活细胞向前遗传方法，识别和分离衣 原体沙眼中的发育突变体
Summary
该协议利用荧光启动子-记者，活细胞显微镜，和个人包含提取在定向前的基因方法，以识别和分离 衣原体沙眼的发育突变体。
Abstract
细胞内细菌病原体 沙眼衣原体 经历一个由两种形态离散发育形式的发育周期。非复制性基本体 （EB） 启动宿主的感染。一旦进入，EB分化成沉默体（RB）。然后，RB 经过多轮复制，然后区分为传染性 EB 形式。由于在细胞类型之间无法切换，无法防止宿主入侵或复制，因此此循环对衣原体生存至关重要。
由于衣原体细胞内性质的强制性 ，基因技术的限制 阻碍了细胞类型发育中分子机制的识别。我们设计了一种新型的双启动子-报告机质粒系统，结合活细胞显微镜，可实现细胞类型实时切换的可视化。为了识别参与细胞类型发育调节的基因，利用活细胞启动-报告器系统，将双报告机菌株的化学突变、 衣原体成像和跟踪与改变的发育动力学 相结合，然后对突变体进行克隆分离，以开发前瞻性遗传方法。这种前瞻性基因工作流程是一个灵活的工具，可以修改为定向审讯到广泛的遗传途径。
Introduction
沙眼衣原体 （Ctr）是一种强制性的细胞内病原体，通过双相发育周期，这是其生存和增殖必不可少的1。这个周期包括两种发育形式，基本身体（EB）和沉默体（RB）。EB是复制无能，但通过效应诱导内分泌2调解细胞入侵。进入主机后，EB 将成熟到复制 RB。RB 在转换回 EB 之前执行多轮复制，以便启动后续的感染。
有限的基因工具将大部分衣原体研究限制在生化研究或使用代孕系统。因此，阐明基因调控与发育周期控制已困难,3、4。衣原体领域一个更重要的挑战是高分辨率时间跟踪衣原发育周期和确定其调控所涉及的蛋白质。在衣原体发育周期期间的基因表达传统上由破坏性的”终点”方法进行，包括RNAseq、qPCR和固定细胞显微镜5,5，6。虽然这些方法提供了宝贵的信息，但采用的技术是费力的，具有低时分辨率5,5，6。
在过去十年中，Ctr的基因操作随着质粒转化和诱变方法的引入而取得进展,8,。在这项研究中，开发了一个基于质粒的系统，用于监测个体内含物在感染过程中实时的衣原体发育。创建一个克拉米转换剂，同时表示 RB 和 EB 细胞类型特定的启动子-执行者。RB具体记者是通过将荧光蛋白三叶草上游的早期RB基因的启动者引信所构建的。EUO是一个转录调节器，抑制后期EB相关基因的子集10。hctB的启动剂，编码一种与EB核素凝结中涉及的组蛋白样蛋白质，直接克隆在mKate2（RFP）的上游，以创建EB特异性记者11。hctB 舞会-mKate2/euo舞会-克洛弗的骨干是p2TK2SW27。hctB 和 euo启动子从 Ctr-L2 基因组 DNA 中扩增。每个启动子序列由指定衣原基因的预测的转录开始位点上游的+100个碱基对以及相应 ORF 前 30 个核苷酸（10 个氨基酸）组成。荧光FP变异被商业地获得为Ctr codon优化基因块，并在框架中克隆与每个氯核基因和启动子前30个核苷酸。incD 终止器直接克隆在 mKate2 的下游。第二个发起人 – 记者入下端接终止器的下游。p2TK2SW2 中的安西林抗药性基因 （bla） 被 pBam4 中的aadA基因 （Spectinomycin 耐药性） 所取代.这导致了最终的构造p2TK2-hctB舞会-mKate2/euo舞会-克洛弗（图1A），被转换成CT-L2hctB7。此 RB/EB 报告器应变允许使用活细胞显微镜观察单个内含物内的发育周期（图 1B，C）。
采用我们的启动器-记者结构结合化学突变，设计了一个协议，跟踪和分离从Ctr血清L2突变种群中表现出发育异常的个体克隆。该协议允许直接监测单个衣原体内含物，跟踪基因表达特征，识别表达改变发育基因表达模式的衣原体克隆，以及 将衣原 体与单个内含物进行克隆分离。
虽然这个协议是专门为识别与衣原体发育有关的基因而创建的，但它可以很容易地适应，以询问任何数量的衣原体遗传途径。
Protocol
Representative Results
Discussion
由于现有遗传工具的局限性，对控制衣原体发育周期的分解机制进行了阻碍。将我们的启动者-记者衣原体与活细胞自动显微镜结合使用，建立了一个系统，可在24小时的时间内监测单个内含物中的细胞类型发展。该系统结合化学诱变和直接内含分离，建立了一种快速和克隆选择表达改变发育特征的衣原体的方法（图5）。
当提供细胞内离子?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
我们感谢华盛顿州立大学的安德斯·奥姆斯兰博士提供CIP-1斧头媒体。这项工作得到了NIH授权R01AI130072、R21AI135691和R21AI113617的支持。爱达荷大学和模拟复杂互动中心的启动资金通过NIH赠款P20GM104420提供了额外支持。
Materials
| 24-well polystyrene plates | Corning | 3524 | Cell culture growth for reinfection of isolates |
| 6-well glass bottom plates | Cellvis | P06-1.5H-N | Cell culture growth for imaging |
| 96-well glass bottom plates | Nunc | 165305 | Cell culture growth for imaging |
| Bold line CO2 Unit | OKO Labs | CO2 UNIT BL | Stage incubator CO2 control |
| Bold line T Unit | OKO Labs | H301-T-UNIT-BL-PLUS | Stage incubator temperature control |
| Borosilicate glass capillary tubes | Sutter Instrument | B1005010 | Capillary tubes |
| BrightLine bandpass emissions filter (514/30nm) | Semrock | FF01-514/30-25 | Fluoescent filter cube |
| BrightLine bandpass emissions filter (641/75nm) | Semrock | FF02-641/75-25 | Fluoescent filter cube |
| CellTram Vario | Eppendorf | 5196000030 | Microinjector |
| Chlamydia trachmatis serovar L2 | ATCC | VR-577 | Chlamydia trachomatis |
