1。化学诱变注:我们发现该复制的RB的形式是比EB形式更适合于化学诱变。在周期中（18至20 HPI），苏格兰皇家银行是人数最多的前RB-EB过渡。由于沙眼衣原体是一种专性细胞内病原体，宿主健康的影响诱变可以限制细菌复苏。 Vero细胞被发现是有抵抗性的高层次的EMS比其他测试的细胞系的不利影响。 通过重组突变分离需要选择对抗生素有抗药性的重组后代。用于产生能够执行重组分析和隔离等基因的菌株的突变体，我们建议使用耐药菌株（ 如利福平，大观霉素，甲氧苄氨嘧啶）。一个逐步选择过程（15，16）所产生的抗生素抗性菌株。 不E: 沙眼衣原体 （株L2 / 434/Bu的/ ATCC VR902B）BSL2病原体。请参考机构的标准作业程序（SOP）处理这类病原体。 <ol><li>培养细胞和感染的制备: <ol><li>种子约1×10 6 Vero细胞（ATCC CCL-81），每25厘米2（T25）在3毫升烧瓶中的Dulbecco改良的Eagle培养基（DMEM）中，用10％胎牛血清（FBS）的补充。在37℃，5％CO 2。细胞应在24小时内形成一个融合的单层。 </li><li>吸介质。感染汇合的T25烧瓶衣原体感染的Vero细胞的感染复数（MOI）5（〜14×10 6）在总体积为3ml的培养基（DMEM，200 ng / ml的cyclohexamide，10％FBS）。 </li></ol></li><li>开始诱变感染后18小时（HPI）: <ol><li>制备20毫克/毫升的EMS（甲磺酸乙酯）的磷酸盐缓冲盐水（PBS），0.9 mM的钙氯和0.49毫米氯化镁作为EMS的化学安全罩是一种易挥发的液体。 注:EMS是一种有效的诱变剂和致癌物质。中性EMS浪费和净化泄漏，塑料，纸张和玻璃器皿用1 M氢氧化钠。 </li><li>吸出培养基，用3ml的PBS / MgCl 2的/ CaCl 2的洗细胞一次。 3毫升20毫克/毫升EMS在PBS / MgCl 2的/ CaCl 2的一个小时在通风橱中于室温下孵育细胞。 Vero细胞是在短期内能够生存这个浓度的EMS和条件外孵化器。请记住，包括对照样品尚未诱变。 </li><li>移除介质包含EMS，并将其放置在一个容器中的1 M NaOH灭活诱变。清洗感染的细胞在3毫升PBS +的MgCl 2 / CaCl 2的 3次，以除去残留的诱变剂。加入6毫升DMEM/10％FBS 200毫微克/毫升环己酰胺和25微克/毫升的庆大霉素，5％CO2孵育<suB&gt; 2，37°C为48-72小时。夹杂出现无诱变后的36小时左右的细菌。在72小时约10％的夹杂物都充满了细菌，而其余保持为空。 </li><li>低渗裂解提取EBS:完全吸介质。添加1.0毫升H 2 O和岩石撞出细胞10分钟。裂解细胞，通过上下吹打至少10倍。在离心收集裂解液添加0.250毫升的5倍蔗糖磷酸谷氨酸钠缓冲液（SPG），使其终浓度1X。储存于-80℃。 注意:可以进行评估，利福平抗性的诱导水平的诱变。为了测定利福平耐药性的频率，斑块1×10 8 7×10倍的系列稀释液中的细菌的存在下，200毫微克/毫升利福平。利福平耐药性的频率被定义作为镀金的细菌总数除以利福平抗性斑块的数量。参见下面的牌匾说明部分。 </li></ol></li></ol> 2。 沙眼衣原体突变株的蚀斑纯化克隆分离<ol><li>设置汇合单层的Vero细胞在6孔板:的种子〜0.4×10 6细胞在3毫升DMEM/10％FBS每口井。让细胞形成一个均匀的单层在未来24小时。 </li><li>解冻冰诱变衣原体原液。 DMEM/10％FBS中，总体积为200μl稀释每执行6×10倍系列稀释，并将它们设置在冰上。 注:细菌滴度包容形成单位（IFU）是一个很好的估计空斑形成单位（PFU）。旨在牌匾10，100，1000 PFU。 </li><li>洗两次Vero细胞用3ml PBS中，每孔。 </li><li>加入3毫升的DMEM到6孔培养板的各孔中加入100μl的细菌稀释液一式两份。旋流板，以确保均匀混合。 </li><li>自旋感染的板以2,700 xg 30分钟，在15°C，然后在37℃孵育在加湿5％CO 2培养箱中培养1-2小时。 </li><li>准备0.54％琼脂糖DMEM（6井）: <ol><li>添加以下成分:18.9毫升冷2X DMEM，4.2毫升FBS 100X NEAA，0.42毫升，0.042毫升200微克/毫升cyclohexamide的50mg/ml的庆大霉素，0.021毫升。拌匀。 </li><li> 18.9毫升热无菌1.2％琼脂糖/ H 2 O混合，搅拌，以防止团块。混合应该是温暖的触摸。请在温水浴在55°C，然后再增加感染的细胞。 </li></ol></li><li>吸菌悬液的菜肴和应用6毫升0.54％琼脂糖/ DMEM每口井。使琼脂糖完全固化在室温15分钟的引擎盖外。擦干盖子去掉，在15分钟的生物安全罩板。 注:从生物防护罩的振动可以扭曲凝固的琼脂糖。 </li><li>在CO 2培养箱在37℃孵育7-10天。借助在解剖显微镜应该是可见的噬菌斑。 （重新参见图3为一个典型的图像的斑块。） </li><li>一天前隔离的突变体，种子1×10 4 Vero细胞在96孔板每孔100μl。细胞将在24小时内汇合。 </li><li>使用解剖显微镜，标志牌匾被拾起。斑块使用无菌1.0毫升屏障枪头，收集插头。 </li><li>将噬斑重新悬浮于100μlDMEM中含10％胎牛血清，400 ng / ml的cyclohexamide，50微克/毫升庆大霉素补充。 </li><li>为了扩大突变株，覆盖暂停汇合单层Vero细胞上。自旋板在2700 XG，15℃30分钟。 </li><li>在37°C / 5％CO 2培养箱孵育。维斯特胚体&gt; 50％的细胞被感染时，它可以发生早在48 HPI迟为14天。这种感染的细胞量可以存储足够的活菌​​。突变株不同在增长率和感染力。允许生长缓慢株NATURALLY再感染邻近的细胞，直到有足够的细胞被感染。 </li><li>提取EBS低渗裂解感染细胞: <ol><li>完全吸介质。 </li><li>加入160微​​升无菌水。在室温下孵育10分钟。 </li><li>上下吹打几次，以确保完全裂解，破碎细胞，使用障碍提示，以避免交叉污染。 </li><li> 160微升裂解转移到离心管。 </li><li>混合在40μl的5倍盛。储存于-80℃。 </li></ol></li><li>含量和屏幕突变体的表型（S）的利益。 </li></ol> 3。选择衣原体突变体的全基因组测序<ol><li>梯度纯化EBS，这在很大程度上是免费的污染宿主DNA提取基因组DNA。文献可以发现衣原体和净化EBS大型文化协议。 （17）。使用商业基因组DNA纯化试剂盒（ 例如:的 QIAGEN猫。69504）分机RACT从2×10 9个细菌的DNA，按照制造商的指示。 </li><li>使用荧光（量子比特，Invitrogen公司猫。Q32866）准确地测量DNA的量。 1-5微克纯化的DNA所需的Illumina公司的测序平台。 注意:几个平台可用于细菌基因组的序列，包括基于Illumina/Solexa-及固体系统。我们选择使用的HiSeq（Illumina公司），因为它产生的高密度短，但高质量测序结果。规模较小的基因组测序，如IonTorrent（Life Technologies公司）和MySeq（Illumina公司）是合适的并且超过足够的复用这些高达4的时间，超过足够的基因组组装的最小覆盖25X 衣原体基因组的测序。下游的DNA的测序文库Illumina的平台描述如下。 </li><li>适当大小的片段DNA（300-400碱基对Illumina的测序）使用自适应聚焦声学S220仪器（Covaris）根据制造商建议的设置。注:DNA片段雾化结果的样品，由于样品的雾化更大的损失。 </li><li>准备使用商业建筑套件（Ill​​umina的FC-102-1001）的基因组测序库，按照制造商的指示。索引库可以使用条形码等多个样品可以同时汇集和测序引物（Illumina的FC-121-1003）。 </li><li>运行测序样品。这一步通常是由商业服务或核心设备，由专门的技术人员操作。按照他们的程序和建议。 </li><li> Illumina公司的读取（FASTQ格式）可以组装到参考基因组中使用的用户友好的图形用户界面软件，如Geneious（Biomatters），也可以用于SNP /突变鉴定。地图突变，设置参数为90％，最小变异频率和50X最小覆盖。开放式源代码MAQ（18）和宽带无线接入（19），如E组汇编程序，也可以被使用。 </li><li> Sanger测序确认突变:使用基因组DNA为模板，进行PCR扩增300-500 bp的侧翼地区发现的变异稀释（1:20）的PCR产物纯化或Sanger测序和序列。 </li></ol> 4。 衣原体重组子的生成注意: 衣原体可以交换DNA在感染过程中产生的重组菌株（12，13，20），它允许。共同感染的细胞有两个独特的抗生素耐药的菌株，重组"后代"之后，可以选择双抗生素抗性（12，13）。利用这种现象，我们采取分开突变并产生合作等基因的菌株。因此，突变株中产生的抗生素有抗药性的背景（ 例如利福平耐药性（里夫R）H471Y为CTL0567的rpoB），使得它们可以划线，野生型菌株轴承，GA不同的抗生素有抗药性的等位基因（ 如霉素抗性（SPC R），:G1197A r01/r02（16SRNA复制1和2））。关于衣原体 DNA交换和重组的详细信息，请参见参考文献（12，13）。 <ol><li>合作centrifuging6×10 5到24孔板上生长的Vero细胞生长单层每一株细菌感染野生型和突变株克隆。同时，每一株单独感染单层。 </li><li>于44 HPI，收获EBS低渗裂解感染细胞: <ol><li>完全吸介质。 </li><li>添加0.4毫升无菌水，静置10分钟。 </li><li>裂解细胞剧烈吹打向上和向下的至少10倍。转让裂解液至微量离心管中。 </li><li> 1X SPG终浓度为0.1毫升的5倍SPG混合。 </li><li>粗裂解物可以立即使用或储存于-80℃。 </li></ol></li><li>斑块50μl的粗EB预科- 5×1:10系列稀释琼脂糖/ DMEM辅以适当的抗生素选择重组子。 （见表典型的抗生素浓度重组体的选择。） </li><li>斑块净化和丰富重组株以上。分数期望表型的重组体的存在或不存在。从重组菌株中提取DNA（下一节）DNAzol试剂治疗的目的基因的突变的存在或不存在。 </li><li>十字架可以重复轴承其他抗生素抗性标记的菌株进一步分离突变，产生同源菌株。合作到另一个轴承不同的抗生素标记的野生型菌株感染选定重组。 </li></ol> 表1:用于选择重组体的抗生素浓度: <table border="1"><tbody><tr><th>终浓度</th><th>编制说明</th></tr><tr><td> 200 ng / ml的利福平</td&gt; <td> 25毫克/毫升存储库存在二甲基亚砜（DMSO）。高达200微克/毫升工作液稀释用H 2 O的存储库存储存在-20°C在黑暗中。 </td></tr><tr><td> 200微克/毫升的甲氧苄氨嘧啶</td><td>股票DMSO 100毫克/毫升。储存在-20°C </td></tr><tr><td> 200微克/毫升的大观霉素</td><td> 100毫克/毫升股票在水中100微升等分。贮存于-20℃。避免反复冻融。 </td></tr></tbody></table> 5。重组菌株进行基因分型基因组DNA的提取注:也可用于基于列的纯化试剂盒。 DNA提取DNAzol试剂处理的一个优点是成本。 <ol><li> 1.2×10 5衣原体感染单层Vero细胞生长在12孔板上。 </li><li>在36-48 HPI，抽吸介质和添加0.375毫升DNAzol试剂（Invitrogen公司10503-027）。轻轻搅动细胞和吸管莱莎TES到1.5 ml离心管。 </li><li>此外，0.188毫升，100％的乙醇沉淀DNA。颠倒混匀。 </li><li>后台处理用枪头，把它放在一个干净的试管中提取DNA。或者，通过离心沉淀DNA以最快的速度为2分钟，在室温或4℃，倾析上清液。 </li><li>用1.0毫升75％乙醇洗涤DNA沉淀两次。 </li><li>空气干燥除去乙醇，持续15秒后，DNA。 </li><li>溶解DNA，然后0.2毫升8毫米的NaOH中和至pH为8.0与20.2微升0.1 M羟乙基。调出与水或TE，至终体积为0.5ml。另外，在TE缓冲液重悬DNA（DNA沉淀不会完全溶解）。 </li><li>使用DNAzol试剂提取DNA为模板，PCR扩增300-500 bp的侧翼地区确定的突变。序列（1:20）的PCR产物纯化或稀释Sanger测序。 </li></ol>

