这项工作得到补助从NIH DJB（AI041930，AI073142，AI075931，AI091461）。

<ol>
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作者宣称，他们有没有竞争经济利益。

在这里，我们提供了一个协议，用于有效的基因定位在ΔKu80的寄生虫株基因操纵拥有对象的基因缺失，基因置换和/或标签的基因的后代，以使有效地回收。的顺序执行这些方法提供了可靠的包含单个或多个目标的遗传操作1-3的寄生虫突变体的分离方法。有针对性的删除而产生取决于多种因素，ΔKu80的应变与所提出的策略和协议的使用极大地提高了工作效率和易于精确定义的突变株T.功能基因组研究中的弓形虫 。 基因组的T。弓形虫在无性阶段的单倍体。因此，考虑规划时删除基因，该基因不得在体外至关重要的。然而，效率的圆盾鼎基因敲除在ΔKU80株是非常高，这使隔离显著受损的复制率的突变株。如果有针对性的基因是必不可少的，寄生虫往往在选择过程中停止复制。针对性的基因操纵的另一个关键因素是必须取得至少620 bp的基因组靶向侧翼检测到有针对性的集成，完美的同源性。较长的同源性区域产生更高的效率目标和使用目标约1,000 bp的DNA侧翼是一个可靠和有效的方法1-4。出于这个原因，重要的是产生T的同一菌株的基因组的目标侧翼刚地弓形虫 （RH，保诚）作为操纵基因的菌株。缺乏足够的同源性，经常导致在1个基因组的定位后刀面的靶向整合，但没有其他。不完全整合或未能取得淘汰赛MAŸ也可以由于游离持久。转染后，立即所有的靶向分子的非集成的附加体，有时靶向分子，可以持续许多世代的附加体。 〜1 KBP使用目标DNA侧翼，并继续通过寄生虫在选择之前，再亚克隆经常解决与游离的持久性有关的问题。 一旦淘汰赛进行验证，并除去的HXGPRT标记，基因打靶策略可以重复1-3在一个单一的寄生虫株产生多个基因缺失的第二GOI。缺失，替换，或标记基因的产生，也可以通过使用不同的可选择的标记物（ 博莱霉素，CAT，乙胺嘧啶抗性DHFR等）。 CAT可选择标记虽然目前存在于不同IIΔKu80的保诚基因组1，此标记可以使用HXGPRT除去</&gt;选择这里描述的协议。在我们的手中，HXGPRT选择是最安全和最有效的靶向效率最高的单拷贝针对性插入标记是足够强大的选择在MPA + X介质。HXGPRT还提供了目前唯一有用的方法，有针对性地删除一个可选择的标记（ 图3）使用6 -硫代黄嘌呤（6TX）选择2,3。因此，该协议提供了目前唯一的方法定义的突变株与一些有针对性的遗传操作。 ΔKu80的弓形虫株进行有针对性的遗传操作，该协议提供了一个有价值的和有效的方法，和适用于一个单一的基因，基因家族，或功能基因组的全基因组研究的分析。 ΔKu80的菌株1,2之前，可用的，这些方法尚未得到广泛应用，由于到该INE这些方法fficiency。因此，该协议是广泛适用于有针对性的遗传操作弓形虫和所有感兴趣在寄生虫宿主反应，生物学，功能基因组学的问题上解决一系列调查访问。

