Name: markerless gene deletion by floxed cassette allelic exchange mutagenesis in chlamydia trachomatis
Uploaded: 2020-01-30T16:00:00
Description: Watch this Scientific Journal Video about Markerless Gene Deletion by Floxed Cassette Allelic Exchange Mutagenesis in Chlamydia trachomatis at JoVE.com

这项工作得到了国家卫生研究所公共卫生服务部（国家卫生研究所）向K.A.Fields提供的赠款（A1065530和Al124649）的支持。

1. pSUmC-4.0的设计和组装，具有与兴趣基因特有的同源武器
<ol><li>识别直接用于删除的基因的上游和下游的 ±3 kb 区域，作为同源重组的 5' 和 3' 同源臂（图 1）。</li>
	<li>设计 PCR 引源到 1） 从衣原基因组 DNA 和 2） 放大 3 kb 5' 同源臂和 2） 包含 15–30 bp 悬伸特定于 pSUmC-4.0 时在 Sall 限制酶位点消化。确保与 pSUmC-4.0 重叠的引基区域的熔融温度为 55°C，并且整个引基的发夹熔合?...

<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=Transformation+of+Chlamydia:+current+approaches+and+impact+on+our+understanding+of+chlamydial+infection+biology.">Transformation of Chlamydia: current approaches and impact on our understanding of chlamydial infection biology.</a> Microbes and Infection. 20 (7-8), 445-450 (2018).</li><li>Kari, L., 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=Generation+of+targeted+Chlamydia+trachomatis+null+mutants.">Generation of targeted Chlamydia trachomatis null mutants.</a> Proceedings of the National Academy of Sciences of the United States of America. 108, 7189-7193 (2011).</li><li>LaBrie, S. D., 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=Transposon+Mutagenesis+in+Chlmaydia+trachomatis+Identifies+CT339+as+a+ComEC+Homolog+Important+for+DNA+Uptake+and+Later+Gene+Transfer.">Transposon Mutagenesis in Chlmaydia trachomatis Identifies CT339 as a ComEC Homolog Important for DNA Uptake and Later Gene Transfer.</a> mBio. 10 (4), 01343 (2019).</li><li>Johnson, C. M., Fisher, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Site-specific,+insertional+inactivation+of+incA+in+Chlamydia+trachomatis+using+a+group+II+intron.">Site-specific, insertional inactivation of incA in Chlamydia trachomatis using a group II intron.</a> PLoS One. 8, 83989 (2013).</li><li>Mueller, K. E., Wolf, K., Fields, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+Deletion+by+Fluorescence-Reported+Allelic+Exchange+Mutagenesis+in+Chlamydia+trachomatis.">Gene Deletion by Fluorescence-Reported Allelic Exchange Mutagenesis in Chlamydia trachomatis.</a> mBio. 7, 01817 (2016).</li><li>Keb, G., Hayman, R., Fields, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Floxed+cassette+Allelic+Exchange+Mutagenesis+Enables+Markerless+Gene+Deletion+in+Chlamydia+trachomatis+and+can+Reverse+Cassette-Induced+Polar+Effects.">Floxed cassette Allelic Exchange Mutagenesis Enables Markerless Gene Deletion in Chlamydia trachomatis and can Reverse Cassette-Induced Polar Effects.</a> Journal of Bacteriology. 200, 00479 (2018).</li><li>Yarmolinsky, M., Hoess, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=THe+legacy+of+Nat+Sternberg:+the+genesis+of+Cre-lox+technology.">THe legacy of Nat Sternberg: the genesis of Cre-lox technology.</a> Annual Review of Virology. 2, 25-40 (2015).</li><li>McKuen, M. J., Mueller, K. E., Bae, Y. S., Fields, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence-Reported+Allelic+Exchange+Mutagenesis+Reveals+a+Role+for+Chlamydia+trachomatis+TmeA+in+Invasion+that+is+Independent+of+Host+AHNAK.">Fluorescence-Reported Allelic Exchange Mutagenesis Reveals a Role for Chlamydia trachomatis TmeA in Invasion that is Independent of Host AHNAK.</a> Infection and Immunity. 85 (12), 00640 (2017).</li><li>Song, L., 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=Chlamydia+trachomatis+Plasmid-Encoded+Pgp4+is+a+Transcriptional+Regulator+of+Virulence-Associated+Genes.">Chlamydia trachomatis Plasmid-Encoded Pgp4 is a Transcriptional Regulator of Virulence-Associated Genes.</a> Infection and Immunity. 81 (3), 636-644 (2013).</li><li>Silayeva, O., Barnes, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gibson+Assembly+facilitates+bacterial+allelic+exchange+mutagenesis.">Gibson Assembly facilitates bacterial allelic exchange mutagenesis.</a> Journal of Microbiological Methods. 144, 157-163 (2017).</li><li>Hackstadt, T., Scidmore, M., Rockey, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipid+Metabolism+in+Chlamydia+trachomatis-Infected+Cells:+Directed+Trafficking+of+Golgi-Derived+Sphingolipids+to+the+Chlamydial+Inclusion.">Lipid Metabolism in Chlamydia trachomatis-Infected Cells: Directed Trafficking of Golgi-Derived Sphingolipids to the Chlamydial Inclusion.</a> Proceedings of the National Academy of Sciences of the United States of America. 92 (11), 4877-4881 (1995).</li><li>Jeffrey, B. M., Suchland, R. J., Eriksen, S. G., Sandoz, K. M., Rockey, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomic+and+phenotypic+characterization+of+in+vitro-generated+Chlamydia+trachomatis+recombinants.">Genomic and phenotypic characterization of in vitro-generated Chlamydia trachomatis recombinants.</a> BMC Microbiology. 13, 142 (2013).</li><li>Suchland, R. J., Bourillon, A., Denamur, E., Stamm, W. E., Rothstein, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rifampin-resistant+RNA+polymerase+mutants+of+Chlamydia+trachomatis+remain+susceptible+to+the+ansamycin+rifalazil.">Rifampin-resistant RNA polymerase mutants of Chlamydia trachomatis remain susceptible to the ansamycin rifalazil.</a> Antimicrobial Agents Chemotherapy. 49, 1120-1126 (2005).</li></ol>