<ol>
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L., Henson, E., McClarty, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+trimethoprim-+and+sulphisoxazole-resistant+Chlamydia+trachomatis.">Characterization of trimethoprim- and sulphisoxazole-resistant Chlamydia trachomatis.</a> Mol Microbiol. 14 (2), 271-281 (1994).</li><li>Scidmore, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cultivation+and+Laboratory+Maintenance+of+Chlamydia+trachomatis.">Cultivation and Laboratory Maintenance of Chlamydia trachomatis.</a> Curr Protoc Microbiol. Chapter 11, Unit 11A 11 (2005).</li><li>Li, H., Ruan, J., Durbin, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+short+DNA+sequencing+reads+and+calling+variants+using+mapping+quality+scores.">Mapping short DNA sequencing reads and calling variants using mapping quality scores.</a> Genome Res. 18 (11), 1851-1858 (2008).</li><li>Li, H., Durbin, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+and+accurate+short+read+alignment+with+Burrows-Wheeler+transform.">Fast and accurate short read alignment with Burrows-Wheeler transform.</a> Bioinformatics. 25 (14), 1754-1760 (2009).</li><li>Suchland, R. J., Sandoz, K. M., Jeffrey, B. M., Stamm, W. E., 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=Horizontal+transfer+of+tetracycline+resistance+among+Chlamydia+spp.+in+vitro.">Horizontal transfer of tetracycline resistance among Chlamydia spp. in vitro.</a> Antimicrob Agents Chemother. 53 (11), 4604-4611 (2009).</li><li>Sladek, F. M., Melian, A., Howard-Flanders, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Incision+by+UvrABC+excinuclease+is+a+step+in+the+path+to+mutagenesis+by+psoralen+crosslinks+in+Escherichia+coli.">Incision by UvrABC excinuclease is a step in the path to mutagenesis by psoralen crosslinks in Escherichia coli.</a> Proc Natl Acad Sci U S A. 86 (11), 3982-3986 (1989).</li><li>Wang, Y., 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=Development+of+a+transformation+system+for+Chlamydia+trachomatis,+restoration+of+glycogen+biosynthesis+by+acquisition+of+a+plasmid+shuttle+vector.">Development of a transformation system for Chlamydia trachomatis, restoration of glycogen biosynthesis by acquisition of a plasmid shuttle vector.</a> PLoS Pathog. 7 (9), e1002258 (2011).</li></ol>