弓形虫病是一种常见的专性细胞内原生动物寄生虫，经常和慢性感染动物和人类的广泛的5。据估计，目前超过1亿人是慢性感染这种病菌。除了​​T所引起的疾病的重要性弓形虫感染的实验工具，强大的基因组学资源，提高可用性，易用性在体外生长和优良的小鼠模型T.弓形虫广泛的研究真核细胞病原体和其他重大apicomplexan寄生虫引起严重的疾病，如疟疾（ 恶性 ）。和隐孢子虫（ 隐孢子虫 ）5,7领先的模型系统。 à重大限制弓形虫作为模式生物的后代进行有针对性的遗传操作一直是低效恢复。这的proble基因打靶米是由于同源重组频率低，相对于非同源重组的频率非常高，在野生型菌株T.广泛的DNA同源性弓形虫即使目标DNA分子遗传学研究2。 最近，我们的基因类型的非同源重组的主要途径受阻I型和II株弓形虫通过删除编码的KU80蛋白质1,2基因。由此产生的类型I和II型ΔKu80的菌株表现出正常的生长速率， 在体外和在体内过程中速殖子和bradyzoite阶段的体外和体内的大小和行为，但是，这些菌株表现出基本上是100％的同源重组频率，这表型的可能性增加迅速隔离所需的后代有针对性的遗传操作由几百到几寿美国和倍1,2,4。最近社会广泛使用的类型，I型和IIΔKu80的菌株有显着提升的步伐，品种，以及T中有针对性的遗传方法的成功率弓形虫1-4,8-13。在这里，我们描述了一个全面的协议，有针对性的基因缺失，基因置换，基因标记ΔKU80株T.弓形虫 。我们演示了如何设计和可靠的目标遗传操作特定的基因或基因位点在ΔKU80株弓形虫。

<table><tbody><tr><td>Overlap Primers</td><td>Integrated DNA Technologies</td><td></td><td>4 nmole Ultramer DNA oligos</td></tr><tr><td>Validation Primers</td><td>Integrated DNA Technologies</td><td></td><td>100 nmole DNA Oligo</td></tr><tr><td>Yeast Strain #90845</td><td>ATCC</td><td></td><td>Designation FY834</td></tr><tr><td>Shuttle Vector pRS416</td><td>ATCC</td><td>87521</td><td></td></tr><tr><td>DH10B &lt;em&gt;E. coli&lt;/em&gt;</td><td>Invitrogen</td><td>12033-015</td><td>SOC broth in kit with &lt;em&gt;E. coli&lt;/em&gt;</td></tr><tr><td>Resuspension Buffer</td><td>Qiagen</td><td></td><td>Buffer P1 in QIAprep Spin Miniprep Kit</td></tr><tr><td>Miniprep Kit</td><td>Qiagen</td><td>27104</td><td>QIAprep Spin Miniprep Kit</td></tr><tr><td>Glass Beads</td><td>Scientific Industries</td><td>SI-BG05</td><td>0.5 mm acid-washed</td></tr><tr><td>Qiacube Automated Robotic Work Station</td><td>Qiagen</td><td></td><td></td></tr><tr><td>Electroporation Cuvette</td><td>USA Scientific</td><td>9104-5050</td><td>2 mm-gap</td></tr><tr><td>BTX600 electroporator</td><td>BTX</td><td></td><td></td></tr><tr><td>Maxiprep Kit</td><td>Qiagen</td><td>12662</td><td>QIAprep Spin Maxiprep Kit</td></tr><tr><td>25 cm&lt;sup&gt;2&lt;/sup&gt; Canted neck plug seal flask</td><td>Corning</td><td>430168</td><td></td></tr><tr><td>150 cm&lt;sup&gt;2&lt;/sup&gt; Canted Neck plug seal flask</td><td>Corning</td><td>430823</td><td></td></tr><tr><td>Human Foreskin Fibroblasts (HFF) cells</td><td>ATCC</td><td>SCRC1041</td><td></td></tr><tr><td>Swin-lok Filter Holder</td><td>Whatman</td><td>420200</td><td>25-mm-diameter</td></tr><tr><td>Membrane</td><td>Whatman</td><td>110612</td><td>Nucleopore Track-etched Membrane 3 mm pore size, 25-mm-diameter</td></tr><tr><td>Mycophenolic acid (MPA)</td><td>Sigma</td><td></td><td></td></tr><tr><td>Xanthine (X)</td><td>Sigma</td><td></td><td></td></tr><tr><td>96-well Tissue Culture Tray</td><td>Corning Costar</td><td></td><td></td></tr><tr><td>24-well Tissue Culture Tray</td><td>Corning Costar</td><td></td><td></td></tr><tr><td>MEM Eagle Media</td><td>Lonza Biowhittaker</td><td>12-611F</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Gibco</td><td>26140-111</td><td></td></tr><tr><td>Antibiotic-Antimycotic (Anti-Anti)</td><td>Gibco</td><td>15240-062</td><td></td></tr><tr><td>Spin Column</td><td>Primm Labs</td><td>PAE-100</td><td>Easy Clean DNA Extraction Spin Kit</td></tr><tr><td>T4 DNA Ligase</td><td>New England Biolabs</td><td></td><td></td></tr><tr><td>6-thioxanthine</td><td>Acros Organics</td><td></td><td></td></tr><tr><td>Tissue DNA Minikit</td><td>Qiagen</td><td>51104</td><td>QIAamp DNA Blood Mini Kit</td></tr><tr><td>Cell Culture Freeze/Recovery Media</td><td>Gibco</td><td>126-48-010</td><td></td></tr><tr><td>Phosphate Buffered Saline</td><td>Hyclone</td><td>SH30028.02</td><td>minus calcium, minus magensium</td></tr></tbody></table>

