Name: forward genetic approaches in chlamydia trachomatis
Uploaded: 2013-10-23T18:30:00
Description: Watch this Scientific Journal Video about Forward Genetic Approaches in Chlamydia trachomatis at JoVE.com

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...

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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...

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

正向遗传途径<em&gt;沙眼衣原体</em

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。

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Forward Genetic Approaches in Chlamydia trachomatis

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

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

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 ...

Large-Scale Purification of Porcine or Bovine Photoreceptor Outer Segments for Phagocytosis Assays on Retinal Pigment Epithelial Cells

猪或牛光感受器外段的细胞吞噬分析对视网膜色素上皮细胞大规模纯化

Analysis of one of the vital functions of retinal pigment epithelial (RPE) cells, the phagocytosis of spent aged distal fragments of photoreceptor outer ...

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

Forward Genetics Screens Using Macrophages to Identify Toxoplasma gondii Genes Important for Resistance to IFN-ÃƒÅ½Ã‚Â³-Dependent Cell Autonomous Immunity

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 ...

Utilizing the Antigen Capsid-Incorporation Strategy for the Development of Adenovirus Serotype 5-Vectored Vaccine Approaches

利用衣壳抗原掺入战略的腺病毒血清型5载体疫苗方法的发展

Adenovirus serotype 5 (Ad5) has been extensively modified with traditional transgene methods for the vaccine development. The reduced efficacies of these ...

Phage Phenomics: Physiological Approaches to Characterize Novel Viral Proteins

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

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

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 ...