此处描述的FLAEM在沙眼病中生成无标记基因缺失的协议允许有针对性地删除非必需基因，并消除盒式极性效应。该协议依赖于对插入pSUmC 4.0自杀载体的5'和3'同源臂的精心设计，对沙眼的有效转化，以及对分离的突变菌株的仔细筛选。通过这种方法成功的基因组工程导致细菌是非荧光的，并在靶向基因删除部位包含单个loxP疤痕序列。此外，该方法有可能适用于同一C型沙眼菌?...

衣原体沙眼是细菌性传播疾病的主要原因，对人类健康构成重大负担。每年有超过1亿人感染沙眼病。尽管对生殖健康有害，如盆腔炎、宫外孕和/或不育，但大约70%的妇女感染无症状。疾病后遗症与由C.沙眼感染2引起的免疫病理学直接相关。有效的疫苗尚未开发;因此，了解细菌毒性因子和其他细菌基因产物的功能是一个重要而紧迫的研究课题。
由于细胞内细菌、宿主细胞入侵、细胞内复制、后代释放和宿主免疫反应的逃避是关键过程。C. 沙眼形成寄生膜结合的气穴，称为内含物，用于细胞内发育。通过III型分泌系统（T3SS）3分泌效应蛋白，可以建立内含物和许多其他关键过程。由于沙眼的遗传性不通性，这些分泌效应器的功能被限制了很多年。与大肠杆菌不同，许多经典的克隆技术不适用于衣原体。几个主要的限制包括转换效率，缺乏反选择记者，如sacB，和质粒维护。大肠杆菌质粒通常可以无限期地维持与复制的起源和适当的选择性压力，C.沙眼质粒需要额外的8个开放读取帧（pgp1-8）的维护，发现在原生pL2质粒在L2血清质粒4。
近年来，已经产生了多种基因工具，以适应衣原体的独特生物学，但仍有限制5，6，7。通过乙基甲酸酯（EMS）处理的化学诱变可以引入失义突变，或者（不太频繁）可能导致核苷酸转化，引入过早停止的柯顿，产生无意义突变8。转位子插入是有效的基因破坏，但目前技术在衣原体研究是费力和费时的9。EMS治疗和转波龙诱变技术都会产生随机突变，需要严格的筛选方法来分离突变菌株。一种通过插入II组内子（例如，TargeTron）来破坏基因的方法允许定向诱变;但是，此方法受效率的限制，并且插入站点并不总是正确预测10。
荧光报告等位基因交换突变（FRAEM）是一种用于靶向基因删除的策略，同时插入一个提供抗生素耐药性的选择盒和荧光报告器11。然而，FRAEM由于盒式极性效应对下游基因的影响而变得复杂，特别是当靶向多cistronic操作体中的基因时。花环状体等位基因交换突变（FLAEM）是一种新型的遗传方法，旨在缓解以前用FRAEM选择盒12观察到的盒式极性效应。FLAEM利用Cre-loxP基因组编辑来去除选择盒和恢复下游基因的表达。含有抗生素耐药性和绿色荧光蛋白（GFP）的选择盒通过侧翼loxP位点进行了重新设计。这些loxP位点可以在存在Cre重组酶的情况下重组，导致从基因组13中切除盒。这一策略已被证明，以减轻盒诱发的极性效应，当目标tmeA删除12，14。
FRAEM 和 FLAEM 方法都使用相同的自杀载体 pSUmC 4.0，可通过pgp6的诱导表达有条件地保持这种载体。pgp6的表达先前已被证明是质粒保留所必需的，因此用于控制质粒维持11，15。当C.沙眼在介质中生长，辅以无水四环素（aTc）诱导pgp6表达时，保持向量。在没有 aTc 的情况下，矢量将丢失。通过选择盒基因的等位交换，实现靶向基因删除。目标基因的上游和下游直接的3 kb区域作为重组的同源臂。这些手臂被克隆到选择盒的两侧pSUmC 4.0矢量中。通过荧光报告观察成功的C.沙眼转化和重组事件。在选带盒中的载体骨干和gfp上表达mCherry会产生红色和绿色荧光内含物。一旦从培养培养物中去除aTc，仅绿色内含物表示成功重组事件，自杀载体的丢失和选择盒与细菌基因组的整合。
FLAEM 代表 FRAEM 的扩展，它通过随后将 Cre 重组酶表达载体 pSU-Cre 转换为新创建的突变菌株。Cre 重组酶有助于loxP位点之间的重组和选择盒的切除。重组事件通过荧光报告表示。pSU-Cre矢量编码mCherry;因此，成功转化通过添加红色荧光到gfp-表达内含物。在没有选择性压力的情况下培养盒式磁带会导致在 loxP位点进行 Cre 介导重组，而盒式磁带的丢失由仅红色内含物指示。与 pSUmC-4.0 一样，pgp6的诱导表达用于有条件地维持 pSU-Cre。一旦消除aTc和抗生素选择，质粒被治愈，由此产生的无标记缺失菌株是非荧光的。该方法解决了盒式极性效应问题。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Agarose</td>
			<td>KSE Scientific</td>
			<td>BMK-A1705</td>
			<td>Molecular Biology Grade</td>
		</tr>
		<tr>
			<td>Anhydrotetracycline hydrochloride</td>
			<td>ACROS Organics</td>
			<td>233131000</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2 Buffer</td>
			<td></td>
			<td></td>
