.1厘米的预冷无菌电穿孔比色皿中。轻轻敲击比色皿以均匀分布混合物，并在冰上孵育 10 到 30 分钟。同时，从完全融合的先前培养的ISE6细胞培养瓶中取出培养基，并通过用1.5毫升含有碳酸氢钠和HEPES缓冲液的新鲜培养基轻轻冲洗细胞来重悬细胞层。产生均匀的细胞悬液后，将整个体积转移到单个无菌的两毫升微量离心管中。接下来，使用电穿孔器电穿孔帕氏立克次氏体和质粒DNA混合物。电穿孔后，使用无菌扩展细尖移液器将少量ISE6细胞悬液转移到比色皿中，然后轻轻上下移液以回收电穿孔的帕氏立克次体。然后，将比色皿的内容物转移到含有ISE6细胞悬液剩余部分的两毫升管中。接下来，在室温下离心样品，首先以700 G离心两分钟以拉下ISE6细胞，然后在13， 600 G下离心一分钟以拉下立克次体。将试管在 34 摄氏度下孵育 15 分钟至 1 小时。孵育后，将一种转化的重悬内容物转移到一个25平方厘米的细胞培养瓶中，该培养瓶含有3.5毫升含有碳酸氢钠和HEPES缓冲液的新鲜培养基。摇动烧瓶以使混合物均匀分布并在 34 摄氏度下孵育。16至24小时后，将大观霉素和链霉素各10微升加入孵育烧瓶中。摇动烧瓶，将抗生素均匀地混合到培养基中，然后将烧瓶放回培养箱中。野生型帕氏立克次体和ISE6细胞的成功增殖通过吉姆萨染色可见。ISE6细胞的细胞核在细胞内和细胞外立克次氏体中被吉姆萨染色为深紫色是可见的。孵育7天后，通过共聚焦显微镜证实了在ISE6细胞中表达红色荧光蛋白的帕氏立克次氏体的转化。10天后观察到感染率大幅增加。在整个过程中保持所有无菌。注意细节并保持耐心，因为某些种类的立克次氏体需要很长时间才能转化，尤其是生长缓慢的内共生体。该协议使立克次体生物学的多个方面及其与节肢动物载体和哺乳动物宿主的相互作用得以研究。例如，表达荧光蛋白的立克次氏体已被用于跟踪运动性和共生体蜱细胞相互作用。

Research

JoVE Journal

Biology

An Electroporation Method to Transform Rickettsia spp. with a Fluorescent Protein-Expressing Shuttle Vector in Tick Cell Lines

Passaging HuES Human Embryonic Stem Cell-lines with Trypsin.

人类胚胎干细胞传代的色调线与胰蛋白酶

In this video we demonstrate how our lab routinely passages HuES human embryonic stem cell lines with trypsin.  Human embryonic stem cells are artifacts ...

科研

生物学

Tracheotomy: A Method for Transplantation of Stem Cells to the Lung

气管切开术：干细胞移植肺的方法

Lung disease is a leading cause of death and likely to become an epidemic given increases in pollution and smoking worldwide. Advances in stem cell ...

In utero and ex vivo Electroporation for Gene Expression in Mouse Retinal Ganglion Cells

In utero and ex vivo Electroporation for Gene Expression in Mouse Retinal Ganglion Cells

在子宫内和前体内的基因表达在小鼠视网膜神经节细胞的电穿孔

The retina and its sole output neuron, the retinal ganglion cell (RGC), comprise an excellent model in which to examine biological questions such as cell ...

Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency

使用基因脉冲MXcell电穿孔系统主细胞的转染效率高

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells (iPSCs) Using Retroviral Vector with GFP

Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells iPSCs Using Retroviral Vector with GFP

人类体细胞重编程为诱导多能干细胞（iPS细胞）用绿色荧光蛋白逆转录病毒载体

Human embryonic stem cells (hESCs) are pluripotent and an invaluable cellular sources for in vitro disease modeling and regenerative medicine1. It has ...

A Guide to Modern Quantitative Fluorescent Western Blotting with Troubleshooting Strategies

指南现代荧光定量免疫印迹与故障排除策略

The late 1970s saw the first publicly reported use of the western blot, a technique for assessing the presence and relative abundance of specific proteins ...

Internalization and Observation of Fluorescent Biomolecules in Living Microorganisms via Electroporation

通过电穿孔内在和生物分子荧光观察生活中的微生物

The ability to study biomolecules in vivo is crucial for understanding their function in a biological context. One powerful approach involves fusing ...

Method for Measuring the Activity of Deubiquitinating Enzymes in Cell Lines and Tissue Samples

测量去泛素酶的活性细胞系和组织样品的方法

The ubiquitin-proteasome system has recently been implicated in various pathologies including neurodegenerative diseases and cancer. In light of this, ...

Comparison of Three Different Methods for Determining Cell Proliferation in Breast Cancer Cell Lines

的三种不同方法对乳腺癌细胞株确定细胞增殖的比较

Measuring cell proliferation can be performed by a number of different methods, each with varying levels of sensitivity, reproducibility and compatibility ...

An Efficient In Vitro Transposition Method by a Transcriptionally Regulated Sleeping Beauty System Packaged into an Integration Defective Lentiviral Vector

An Efficient In Vitro Transposition Method by a Transcriptionally Regulated Sleeping Beauty System Packaged into an Integration Defective Lentiviral Vector

一种有效的体外转位方法由转录调节的睡眠美容系统封装到一个集成缺陷的慢向量

The Sleeping Beauty (SB) transposon is a non-viral integrating system with proven efficacy for gene transfer and functional genomics. To optimize the SB ...

一种在蜱细胞系中用荧光蛋白表达穿梭载体转化立 克次 体属的电穿孔方法

一种在蜱细胞系中用荧光蛋白表达穿梭载体转化立克次体属的电穿孔方法