有没有透露。

这种方法符合遗传分析的基本要求，因为它建立了基因型和表现型之间的联动。重要的是，这是实现不借助常规的分子工具的重组DNA转化的插入失活的基因在细菌中，这通常是在非模型的微生物的基因的功能分析的限速步骤。 一个关键的步骤是，以确保克隆的噬菌斑纯化的突变体。与野生型或突变"钳工"的交叉污染，可以迅速导致突变株被outcompeted。同样，测绘非克隆样本的?...

有一个独特的专性细胞内细菌沙眼衣原体占估计有280万生殖道感染，每年在美国（疾病控制中心），相关的后遗症，如盆腔炎，异位妊娠，不孕不育（1） 衣原体属生理发育周期的两相组成的两种形式:感染性但非复制生小体（EB）和非感染性的，但复制的网状体（RB）。感染开​​始，随后者endoctyotosis（2）的上皮细胞的胚体附件。称为膜结合液泡内夹杂物，EBS分化成RB形式，然后复制二进制裂变的。时周期中，资源块的转换回成胚体，然后将其排出到细胞外空间，开始新一轮的感染的宿主细胞裂解（3）。 沙眼衣原体是难治性标准的分子遗传工具，如针对性的基因置换，转座子，转导噬菌体，大多数细菌遗传学的研究中心一直与常规操作，目前还不清楚在何种程度上个别衣原体基因有助于先天免疫逃避的，营养的收购，发育的过渡，或其他进程在真核宿主（4）重要的病原体的生存。因此，这种病原体仍然很差的特点，尽管其临床重要性。 衣原体菌的基因组。都比较小（〜1 MB）（5）采用新一代测序技术测序的多个物种和生物型。比较基因组分析由WGS衣原体种的演变，并使其适应人类（6-8），并在一定程度上提供了独特的见解，提供了一些有关的潜在的毒力因子的功能（9，10）。 Ť他显示临床分离株的遗传多样性并不提供系统最毒力因子的功能映射所需的分辨率，这大概是因为在这样的基因的突变会被容易地选择反对。如果没有从自然选择，诱变诱发的基因变异，加上有明确的检测措施缺陷致病，可扩展的突变谱，可以调查的混杂影响。化学诱变剂，尤其是有用的，因为它们可以产生空的，有条件的，减少亚效等位基因（功能），和hypermorphic（增益函数）等位基因。随着功能强大的下一代基因组测序技术的到来，这种突变可以很容易地识别和映射。以这种方式，可以强关联之间的突变的基因或遗传途径和一个共同的表型，使正向遗传学的方法中的应用。 临床分离株的基因组序列的嵌合透露between血清型和频繁的重组位点（11）。重组的实证证据证明，通过共同感染两种不同的抗生素有抗药性的菌株和选择双抗重组的后代，这是显露有两种菌株的遗传贡献（12，13）。因此，在一个共同感染的设置的野生型和突变株之间的遗传交换允许针对受影响的偏析化学诱导突变导致所观察到的表型的基因，该基因。 这里，我们描述了一种方法来执行衣原体基因分析在化学诱变，WGS感染细胞内的DNA交换（14）（ 图1），以及系统的基础上的。

<table border="0" cellspacing="0" cols="4">
	<colgroup width="291">
	</colgroup>
	<colgroup width="123">
	</colgroup>
	<colgroup width="144">
	</colgroup>
	<colgroup width="84">
	</colgroup>
	<tbody>
		<tr>
			<td>Dulbecco&#8217;s Modified Eagle Medium (DMEM)</td>
			<td>Life Technologies</td>
			<td>11995-073</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Cellgro</td>
			<td>35-010-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethyl methanesulfonate (EMS)</td>
			<td>Sigma</td>
			<td>M0880</td>
			<td></td>
		</tr>
		<tr>
			<td>Cyclohexamide</td>
			<td>Sigma</td>
			<td>C4859-1ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Gentamicin</td>
			<td>Life Technologies</td>
			<td>15750-060</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS)</td>
			<td>Life Technologies</td>
			<td>14190-144</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS) solution with 0.493 mM MgCl2 and 0.901 mM CaCl2 (PBS+MgCl2/CaCl2)</td>
			<td>Life Technologies</td>
			<td>14040-133</td>
			<td></td>
		</tr>
		<tr>
			<td>1 M NaOH</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>5x SPG buffer (1.25 M sucrose, 50 mM sodium phosphate, 25 mM glutamic acid)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SPG buffer (0.25 M sucrose, 10 mM sodium phosphate, 5 mM glutamic acid)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Water (sterile, tissue culture grade)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>2x DMEM (prepared from powder, buffered with 7.4 g/L Sodium Bicarbonate</td>
			<td>Sigma</td>
			<td>D7777</td>
			<td></td>
		</tr>
		<tr>
			<td>Nonessential amino acids (NEAA)</td>
			<td>Life Technologies</td>
			<td>11140-050</td>
			<td></td>
		</tr>
		<tr>
			<td>1.2% GTG Agarose , autoclaved</td>
			<td>Lonzo</td>
			<td>50070</td>
			<td></td>
		</tr>
		<tr>
			<td>Genomic DNA purification kits</td>
			<td>Qiagen</td>
			<td>69504</td>
			<td></td>
		</tr>
		<tr>
			<td>DNAzol</td>
			<td>Life Technologies</td>
			<td>10503-027</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol (molecular biology grade)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>8 mM NaOH</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>0.1 M HEPES ( 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) buffer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>25 cm2 tissue culture flasks (T25 flasks)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>6-well tissue culture plates</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>12-well tissue culture plates</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>96-well tissue culture plates</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Chemical safety hood</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Biological safety hood</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>&#62;Centrifuge and adaptors for spinning tissue plates</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>&#62;Dissection microscope</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorometer (Qubit)</td>
			<td>Invitrogen</td>
			<td>Q32866</td>
			<td></td>
		</tr>
		<tr>
			<td>Adaptive Focused Acoustics S220 instrument</td>
			<td>Covaris</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