			<td>10 mM Tris pH 7.4, 50 mM Calcium Chloride Dihydrate</td>
		</tr>
		<tr>
			<td>Calcium Chloride Dihydrate</td>
			<td>Sigma</td>
			<td>C7902-500G</td>
			<td>Suitable for cell culture</td>
		</tr>
		<tr>
			<td>Cycloheximide</td>
			<td>Sigma</td>
			<td>7698-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>dam-/dcm- Competent E. coli</td>
			<td>New England BioLabs</td>
			<td>C2925H</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>ATCC</td>
			<td>4-X</td>
			<td>Sterile filtered cell culture tested</td>
		</tr>
		<tr>
			<td>Glutamic acid</td>
			<td>Sigma</td>
			<td>G8415-100G</td>
			<td>L-Glutamic acid</td>
		</tr>
		<tr>
			<td>Growth Media #1</td>
			<td></td>
			<td></td>
			<td>RPMI 1640 media supplemented with 10 % (vol/vol) heat-inactivated fetal bovine serum (FBS).</td>
		</tr>
		<tr>
			<td>Growth Media #2</td>
			<td></td>
			<td></td>
			<td>RPMI 1640 media supplemented with 10 % (vol/vol) heat-inactivated fetal bovine serum (FBS) and 1 &#181;g/mL cycloheximide</td>
		</tr>
		<tr>
			<td>Hanks&#39; Balanced Salt Solution (HBSS) (1x)</td>
			<td>Gibco</td>
			<td>24020-117</td>
			<td></td>
		</tr>
		<tr>
			<td>Heat Inactivated Fetal Bovine Serum Qualified One Shot (FBS)</td>
			<td>Gibco</td>
			<td>A38402-02</td>
			<td></td>
		</tr>
		<tr>
			<td>McCoy Cells</td>
			<td>ATCC</td>
			<td>CRL-1696</td>
			<td></td>
		</tr>
		<tr>
			<td>Monarch Plasmid Miniprep Kit</td>
			<td>New England BioLabs</td>
			<td>T1010S</td>
			<td>Small scale DNA purification</td>
		</tr>
		<tr>
			<td>NaH2PO4</td>
			<td>Sigma</td>
			<td>S3139-250G</td>
			<td>Sodium phosphate monobasic</td>
		</tr>
		<tr>
			<td>Na2HPO4</td>
			<td>Sigma</td>
			<td>S5136-500G</td>
			<td>Sodium phosphate dibasic</td>
		</tr>
		<tr>
			<td>NEB 10-beta Electrocompetent E. coli Cells</td>
			<td>New England BioLabs</td>
			<td>C3020K</td>
			<td></td>
		</tr>
		<tr>
			<td>NEBuilder HiFi DNA assembly Cloning Kit</td>
			<td>New England BioLabs</td>
			<td>E5520S</td>
			<td>Gibson Assembly Kit</td>
		</tr>
		<tr>
			<td>Penicillin G sodium salt</td>
			<td>Sigma</td>
			<td>P3032-10MU</td>
			<td>Bioreagent suitable for cell culture</td>
		</tr>
		<tr>