| CIP-1 media | In house | NA | Axenic media. IPB supplemented with 1% FBS, 25 μM amino acids, 0.5 mM G6P, 1.0 mM ATP, 0.5 mM DTT, and 50 μM GTP, UTP, and CTP. (Omsland, A. 2012) made in-house. |
| Cos-7 cells (ATCC) | ATCC | CRL-1651 | African green monkey kidney cell (host cells) |
| Cycloheximide | MP Biomedicals | 194527 | Host cell growth inhibitor |
| Ethyl methanesulfonate, 99% | Acros Organics | AC205260100 | Mutagen |
| Fetal Plex | Gemini Bio-Products | 100-602 | Supplement for base growth media |
| Fiji/ImageJ | https://imagej.net/Fiji | NA | Open sourse Image analysis software. https://imagej.net/Fiji |
| Galaxy 170 S CO2 incubator | Eppendorf | CO1700100X | Cell culture incubation |
| gblocks (Fluorescent FP variants: Clover and mKate2) | Integrated DNA Technologies | NA | gblock ORFs of Ctr optimized FP varients for cloning into p2TK2SW2 |
| Gentamycin 10mg/ml | Gibco | 15710-064 | Antibiotic for growth media |
| HBSS (Hank's Balanced Salt Solution) | Corning | 21-020-CM | Host cells rinse |
| Heparin sodium | Amersham Life Science | 16920 | inhibits and reverses the early electrostatic interactions between the host cell and EBs |
| HEPES 1M | GE Life Sciences | SH30237.01 | pH buffer for growth media |
| InjectMan | Eppendorf | 5179 000.018 | Micromanipulator |
| Jupyter Notebook | https://jupyter.org/ | NA | Visualization of inclusion traces. https://jupyter.org/ |
| Lambda 10-3 | Sutter Instrument | LB10-3 | Filter wheel controler |
| Oko Touch | OKO Labs | Oko Touch | Interface to control the Bold line T and CO2 Unit |
| Prior XY stage | Prior | H107 | Motorized XY microscope stage |
| PrismR Centrifuge | Labnet | C2500-R | Temperature controlled microcentrifuge |
| Problot Hybridization oven | Labnet | H1200A | Rocking Incubator for infection with Chlamydia |
| Proscan II | Prior | H30V4 | XYZ microscope stage controler |
| Purifier Class 2 Biosafety Cabinet | Labconco | 362804 | Cell culture work |
| RPMI-1640 (no phenol red) | Gibco | 11835-030 | Base growth media for imaging |
| RPMI-1640 (phenol red) | GE Life Sciences | SH30027.01 | Base growth media |
| scopeLED excitation LEDs (470nm,595nm) | scopeLED | F140 | Excitation light |
| Sonic Dismembrator Model 500 | Fisher Scientific | 15-338-550 | Sonicator, resuspending chlamydial pellet |
| Stage incubator | OKO Labs | H301-K-FRAME | Cluster well plate incubation chamber |
| sucrose-phosphate-glutamate buffer 1X (SPG) | In house | NA | Chlamydial storage buffer. (10 mM sodium phosphate [8 mM K 2HPO 4, 2 mM KH 2PO 4], 220 mM sucrose, 0.50 mM L-glutamic acid; pH 7.4) |
| T-75 Flasks | Thermo Scientific | 156499 | Cell culture growth |
| TE 300 inverted microscope | Nikon | 16724 | microscope |
| THOR LED | Thor Labs | LEDD1B | White light |
| Trypsin | Corning | 25-052-CI | Dislodges host cells from flask for seeding into plates |
| Zyla sCMOS | Andor | ZYLA-5.5-USB3 | imaging camera |
| µManager 2.0gamma | https://github.com/micro-manager/micro-manager | NA | Open sourse automated microscope control software package |
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