genetic manipulation in &delta;ku80 strains for functional genomic analysis of toxoplasma gondii

针对性的遗传操作使用同源重组的方法的选择功能基因组学分析，以获得基因的功能和表型（次）的详细视图。与靶基因缺失，有针对性的突变，传统的基因的功能，和/或标签的基因的突变株的发展提供了强有力的战略，以处理基因的功能，尤其是当这些基因操作可以有效地针对感兴趣的使用整合介导的基因位点，通过双击跨越同源重组。 由于非同源重组，功能基因组学分析弓形虫率非常高的情况下有效的方法，针对特定基因位点的基因缺失和基因替换先前已限制。最近，我们取消了非同源重组的主要途径，在I型和II型菌株T.弓形虫删除基因encodin克的KU80蛋白质1,2。 Δku80株正常工作过程中速殖子（急性）和bradyzoite（慢性） 在体外和体内的阶段，并表现出基本上是一个100％的同源重组频率。 ΔKu80的菌株进行功能基因组学研究可行的单一基因，以及基因组规模1-4。 在这里，我们报告的方法使用类型I型和IIΔku80Δhxgprt株推进T中的基因打靶方法弓形虫 。我们概述了用于产生对象的次黄嘌呤-黄嘌呤-鸟嘌呤磷酸核糖转移酶（HXGPRT）可选择标记的插入或缺失的基因缺失，基因置换和标记基因的有效方法。所描述的基因打靶协议可以用在多种方式推进Δku80株寄生虫基因组功能分析和开发单一菌株进行多个有针对性的遗传操作。这种遗传方法的应用和随后的表型分析将揭示T.生物学方面的基础和独特的弓形虫和相关重大人类病原体引起的疟疾（ 恶性 ）。和隐孢子虫病（ 隐孢子虫 ）。