			<td>QIAGEN Plasmid Maxi Kit</td>
			<td>QIAGEN</td>
			<td>12162</td>
			<td>Large scale DNA purification</td>
		</tr>
		<tr>
			<td>Q5 Hot Start High-Fidelity DNA Polymerase</td>
			<td>New England BioLabs</td>
			<td>M0515</td>
			<td>Fragment PCR Polymerase</td>
		</tr>
		<tr>
			<td>RPMI 1640 Medium (1x)</td>
			<td>Gibco</td>
			<td>11875-093</td>
			<td>Containing 2mM L-glutamine</td>
		</tr>
		<tr>
			<td>Sall-HF</td>
			<td>New England BioLabs</td>
			<td>R3138S</td>
			<td></td>
		</tr>
		<tr>
			<td>Sbfl-HF</td>
			<td>New England BioLabs</td>
			<td>R3642S</td>
			<td></td>
		</tr>
		<tr>
			<td>Selection Media #1</td>
			<td></td>
			<td></td>
			<td>RMPI 10 % FBS, 1 &#181;g/mL cycloheximide, 500 &#181;g/mL spectinomycin, and 50 ng/mL anhydrous tetracycline dissolved in DMSO</td>
		</tr>
		<tr>
			<td>Selection Media #2</td>
			<td></td>
			<td></td>
			<td>RMPI 10 % FBS, 1 &#181;g/mL cycloheximide, 500 &#181;g/mL spectinomycin</td>
		</tr>
		<tr>
			<td>Selection Media #3</td>
			<td></td>
			<td></td>
			<td>RPMI 10 % FBS, 1 &#181;g/mL cycloheximide, 50 ng/mL aTc, and 0.6 &#181;g/mL penicillin</td>
		</tr>
		<tr>
			<td>Sodium Acetate Buffer Solution</td>
			<td>Sigma</td>
			<td>S7899-100ML</td>
			<td>3M</td>
		</tr>
		<tr>
			<td>SOC Outgrowth Medium</td>
			<td>New England BioLabs</td>
			<td>B9020SVIAL</td>
			<td></td>
		</tr>
		<tr>
			<td>Spectinomycin dihydrochloride pentahydrate, Cell Culture Grade</td>
			<td>Alfa Aesar</td>
			<td>J61820</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma</td>
			<td>S1888-1KG</td>
			<td>Bioreagent suitable for cell culture</td>
		</tr>
		<tr>
			<td>Sucrose-Phosphate-Glutamate Buffer (SPG)</td>
			<td></td>
			<td></td>
			<td>37.5g sucrose, 1.25 g Na2HPO4, 0.18 g NaH2PO4, 0.36 glutamic acid for 500 ml tissue culture grade water</td>
		</tr>
		<tr>
			<td>Tris</td>
			<td>AMRESCO</td>
			<td>0497-5KG</td>
			<td>Ultrapure grade</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (1x)</td>
			<td>Gibco</td>
			<td>25200-056</td>
			<td>0.25%</td>
		</tr>
		<tr>
			<td>Water</td>
			<td>Sigma</td>
			<td>W3500-500ML</td>
			<td>Sterile-filtered, BioReagent, Suitable for cell culture</td>
		</tr>
	</tbody>
</table>