forward genetic approaches in chlamydia trachomatis

沙眼衣原体 ，性病和眼部感染的病原体，仍然差的特点，由于其难解实验转型与重组DNA。我们开发了一种方法来进行遗传分析，C.尽管缺乏分子遗传工具衣原体 。我们的方法包括:）。化学诱变快速生成全面定义基因的突变体库，具有不同表型，II。）全基因组测序（WGS）映射潜在的遗传病变，找到突变基因（S）之间的关联一个共同的表型; III）代重组菌株通过哺乳动物细胞突变体和野生型细菌感染的合作。因此，我们能够建立基因型和表现型之间的因果关系。耦合化学诱导的基因变异与WGS建立相关的基因型 - 表型关联寿LD是广泛适用于大名单，医疗和环境重要的微生物，目前棘手的遗传分析。

暴露于诱变剂会导致夹杂物出现没有细菌，大概是由于细菌细胞死亡。通常情况下，血清型LGV-L2将完全溶解感染后48小时内受感染的细胞，但治疗与诱变这个周期延长到90小时。大约10％的夹杂物有望复苏。在我们的实验中，用20毫克/毫升EMS感染的Vero细胞导致在回收感染性子代，与未处理的对照组相比，减少99％。诱变剂治疗也导致斑块的出现改变形态，包括小斑块（SPQ）（ 图3）。其...

Watch this Scientific Journal Video about Forward Genetic Approaches in Chlamydia trachomatis at JoVE.com

Forward Genetic Approaches in Chlamydia trachomatis

我们描述了一种方法来进行遗传分析化学诱变和全基因组测序的基础上衣原体 。此外，感染细胞内的DNA交换系统可用于遗传作图描述。此方法可广泛适用于微生物系统，缺乏改造系统和分子遗传工具。

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 ...

forward-genetic-approaches-in-chlamydia-trachomatis

Research

JoVE Journal

Immunology and Infection

Forward Genetic Approaches in Chlamydia trachomatis

12.8K 次观看.  Duke University Medical Center. 我们描述了一种方法来进行遗传分析化学诱变和全基因组测序的基础上衣原体 。此外，感染细胞内的DNA交换系统可用于遗传作图描述。此方法可广泛适用于微生物系统，缺乏改造系统和分子遗传工具。 

视频: Forward Genetic Approaches in Chlamydia trachomatis

A Genetic Screen to Isolate Toxoplasma gondii Host-cell Egress Mutants

The widespread, obligate intracellular, protozoan parasite Toxoplasma gondii causes opportunistic disease in immuno-compromised patients and causes birth defects upon congenital infection. The lytic replication cycle is characterized by three stages: 1. active invasion of a nucleated host cell; 2. replication inside the host cell; 3. active egress from the host cell. The mechanism of egress is increasingly being appreciated as a unique, highly regulated process, which is still poorly understood at the molecular level. The signaling pathways underlying egress have been characterized through the use of pharmacological agents acting on different aspects of the pathways1-5. As such, several independent triggers of egress have been identified which all converge on the release of intracellular Ca2+, a signal that is also critical for host cell invasion6-8. This insight informed a candidate gene approach which led to the identification of plant like calcium dependent protein kinase (CDPK) involved in egress9. In addition, several recent breakthroughs in understanding egress have been made using (chemical) genetic approaches10-12. To combine the wealth of pharmacological information with the increasing genetic accessibility of Toxoplasma we recently established a screen permitting the enrichment for parasite mutants with a defect in host cell egress13. Although chemical mutagenesis using N-ethyl-N-nitrosourea (ENU) or ethyl methanesulfonate (EMS) has been used for decades in the study of Toxoplasma biology11,14,15, only recently has genetic mapping of mutations underlying the phenotypes become routine16-18. Furthermore, by generating temperature-sensitive mutants, essential processes can be dissected and the underlying genes directly identified. These mutants behave as wild-type under the permissive temperature (35 &deg;C), but fail to proliferate at the restrictive temperature (40 &deg;C) as a result of the mutation in question. Here we illustrate a new phenotypic screening method to isolate mutants with a temperature-sensitive egress phenotype13. The challenge for egress screens is to separate egressed from non-egressed parasites, which is complicated by fast re-invasion and general stickiness of the parasites to host cells. A previously established egress screen was based on a cumbersome series of biotinylation steps to separate intracellular from extracellular parasites11. This method also did not generate conditional mutants resulting in weak phenotypes. The method described here overcomes the strong attachment of egressing parasites by including a glycan competitor, dextran sulfate (DS), that prevents parasites from sticking to the host cell19. Moreover, extracellular parasites are specifically killed off by pyrrolidine dithiocarbamate (PDTC), which leaves intracellular parasites unharmed20. Therefore, with a new phenotypic screen to specifically isolate parasite mutants with defects in induced egress, the power of genetics can now be fully deployed to unravel the molecular mechanisms underlying host cell egress.