提供的模板的详细构造一个目标质粒删除的基因，包括限制性酶切位点的位置和生成，促进基因定位和基因打靶技术的验证，以及为随后删除HXGPRT，在质粒构建的引物单步的过程（ 图1A-C，图2A，图3A）。作出有针对性的基因缺失（ 图2A），用来验证的淘汰赛删除的引物对，例如，I型rop18（ 图2B），以及具有代表性的PCR验证结果的一般原理图 （ 图2C）。此代表性的结果示出为说明结果的范围内，可以得到在基因位点是比较困难的目标。成功的目标的基因的缺失将导致在没有的基因，在感兴趣的PCR产物（PCR 1），3&#39;的基因组的目标侧翼（PCR2）的存在下，以及存在的HXGPRT适当整合的可选择的标记之间的5&#39;和3&#39;的基因组的目标侧翼定义的删除（PCR3及PCR4） 。验证克隆3，4，5，6，8，9，和11有针对性的基因缺失（击倒）HXGPRT已经取代了印度政府（ 图2C）。 表示不包含基因缺失的克隆，可以由各种不同的条带模式。通常情况下，没有基因缺失的克隆将镜像亲本株（亲柄）与PCR1和PCR2的观察到的波段，但不为PCR3或PCR4看到的克隆1和2（ 图2C）。这种"家长模式"源于一些潜在的机制。有时候，MPA抗寄生虫进行非集成坚持附加体pΔGOI目标质粒，我们注意到，继续选择常常迫使这些附加体集成。我们也观察到一些罕见的背景不同IΔKu80的菌株由于集成的pΔGOI意外定位质粒其中包含一个的HXGPRT cDNA的通过表达DHFR的 5&#39;和3&#39;的启动子元件，到DHFR基因座或到部分删除的HXGPRT的轨迹14。虽然这种罕见的事件是有针对性的整合，它不是旨在整合和作为一个关键点要记住在设计任何基因替换策略。质粒DNA同源性进行您的pΔGOI针对大于120个基点可能会提供一个替代网站重组在ΔKU80株2，可以给一个不受欢迎的背景中。在这样的背景下，减少或不存在的相比，I型II型保诚ΔKu80的菌株因为是基于I型序列核苷酸多态性时相比，II型DNA的HXGPRT可选择标记大大降低了这一难得的背景在实验中使用II型PRUΔKU80应变1。如果一个基因位点是必不可少的（不能删除），或有一个极低的基因靶向频率轨迹，或如果坚持[]附加体都是不容易消除了通过增长和选择非集成，这种父母的格局将主宰观察MPA的图案抗性克隆。另外，有时可观察到靶向DNA分子只在5&#39;或3&#39;的基因组的目标侧翼和一个带观察为PCR3或PCR4（但不是两者）看到随着PCR1和PCR2在克隆10（5集成集成）和克隆7（3&#39;整合）（ 图2C）。这些模式表明单个交叉的不经常发生的，在上面的轨迹，集成的HXGPRT，但此对象的集成不删除感兴趣的基因为目标的质粒整合。这种模式（泳道7＆10 图2C）EM强调说需要报告PCR数据验证，定点整合的发生，而不是一个单一的同源跨越的目标分子的第二非同源整合。在极少数情况下，我们观察到基因的克隆12（ 图2C），表示父母图案和有针对性的删除模式表示的混合物。这种模式最有可能产生有时从两个克隆以及在同一地点存在的寄生虫基因型。 示出的一般原理用于去除的HXGPRT来自Δku80Δgoi的:: HXGPRT删除HXGPRT的应变（ 图3A）。的引物对用于验证从ΔKu80的ΔGRA2 :: HXGPRT（ 图3B）的去除HXGPRT PCR5的一个独特〜1.2 kbp的带放大看到克隆4，第7，第9，第11和12（ 图3C）。如果不从HXGPRT的轨迹，3〜。4 KBP波段（克隆1，2，3，6，8，10，和父母控制）。几种机制的作用，使去除HXGPRT（6TX选择）更具挑战性和一体化的HXGPRT（MPA + X选择）的效率比。 MPA的选择是简单更有效率大于6TX选择14,16。此外，更高的水平的HXGPRT表达需要为的挑选6TX比MPA选择16,24。因此，可能减少HXGPRT的酶活性或基因突变或表观遗传学变化，减少在一个基因的基因表达水平的HXGPRT的突变，有可能取消的有效性选择6TX。即使有了这些挑战6TX选择的的挑选6TX在ΔKu80的菌株，成功率大于90％1-3在第一次尝试。 