markerless gene deletion by floxed cassette allelic exchange mutagenesis in chlamydia trachomatis

衣原体沙眼是一种强制性的细胞内病原体，在历史上一直难以进行基因操作。由于缺乏遗传工具，在阐明沙眼菌用来创造和维持一个特权细胞内利基的机制方面，进展有限。幸运的是，基因操纵技术最近有了新的进展。其中包括荧光报告等位基因突变（FRAEM）的发展。该方法允许靶向基因删除，同时插入编码抗生素耐药性和绿色荧光蛋白（GFP）的选择盒。由于极性对下游基因的影响，在靶向多cistronic操作器中的基因时，依赖这种策略可能会很复杂。浮环盒等位基因交换突变（FLAEM），本文描述的方案，是为缓解盒引起极性效应而开发的。FLAEM利用Cre-loxP基因组编辑，在以等位交换定向删除后去除选择盒。生成的菌株包含一个或多个编码序列的无标记基因删除。该技术有助于直接评估基因功能，并扩展了C.沙眼遗传操作工具的系列。

使用FLAEM在C.沙眼中无标记基因删除的方法依赖于谨慎的克隆和转化技术。成功的等位基因重组是一个基本的第一步，需要识别和插入同源臂到pSUmC-4.0克隆载体（图1）。无标记基因删除的一个基本第二步是通过Cre-lox基因组编辑去除荧光报告器和抗生素选择盒，如图2所示。用于完成每个步骤的矢量在图 3中进行了批出。<strong...

