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
Summary
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。
Abstract
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。
Introduction
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。
Protocol
Representative Results
Discussion
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?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| Agarose | KSE Scientific | BMK-A1705 | Molecular Biology Grade |
| Anhydrotetracycline hydrochloride | ACROS Organics | 233131000 | |
| CaCl2 Buffer | 10 mM Tris pH 7.4, 50 mM Calcium Chloride Dihydrate | ||
| Calcium Chloride Dihydrate | Sigma | C7902-500G | Suitable for cell culture |
| Cycloheximide | Sigma | 7698-1G | |
| dam–/dcm– Competent E. coli | New England BioLabs | C2925H | |
| DMSO | ATCC | 4-X | Sterile filtered cell culture tested |
| Glutamic acid | Sigma | G8415-100G | L-Glutamic acid |
| Growth Media #1 | RPMI 1640 media supplemented with 10 % (vol/vol) heat-inactivated fetal bovine serum (FBS). | ||
| Growth Media #2 | RPMI 1640 media supplemented with 10 % (vol/vol) heat-inactivated fetal bovine serum (FBS) and 1 µg/mL cycloheximide | ||
| Hanks' Balanced Salt Solution (HBSS) (1x) | Gibco | 24020-117 | |
| Heat Inactivated Fetal Bovine Serum Qualified One Shot (FBS) | Gibco | A38402-02 | |
| McCoy Cells | ATCC | CRL-1696 | |
| Monarch Plasmid Miniprep Kit | New England BioLabs | T1010S | Small scale DNA purification |
| NaH2PO4 | Sigma | S3139-250G | Sodium phosphate monobasic |
| Na2HPO4 | Sigma | S5136-500G | Sodium phosphate dibasic |
| NEB 10-beta Electrocompetent E. coli Cells | New England BioLabs | C3020K | |
| NEBuilder HiFi DNA assembly Cloning Kit | New England BioLabs | E5520S | Gibson Assembly Kit |
| Penicillin G sodium salt | Sigma | P3032-10MU | Bioreagent suitable for cell culture |
| QIAGEN Plasmid Maxi Kit | QIAGEN | 12162 | Large scale DNA purification |
| Q5 Hot Start High-Fidelity DNA Polymerase | New England BioLabs | M0515 | Fragment PCR Polymerase |
| RPMI 1640 Medium (1x) | Gibco | 11875-093 | Containing 2mM L-glutamine |
| Sall-HF | New England BioLabs | R3138S | |
| Sbfl-HF | New England BioLabs | R3642S | |
| Selection Media #1 | RMPI 10 % FBS, 1 µg/mL cycloheximide, 500 µg/mL spectinomycin, and 50 ng/mL anhydrous tetracycline dissolved in DMSO | ||
| Selection Media #2 | RMPI 10 % FBS, 1 µg/mL cycloheximide, 500 µg/mL spectinomycin | ||
| Selection Media #3 | RPMI 10 % FBS, 1 µg/mL cycloheximide, 50 ng/mL aTc, and 0.6 µg/mL penicillin | ||
| Sodium Acetate Buffer Solution | Sigma | S7899-100ML | 3M |
| SOC Outgrowth Medium | New England BioLabs | B9020SVIAL | |
| Spectinomycin dihydrochloride pentahydrate, Cell Culture Grade | Alfa Aesar | J61820 | |
| Sucrose | Sigma | S1888-1KG | Bioreagent suitable for cell culture |
| Sucrose-Phosphate-Glutamate Buffer (SPG) | 37.5g sucrose, 1.25 g Na2HPO4, 0.18 g NaH2PO4, 0.36 glutamic acid for 500 ml tissue culture grade water | ||
| Tris | AMRESCO | 0497-5KG | Ultrapure grade |
| Trypsin-EDTA (1x) | Gibco | 25200-056 | 0.25% |
| Water | Sigma | W3500-500ML | Sterile-filtered, BioReagent, Suitable for cell culture |
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