直接的C-末端标记的蛋白编码基因的一般方案（ 图4）所示。这计划采用直接集成通过3 HXGPRT&#39;的标记基因同源重组的双重跨越，也采用类似上述的验证策略。当一个基因被怀疑是一个不可缺少的基因，这可以使用第二个独立隔离的目标DNA质粒转染验证。替代的方法，如计划调控基因的表达，可用于进一步核实是必不可少的，如果一个基因25。 <img alt="图1" fo:content-width="6.5in" fo:src="/files/ftp_upload/50598/50598fig1highres.jpg" src="/files/ftp_upload/50598/50598fig1.jpg" /> 图1。靶向DNA质粒的设计概述。 。设计示意图重叠引物PCR扩增5&#39;和3&#39;目标侧翼的pRS416穿梭载体和HXGPRT的小基因盒的33 bp的重叠。在原理图中所示的引物为用于产生的5&#39;和3&#39;靶的侧翼pΔROP1810 B.设计目标DNA分子的整体策略。骨干pΔGOIpRS416穿梭载体含尿嘧啶（市建局）和氨苄青霉素（AMP）可选择的标记。 pRS416插入的是一个1〜的KBP 5的DNA扩增基因组DNA的HXGPRT小基因盒和pRS416使用F1和R1引物重叠的目标侧翼，1〜3 KBP&#39;从基因组DNA扩增目标DNA侧翼重叠HXGPRT小基因盒pRS416使用F2和R2引物和HXGPRT的小基因盒。以下独特的限制性内切酶消化站点被添加到引物:RE.X中的F1引物（限制性内切酶的X剪切站点）切割质粒在目标DNA的5&#39;侧翼的5&#39;末端，在R2 RE.Y切割的质粒的引物中的R1和F2的引物在3&#39;端的3&#39;的目标DNA侧翼和RE.Z切除HXGPRT分钟盒式igene C.引物序列，用于生成靶向构建体删除rop18。 黑体区域对应到T。刚地弓形虫基因组序列，以及的nonbold区域对应的限制酶位点重叠与pRS416在HXGPRT小基因盒的序列。 HX_F和HX_R底漆对放大HXGPRT的小基因盒14。读取的引物从5&#39;至3&#39;。表改编从芬特雷斯等[10]。 <a href="https://www.jove.com/files/ftp_upload/50598/50598fig1large.jpg" target="_blank">点击这里查看大图</a> 。 <img alt="图2" fo:content-width="6.5in" fo:src="/files/ftp_upload/50598/50598fig2highres.jpg" src="/files/ftp_upload/50598/50598fig2.jpg" /> 图2。概述删除使用HXGPRT基因的协议。 Disruptio。n的双交叉同源重组事件使用〜的KBP 5 DNA目标侧翼〜1 KBP 3&#39;目标DNA侧翼质粒pΔGOI的的GOI的Δku80Δhxgprt株。 PCR1的PCR策略，PCR2，PCR3，PCR4（不按比例），使基因型验证，验证克隆有针对性地整合HXGPRT印尼政府轨迹和删除。B.代表底漆对设计验证删除rop18 。ROP18_DF ROP18_DR放大PCR1，ROP18_ExF ROP18_CxR的扩增PCR2，ROP18_CxF DHFR_CxR的放大PCR3 DHFR_CxF的和ROP18_CxR放大PCR4（ 见图2A）。读取的引物从5&#39;至3&#39;。表改编从芬特雷斯等:10 C.代表结果验证靶基因的缺失（rop18）使用HXGPRT的。以下转染质粒pΔROP18的成Δku80Δhxgprt和选择在MPA +X，MPA +所述抗性克隆父母应变控制删减rop18。PCR 1（400个基点）和PCR（〜650 bp）的产品是积极的和消极（〜1200 bp的PCR 3）和PCR检测4（约1,300 bp）的产品。 GOI淘汰赛是有针对性积极为PCR2，PCR3 PCR4的产品和为负的PCR 1产品（ 见图2A）。 PCR1和PCR2（ 上图 ）的结果，代表面板PCR3（ 中图 ），和PCR4（ 底部面板）所示。