A Genetic Screen to Isolate Toxoplasma gondii Host-cell Egress Mutants

Forward genetics is a powerful method to unravel the molecular level of how Toxoplasma egresses from its host cell. Protocols are provided to chemically mutagenize parasites, enrich for mutants with defects in induced egress, and validate the phenotype of cloned mutants.

遗传筛选隔离弓形虫宿主细胞的出口突变

The widespread, obligate intracellular, protozoan parasite Toxoplasma gondii causes opportunistic disease in immuno-compromised patients and causes birth ...

科研

免疫与感染

Genetic Manipulation in &Delta;ku80 Strains for Functional Genomic Analysis of Toxoplasma gondii

Targeted genetic manipulation using homologous recombination is the method of choice for functional genomic analysis to obtain a detailed view of gene function and phenotype(s). The development of mutant strains with targeted gene deletions, targeted mutations, complemented gene function, and/or tagged genes provides powerful strategies to address gene function, particularly if these genetic manipulations can be efficiently targeted to the gene locus of interest using integration mediated by double cross over homologous recombination.
Due to very high rates of nonhomologous recombination, functional genomic analysis of Toxoplasma gondii has been previously limited by the absence of efficient methods for targeting gene deletions and gene replacements to specific genetic loci. Recently, we abolished the major pathway of nonhomologous recombination in type I and type II strains of T. gondii by deleting the gene encoding the KU80 protein1,2. The &Delta;ku80 strains behave normally during tachyzoite (acute) and bradyzoite (chronic) stages in vitro and in vivo and exhibit essentially a 100% frequency of homologous recombination. The &Delta;ku80 strains make functional genomic studies feasible on the single gene as well as on the genome scale1-4.
Here, we report methods for using type I and type II &Delta;ku80&Delta;hxgprt strains to advance gene targeting approaches in T. gondii. We outline efficient methods for generating gene deletions, gene replacements, and tagged genes by targeted insertion or deletion of the hypoxanthine-xanthine-guanine phosphoribosyltransferase (HXGPRT) selectable marker. The described gene targeting protocol can be used in a variety of ways in &Delta;ku80 strains to advance functional analysis of the parasite genome and to develop single strains that carry multiple targeted genetic manipulations. The application of this genetic method and subsequent phenotypic assays will reveal fundamental and unique aspects of the biology of T. gondii and related significant human pathogens that cause malaria (Plasmodium sp.) and cryptosporidiosis (Cryptosporidium).

Genetic Manipulation in &lt;em&gt;&amp;Delta;ku80&lt;/em&gt; Strains for Functional Genomic Analysis of &lt;em&gt;Toxoplasma gondii&lt;/em&gt;

Here we report a method for using type I and type II &Delta;ku80 strains of Toxoplasma gondii to efficiently generate targeted gene deletions and gene replacements for functional genomic analysis.

遗传操纵<em&gt;Δku80</em&gt;株功能基因组分析<em&gt;弓形虫</em

Targeted genetic manipulation using homologous recombination is the method of choice for functional genomic analysis to obtain a detailed view of gene ...

VIGS-Mediated Forward Genetics Screening for Identification of Genes Involved in Nonhost Resistance

Nonhost disease resistance of plants against bacterial pathogens is controlled by complex defense pathways. Understanding this mechanism is important for developing durable disease-resistant plants against wide range of pathogens. Virus-induced gene silencing (VIGS)-based forward genetics screening is a useful approach for identification of plant defense genes imparting nonhost resistance. Tobacco rattle virus (TRV)-based VIGS vector is the most efficient VIGS vector to date and has been efficiently used to silence endogenous target genes in Nicotiana benthamiana.

	In this manuscript, we demonstrate a forward genetics screening approach for silencing of individual clones from a cDNA library in N. benthamiana and assessing the response of gene silenced plants for compromised nonhost resistance against nonhost pathogens, Pseudomonas syringae pv. tomato T1, P. syringae pv. glycinea, and X. campestris pv. vesicatoria. These bacterial pathogens are engineered to express GFPuv protein and their green fluorescing colonies can be seen by naked eye under UV light in the nonhost pathogen inoculated plants if the silenced target gene is involved in imparting nonhost resistance. This facilitates reliable and faster identification of gene silenced plants susceptible to nonhost pathogens. Further, promising candidate gene information can be known by sequencing the plant gene insert in TRV vector. Here we demonstrate the high throughput capability of VIGS-mediated forward genetics to identify genes involved in nonhost resistance. Approximately, 100 cDNAs can be individually silenced in about two to three weeks and their relevance in nonhost resistance against several nonhost bacterial pathogens can be studied in a week thereafter. In this manuscript, we enumerate the detailed steps involved in this screening. VIGS-mediated forward genetics screening approach can be extended not only to identifying genes involved in nonhost resistance but also to studying genes imparting several biotic and abiotic stress tolerances in various plant species.

Virus-induced gene silencing is an useful tool for identifying genes involved in nonhost resistance of plants. We demonstrate the use of bacterial pathogens expressing GFPuv in identifying gene silenced plants susceptible to nonhost pathogens. This approach is easy, fast and facilitates large scale screening and similar protocol can be applied to studying various other plant-microbe interactions.

VIGS介导的正向遗传学筛选参与非寄主抗性基因鉴定


	Nonhost disease resistance of plants against bacterial pathogens is controlled by complex defense pathways. Understanding this mechanism is important ...