Watch this Scientific Journal Video about Markerless Gene Deletion by Floxed Cassette Allelic Exchange Mutagenesis in Chlamydia trachomatis at JoVE.com

Markerless Gene Deletion by Floxed Cassette Allelic Exchange Mutagenesis in Chlamydia trachomatis

本文描述的是使用絮状盒状体等位基因突变（FLAEM）在衣原体沙眼中进行靶向、无标记基因删除的方法。

无标记基因删除由絮凝血盒等位基因交换突变在衣原体沙眼

Chlamydia trachomatis is an obligate intracellular pathogen that has been historically difficult to genetically manipulate. Definitive progress in ...

Floxed盒式细胞细胞交换突变是一种重要的方法，因为基因缺失是一种有价值的工具，可用于了解基因产品的功能。这项技术的主要优点是，它允许靶向基因删除，而不会对下游基因造成极性影响。确定直接上下游大约三个千基区域，作为用于同源重组的五个主源和三个主同源臂，并按文本协议所示分配底像。在一到两个反应中，通过 PCR 将新提取的衣原体基因组 DNA 中的三千基五原和三个原源和三个原源臂放大，从而产生足够的片段，供以后的 DNA 组装。在按照文本协议中描述的进行PCR片段的净化和浓缩后，根据制造商的协议，在20微升反应中消化500纳米的pSUmC-4.0向量，其中一个单元为一单元，一个单元为SBF。将消化和纯化的 pSUmC-4.0 向量和五个主源和三个主同源臂 PCR 产品组合在一起，其比例为 0.01 皮毛量向量与每个同源臂的 0.09 象形体。根据制造商的协议，在DNA组装反应中组装碎片。使用一个微升的DNA组装反应，转化50微升的电力大肠杆菌。将转化后的大肠杆菌板板上，每毫升含有100微克的斑点霉素。在37摄氏度下孵育板过夜。第二天，使用荧光倒置显微镜筛选绿色和红色荧光的菌落。预计每个阿加板总共有5到30个菌落。在选定菌落的隔夜液体培养孵育后，使用 PCR 筛选底法确认将每个同源臂插入载体，以放大每次插入。种子麦考伊细胞是小鼠成纤维细胞，通常用于培养衣原体成两个六孔板，如文本协议所述。在1.5毫升管中，加入C.沙眼野生型血清L2基本体，或EBs，体积足以感染12孔的六孔板在2的MOI。将 EBs 以大于 20，000 倍 g 的价格进行 30 分钟，然后丢弃上一液。在 600 微升氯化钙缓冲液中轻轻重新吸收 600 微升的 EB，启动菊花转化。然后，将 12 微克的 pSUMC-4.0 加同源臂添加到管子中，然后轻拂混合溶液。在室温下孵育管子30分钟，每10分钟混合一次。将转化溶液加入 24 毫升 HBSS，然后轻轻移液混合。将两毫升的 inulum 转移到六孔板中每一个汇合麦考伊细胞井中。在20摄氏度下以900次g离心感染，一小时。取出 inoulum，并更换为每井两毫升 RPMI 生长介质 2 号。用5%的二氧化碳在37摄氏度下孵育板。至少7小时后，但不超过12小时，更换介质与两毫升每井选择介质1号。从感染时起，在37摄氏度下继续孵育48小时。使用细胞刮刀轻轻刮擦麦考伊单层将细胞提升至介质中，将细菌收集并传递到麦考伊细胞的新鲜单层上。将材料从每一个井转移到两毫升管中。在4摄氏度的微离心中，在大于20，000倍g的细胞材料中，在30分钟内进行。丢弃上一提液后，通过轻轻移液将细胞颗粒重新在一毫升的HSSS中。在4摄氏度下以200倍g的温度下将细胞碎片取出5分钟。将上流液转移到一个新鲜井的汇合麦考伊细胞。在每个井中再加一毫升的HS，每井的总体积为2毫升。在20摄氏度下以900次g离心感染，一小时。感染后立即将选种介质更换为选择介质。