克隆3，4，5，6，8，9和11表现出正确的带型的PCR 1，PCR2，PCR3和PCR4定义有针对性的基因缺失事件在克隆。克隆1，2，7，10和12是没有基因缺失，对应于其他潜在的有代表性的结果。 （C =家长控制应变Δku80Δhxgprt，M =大小标记） <a href="https://www.jove.com/files/ftp_upload/50598/50598fig2large.jpg" target="_blank">点击此处查看大图</a> 。 <img alt="图3" fo:content-width="6.5in" fo:src="/files/ftp_upload/50598/50598fig3highres.jpg" src="/files/ftp_upload/50598/50598fig3.jpg" /> 图3。的协议重新定位感兴趣的基因删除HXGPRT的概述。 A.战略的HXGPRT去除选择标记的双交换同源重组事件在应变Δku80Δgoi的:: HXGPRT使用〜的KBP 5 DNA目标侧翼和一个1〜的KBP 3的DNA目标侧翼质粒pΔGOIc的的。 PCR基因型验证策略描绘的检测用引物对的PCR产物（PCR5）跨越删除（不按比例）。B.甲代表性的设计的引物对从GRA2验证去除可选择标记HXGPRT轨迹应变Δku80Δgra2中:: HXGPRT引物GRA2 _CLF GRA2_CxR扩增PCR5。引物<html>读取从5&#39;至3&#39;。C.代表面板，可以得到下面的转染与Δku80Δgoi:: HXGPRT和选择6TX质粒pΔGOIc到的耐6TX克隆。无性系4，第7，第9，第11和12表现出正确的带型的PCR5对应的对象删除的HXGPRT标记（〜1.2 kbp的产品，跨越的3&#39;与5&#39;侧翼和以外的3&#39;侧翼有轻微的重叠侧翼）。克隆1，2，3，6，8和10（该频带光）代表预期的条带图案的周围〜3.4 kbp的亲本克隆基因型对应，即使这些克隆表现出一定程度的阻力，容许6TX他们的选择（此型可偶尔油然而生关机的HXGPRT表达（ 见附注协议2.1（第8步）和代表性的成果部分）（C =家长控制应变Δku80Δgoi:: HXGPRT的 ，M =大小标记）。w.jove.com/files/ftp_upload/50598/50598fig3large.jpg"目标="_blank"&gt;点击这里查看大图。 <img alt="图4" fo:content-width="5in" fo:src="/files/ftp_upload/50598/50598fig4highres.jpg" src="/files/ftp_upload/50598/50598fig4.jpg" /> 图4。设计的一个目标标记的蛋白质的C-末端的DNA分子。策略是基于的能力的HXGPRT标记，也可作为3&#39;下游的调节区。的骨干pΔGOItag的pRS416穿梭载体含有尿嘧啶（市建局）及氨苄青霉素（AMP）选择标记。 〜1 5 KBP&#39;目标DNA扩增基因组DNA的侧翼重叠HXGPRT小基因盒和pRS416使用Fgoi引物RTAG，重叠了〜1 KBP 3&#39;从基因组DNA扩增目标DNA侧翼插入pRS416 HXGPRT小基因盒和pRS416使用F2和R2引物和在HXGPRT小基因盒。的5&#39;DNA靶侧翼带有标签（HA，Myc基因，他等），然后由一个替换的终止密码子，终止密码子替换。 MPA + X选择集成的C-末端的标签和的下游HXGPRT，和移动的3&#39;端非编码区的位置后HXGPRT标记。以下独特的限制性内切酶消化网站已加入引物:RE.X在Fgoi引物（限制性内切酶的X剪切站点）切割质粒在目标DNA的5&#39;侧翼的5&#39;末端，并在RE.Y的R2引物切割质粒的3&#39;末端的3&#39;DNA靶侧翼，在rtag命令和F2引物和RE.Z切除HXGPRT的的小基因盒用于质粒构建重新定位标记的基因位点删除HXGPRT标记将恢复安置的3&#39;UTR。