Discovery of New Intracellular Pathogens by Amoebal Coculture and Amoebal Enrichment Approaches

Intracellular pathogens such as legionella, mycobacteria and Chlamydia-like organisms are difficult to isolate because they often grow poorly or not at all on selective media that are usually used to cultivate bacteria. For this reason, many of these pathogens were discovered only recently or following important outbreaks. These pathogens are often associated with amoebae, which serve as host-cell and allow the survival and growth of the bacteria. We intend here to provide a demonstration of two techniques that allow isolation and characterization of intracellular pathogens present in clinical or environmental samples: the amoebal coculture and the amoebal enrichment. Amoebal coculture allows recovery of intracellular bacteria by inoculating the investigated sample onto an amoebal lawn that can be infected and lysed by the intracellular bacteria present in the sample. Amoebal enrichment allows recovery of amoebae present in a clinical or environmental sample. This can lead to discovery of new amoebal species but also of new intracellular bacteria growing specifically in these amoebae. Together, these two techniques help to discover new intracellular bacteria able to grow in amoebae. Because of their ability to infect amoebae and resist phagocytosis, these intracellular bacteria might also escape phagocytosis by macrophages and thus, be pathogenic for higher eukaryotes.

Amoebal coculture is a cell culture system using adherent amoebae to selectively grow intracellular pathogens able to resist phagocytic cells such as amoebae and macrophages. It thus represents a key tool to discover new infectious agents. Amoebal enrichment allows discovery of new amoebal species and of their specific intracellular bacteria.

新的细胞内病原体由Amoebal共培养和Amoebal富集途径探索

Intracellular pathogens such as legionella, mycobacteria and Chlamydia-like organisms are difficult to isolate because they often grow poorly or not at ...

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

The obligate intracellular bacterium Chlamydia elicits a great burden on global public health. C.&#160;trachomatis is the leading bacterial cause of sexually transmitted infection and also the primary cause of preventable blindness in the world. An essential determinant for successful infection of host cells by Chlamydia is the bacterium's ability to manipulate host cell signaling from within a novel, vacuolar compartment called the inclusion. From within the inclusion, Chlamydia acquire nutrients required for their 2-3 day developmental growth, and they additionally secrete a panel of effector proteins onto the cytosolic face of the vacuole membrane and into the host cytosol. Gaps in our understanding of Chlamydia biology, however, present significant challenges for visualizing and analyzing this intracellular compartment. Recently, a reverse-imaging strategy for visualizing the inclusion using GFP expressing host cells was described. This approach rationally exploits the intrinsic impermeability of the inclusion membrane to large molecules such as GFP. In this work, we describe how GFP- or mCherry-expressing host cells are generated for subsequent visualization of chlamydial inclusions. Furthermore, this method is shown to effectively substitute for costly antibody-based enumeration methods, can be used in tandem with other fluorescent labels, such as GFP-expressing Chlamydia, and can be exploited to derive key quantitative data about inclusion membrane growth from a range of Chlamydia species and strains.

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

A live cell fluorescent protein based method for illuminating cellular vacuoles (inclusions) containing Chlamydia is described. This strategy enables rapid, automated determination of Chlamydia infectivity in samples and can be used to quantitatively investigate inclusion growth dynamics.

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 ...

Forward Genetics Screens Using Macrophages to Identify Toxoplasma gondii Genes Important for Resistance to IFN-&#947;-Dependent Cell Autonomous Immunity

Toxoplasma gondii, the causative agent of toxoplasmosis, is an obligate intracellular protozoan pathogen. The parasite invades and replicates within virtually any warm blooded vertebrate cell type. During parasite invasion of a host cell, the parasite creates a parasitophorous vacuole (PV) that originates from the host cell membrane independent of phagocytosis within which the parasite replicates. While IFN-dependent-innate and cell mediated immunity is important for eventual control of infection, innate immune cells, including neutrophils, monocytes and dendritic cells, can also serve as vehicles for systemic dissemination of the parasite early in infection. An approach is described that utilizes the host innate immune response, in this case macrophages, in a forward genetic screen to identify parasite mutants with a fitness defect in infected macrophages following activation but normal invasion and replication in na&#239;ve macrophages. Thus, the screen isolates parasite mutants that have a specific defect in their ability to resist the effects of macrophage activation. The paper describes two broad phenotypes of mutant parasites following activation of infected macrophages: parasite stasis versus parasite degradation, often in amorphous vacuoles. The parasite mutants are then analyzed to identify the responsible parasite genes specifically important for resistance to induced mediators of cell autonomous immunity. The paper presents a general approach for the forward genetics screen that, in theory, can be modified to target parasite genes important for resistance to specific antimicrobial mediators. It also describes an approach to evaluate the specific macrophage antimicrobial mediators to which the parasite mutant is susceptible. Activation of infected macrophages can also promote parasite differentiation from the tachyzoite to bradyzoite stage that maintains chronic infection. Therefore, methodology is presented to evaluate the importance of the identified parasite gene to establishment of chronic infection.

Forward Genetics Screens Using Macrophages to Identify Toxoplasma gondii Genes Important for Resistance to IFN-&amp;#947;-Dependent Cell Autonomous Immunity

Forward genetics is a powerful approach to identify genes in intracellular pathogens important for resistance to cell autonomous immunity. The current approach uses innate immune cells, specifically macrophages, to identify novel Toxoplasma gondii genes important for immune evasion.