继续每48小时将细菌传到麦考伊细胞的新鲜单层上，直到感染后24小时使用荧光倒置显微镜检测到红色和绿色内含物。检测到红色和绿色内含物后，继续像使用选择介质 2 之前一样传递单层。继续传递单层，直到感染后24小时检测到仅绿色内含物，表明pSUMC-4.0载体丢失，并加入loxP选择盒到基因组中。收获并冻结 SPG 中仅绿色内含物。将绿色包含物丰富到介于 0.5 和 1.0 之间的 MOI 中，并像本文中所述将单层收获到 SPG 中。在1.5毫升管中获得50微升的等分，并在零下80摄氏度下冷冻。种子384井组织培养板与麦考伊细胞，如文本协议所述。稀释HBSS中仅绿色细菌的等分，达到每20毫升约50微克。将稀释的 inulum 转移到储液罐托盘中。将整个盘子倒置到废物容器中，从384井的汇合McCoy细胞板中去除介质。使用多通道移液器，在每孔中加入50微升的C.沙眼 inulum。在20摄氏度下以900次g离心感染，一小时。通过轻拂去除 inoulum，并更换为每井 50 微升的生长介质 2 号。用5%的二氧化碳在37摄氏度下孵育板。在孵育板5至12天后，使用荧光倒置显微镜或高含量筛选平台识别具有绿色荧光内含物的单个孔。通过用 P10 尖端刮刮单层，收获带绿色内含物的井。将整个井内装物转移到含有两毫升HSS的管子中。轻轻混合并应用两毫升的 inoculum 到六井板中新鲜汇合的麦考伊单层，以进行感染。丰富、冻结和滴度克隆种群。使用定量PCR确认从克隆分离的C.沙眼中删除靶向基因。使用克隆分离的 C.沙眼 FRAEM 突变体、pSU-Cre 向量和选择介质三重复转换过程。通过单层，直到检测到仅红色内含物，表示选择盒丢失。与以前一样，通过限制 384 井板中的稀释，克隆分离仅红色内含物。通过传递到0.1的MOI来丰富克隆种群。一旦丰富，将选择介质替换为生长介质 2，以启动 pSU-Cre 矢量的损失。一样克隆分离非荧光细菌。但是，使用 Brightfield 显微镜手动扫描板以检测内含物。丰富和冷冻细菌。使用定量实时PCR、西印迹和全基因组DNA测序（如前所述）筛选突变菌株。代表性数据显示，tmeA 是基因删除的目标。C.沙眼tmeA-lx菌株使用FRAEM生成，C.沙眼tmeA-lx菌株使用FLAEM生成。显示 pSUC-4.0 选择盒中包含的 tmeA 和下游 tmeB 的相对 DNA 拷贝数和 2000 年图的 cre。两种突变菌株都含有 tmeA 位点的缺失。然而，tmeA-lx不包含选择盒，如没有g9p DNA所示。tmeA突变菌株减少了tmeB的表达，如mRNA和蛋白质水平所示。当FLAEM用于生成tmeA-lx突变菌株时，tmeB的表达被mRNA和蛋白质水平检测到。仔细构建具有同源臂的 pSUmC 载体对于有针对性地删除基因至关重要。在衣原体变换之前，应花时间确保准确的矢量装配。FLAEM为研究人员提供了一个工具，可以研究特定基因，在不减少目标基因表达的情况下进行有针对性的删除，这可能混淆了研究。FLAEM产生的C.沙眼缺失突变体可用于研究衣原体生物学的各个方面，包括发育和致病机制。

Research

JoVE Journal

Immunology and Infection

Markerless Gene Deletion by Floxed Cassette Allelic Exchange Mutagenesis in Chlamydia trachomatis

Transfection and Mutagenesis of Target Genes in Mosquito Cells by Locked Nucleic Acid-modified Oligonucleotides

酸修饰的寡核苷酸锁定核酸转染和蚊虫细胞的靶基因突变

Plasmodium parasites, the causative agent of malaria, are transmitted through the bites of infected Anopheles mosquitoes resulting in over 250 million new ...