正向遗传学屏风使用巨噬细胞识别<em&gt;弓形虫</em&gt;基因的重要的抗IFN-γ依赖性细胞免疫自主

Toxoplasma gondii, the causative agent of toxoplasmosis, is an obligate intracellular protozoan pathogen. The parasite invades and replicates within ...

Phage Phenomics: Physiological Approaches to Characterize Novel Viral Proteins

Current investigations into phage-host interactions are dependent on extrapolating knowledge from (meta)genomes. Interestingly, 60 - 95% of all phage sequences share no homology to current annotated proteins. As a result, a large proportion of phage genes are annotated as hypothetical. This reality heavily affects the annotation of both structural and auxiliary metabolic genes. Here we present phenomic methods designed to capture the physiological response(s) of a selected host during expression of one of these unknown phage genes. Multi-phenotype Assay Plates (MAPs) are used to monitor the diversity of host substrate utilization and subsequent biomass formation, while metabolomics provides bi-product analysis by monitoring metabolite abundance and diversity. Both tools are used simultaneously to provide a phenotypic profile associated with expression of a single putative phage open reading frame (ORF). Representative results for both methods are compared, highlighting the phenotypic profile differences of a host carrying either putative structural or metabolic phage genes. In addition, the visualization techniques and high throughput computational pipelines that facilitated experimental analysis are presented.

Here, we present phenomic approaches for the functional characterization of putative phage genes. Techniques include a developed assay capable of monitoring host anabolic metabolism, the Multi-phenotype Assay Plates (MAPs), in addition to the established method of metabolomics, capable of measuring effects to catabolic metabolism.

噬菌体型组学:生理途径来表征新型病毒蛋白

Current investigations into phage-host interactions are dependent on extrapolating knowledge from (meta)genomes. Interestingly, 60 - 95% of all phage ...

A Simple Red Blood Cell Lysis Method for the Establishment of B Lymphoblastoid Cell Lines

A number of methods exist for the transformation of B lymphocytes by the Epstein Barr virus in vitro into immortalized cell lines. We have developed a new method with a powerful and simple strategy for the establishment of B-LCLs, the red blood cell lysis method. This method simplified the PBMC separation procedure with red blood cell removal, and used as little as 0.5 mL of whole blood for establishing EBV-immortalized cell lines, which can proliferate to large cell numbers in a relatively short amount time with a 100% success rate. The method is simple, reliable, time saving, and applicable to treating a large number of the clinical samples.

A simple red blood cell lysis method for establishing B-LCLs was developed with high immortalization efficiency, requiring a small amount of blood and saving time from initiation to cryopreservation.

一个简单的红细胞溶解法对B淋巴母细胞系的建立

A number of methods exist for the transformation of B lymphocytes by the Epstein Barr virus in vitro into immortalized cell lines. We have developed a new ...

An Efficient and Simple Method to Establish NK and T Cell Lines from Patients with Chronic Active Epstein-Barr Virus Infection

A number of methods have been described to establish NK/T cell lines from patients with lymphoma or lymphoproliferative syndrome. These methods employed feeder cells, purified NK or T cells with as much as 10 mL of blood, or a high-dose of IL-2. This study presents a new method with a powerful and simple strategy to establish NK and T cell lines by culturing the peripheral blood mononuclear cells (PBMC) with the addition of recombinant human IL-2 (rhIL-2), and uses as little as 2 mL of whole blood. The cells can proliferate quickly in two weeks and be maintained for more than 3 months. With this method, 7 NK or T cell lines have been established with a high success rate. This method is simple, reliable, and applicable to establishing cell lines from more cases of CAEBV or NK/T cell lymphoma.

A simple method for obtaining NK and T cell clones from CAEBV patients was developed with high efficiency, a small amount of peripheral blood, and a low-dose of IL-2.

慢性活性泼斯坦-巴尔病毒感染患者 NK 和 T 细胞系建立的高效简便方法

A number of methods have been described to establish NK/T cell lines from patients with lymphoma or lymphoproliferative syndrome. These methods employed ...

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

Chlamydia trachomatis is an obligate intracellular pathogen that has been historically difficult to genetically manipulate. Definitive progress in elucidating the mechanisms that C. trachomatis use to create and maintain a privileged intracellular niche has been limited due to a lack of genetic tools. Fortunately, there have recently been several new advances in genetic manipulation techniques. Among these is the development of fluorescence-reported allelic exchange mutagenesis (FRAEM). This method allows targeted gene deletion coupled with insertion of a selection cassette encoding antibiotic resistance and green fluorescent protein (GFP). Reliance on this strategy can be complicated when targeting genes within polycistronic operons due to the potential of polar effects on downstream genes. Floxed cassette allelic exchange mutagenesis (FLAEM), the protocol for which is described here, was developed to alleviate cassette-induced polar effects. FLAEM utilizes Cre-loxP genome editing to remove the selection cassette after targeted deletion by allelic exchange. The resulting strains contain markerless gene deletions of one or more coding sequences. This technique facilitates direct assessment of gene function and expands the repertoire of tools for genetic manipulation in C. trachomatis.

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

Described here is a method for targeted, markerless gene deletion in Chlamydia trachomatis using floxed cassette allelic exchange mutagenesis, FLAEM.