科研

免疫与感染

Differentiating Functional Roles of Gene Expression from Immune and Non-immune Cells in Mouse Colitis by Bone Marrow Transplantation

区分功能基因表达的作用，通过骨髓移植免疫和非免疫细胞在小鼠结肠炎

To understand the role of a gene in the development of colitis, we compared the responses of wild-type mice and gene-of-interest deficient knockout mice ...

Forward Genetic Approaches in Chlamydia trachomatis

Forward Genetic Approaches in Chlamydia trachomatis

正向遗传途径<em&gt;沙眼衣原体</em

Chlamydia trachomatis, the etiological agent of sexually transmitted diseases and ocular infections, remains poorly characterized due to its ...

Multiplex PCR Assay for Typing of Staphylococcal Cassette Chromosome Mec Types I to V in Methicillin-resistant Staphylococcus aureus

Multiplex PCR Assay for Typing of Staphylococcal Cassette Chromosome Mec Types I to V in Methicillin-resistant Staphylococcus aureus

??PCR?????????????????????MEC??????V<em&gt;???????</em

Staphylococcal Cassette Chromosome mec (SCCmec) typing  is a very important molecular tool for understanding the epidemiology and  clonal strain ...

Cell-based Flow Cytometry Assay to Measure Cytotoxic Activity

基于细胞的流式细胞仪检测，以测量细胞毒活性

Cytolytic activity of CD8+ T cells is rarely evaluated. We describe here a new cell-based assay to measure the capacity of antigen-specific CD8+ T cells ...

Using Fluorescent Proteins to Visualize and Quantitate Chlamydia Vacuole Growth Dynamics in Living Cells

Using Fluorescent Proteins to Visualize and Quantitate Chlamydia Vacuole Growth Dynamics in Living Cells

利用荧光蛋白可视化和定量<em&gt;衣原体</em&gt;液泡生长动态的活细胞

The obligate intracellular bacterium Chlamydia elicits a great burden on global public health. C.&#160;trachomatis is the leading bacterial cause of ...

Identification of Novel Genes Associated with Alginate Production in Pseudomonas aeruginosa Using Mini-himar1 Mariner Transposon-mediated Mutagenesis

Identification of Novel Genes Associated with Alginate Production in Pseudomonas aeruginosa Using Mini-himar1 Mariner Transposon-mediated Mutagenesis

在伴有海藻酸钠生产新基因的鉴定<em&gt;铜绿假单胞菌</em&gt;使用小型<em&gt; himar1</em&gt;水手转座子介导的诱变

Pseudomonas aeruginosa is a Gram-negative, environmental bacterium with versatile metabolic capabilities. P. aeruginosa is an opportunistic bacterial ...

Identification of Rare Bacterial Pathogens by 16S rRNA Gene Sequencing and MALDI-TOF MS

由16S rRNA基因测序和MALDI-TOF MS罕见的细菌病原鉴定

There are a number of rare and, therefore, insufficiently described bacterial pathogens which are reported to cause severe infections especially in ...

A Simple and Efficient Approach to Construct Mutant Vaccinia Virus Vectors

一种简单有效的方法构建突变牛痘病毒载体

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been ...

A Method to Assess Fc-mediated Effector Functions Induced by Influenza Hemagglutinin Specific Antibodies

流感血凝素特异抗体诱导的 Fc 介导效应函数的一种评估方法

Antibodies play a crucial role in coupling the innate and adaptive immune responses against viral pathogens through their antigen binding domains and ...