Watch this Scientific Journal Video about Genetic Manipulation in Î”ku80 Strains for Functional Genomic Analysis of Toxoplasma gondii at JoVE.com

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

在这里，我们报道了一种采用I型和II型<em&gt;Δku80</em菌株<em&gt;弓形虫</em&gt;高效的生成有针对性的基因缺失和基因替换为功能基因组学分析。

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 ...

genetic-manipulation-ku80-strains-for-functional-genomic-analysis

Research

JoVE Journal

Immunology and Infection

17.0K 次观看.  The Geisel School of Medicine at Dartmouth. 在这里，我们报道了一种采用I型和II型Δku80

视频: Genetic Manipulation in Δku80 Strains for Functional Genomic Analysis of Toxoplasma gondii

Obtaining Highly Purified Toxoplasma gondii Oocysts by a Discontinuous Cesium Chloride Gradient

Toxoplasma gondii is an obligate intracellular protozoan pathogen that commonly infects humans. It is a well characterized apicomplexan associated with causing food- and water-borne disease outbreaks. The definitive host is the feline species where sexual replication occurs resulting in the development of the highly infectious and environmentally resistant oocyst. Infection occurs via ingestion of tissue cysts from contaminated meat or oocysts from soil or water. Infection is typically asymptomatic in healthy individuals, but results in a life-long latent infection that can reactivate causing toxoplasmic encephalitis and death if the individual becomes immunocompromised. Meat contaminated with T. gondii cysts have been the primary source of infection in Europe and the United States, but recent changes in animal management and husbandry practices and improved food handling and processing procedures have significantly reduced the prevalence of T. gondii cysts in meat1, 2. Nonetheless, seroprevalence in humans remains relatively high suggesting that exposure from oocyst contaminated soil or water is likely. Indeed, waterborne outbreaks of toxoplasmosis have been reported worldwide supporting the theory exposure to the environmental oocyst form poses a significant health risk3-5. To date, research on understanding the prevalence of T. gondii oocysts in the water and environment are limited due to the lack of tools to detect oocysts in the environment 5, 6. This is primarily due to the lack of efficient purification protocols for obtaining large numbers of highly purified T gondii oocysts from infected cats for research purposes. This study describes the development of a modified CsCl method that easily purifies T. gondii oocysts from feces of infected cats that are suitable for molecular biological and tissue culture manipulation7.

Obtaining Highly Purified Toxoplasma gondii Oocysts by a Discontinuous Cesium Chloride Gradient

This study describes the development of a modified CsCl method that easily purifies T. gondii oocysts from feces of infected cats that are suitable for molecular biological and tissue culture manipulation

取得高纯度弓形虫</em一个不连续的氯化铯梯度>卵囊

Toxoplasma gondii is an obligate intracellular protozoan pathogen that commonly infects humans. It is a well characterized apicomplexan associated with ...

科研

免疫与感染

Toxoplasma gondii Cyst Wall Formation in Activated Bone Marrow-derived Macrophages and Bradyzoite Conditions

Toxoplasma gondii is an obligate intracellular parasite that can invade any nucleated cell of warm-blooded animals. During infection, T. gondii disseminates as a fast replicating form called the tachyzoite. Tachyzoites convert into a slow-growing encysted form called the bradyzoite by a signaling process that is not well characterized. Within animals, bradyzoite cysts are found in the central nervous system and muscle tissue and represent the chronic stage of infection. Conversion to bradyzoites can be simulated in tissue culture by CO2 starvation, using medium with high a pH, or the addition of interferon gamma (IFN&gamma;). Bradyzoites are characterized by the presence of a cyst wall, to which the lectin Dolichos biflorus agglutinin (DBA) binds. Fluorescently labeled DBA is used to visualize the cyst wall in parasites grown in human foreskin fibroblasts (HFFs) that have been exposed to low CO2 and high pH medium. Similarly, parasites residing in murine bone marrow-derived macrophages (BMMs) display a cyst wall detectable by DBA after the BMMs are activated with IFN&gamma; and lipopolysaccharide (LPS). This protocol will demonstrate how to induce conversion of T. gondii to bradyzoites using a high pH growth medium with low CO2 and activation of BMMs. Host cells will be cultured on coverslips, infected with tachyzoites and either activated with addition of IFN&gamma; and LPS (BMMs) or exposed to a high pH growth medium (HFFs) for three days. Upon completion of infections, host cells will be fixed, permeabilized, and blocked. Cyst walls will be visualized using rhodamine DBA with fluorescence microscopy.

Toxoplasma gondii Cyst Wall Formation in Activated Bone Marrow-derived Macrophages and Bradyzoite Conditions

Toxoplasma gondii converts to a cyst form in response to environmental stresses, which can be mimicked in tissue culture models. This video demonstrates techniques to examine cyst wall formation by activating bone marrow-derived macrophages or changing growth medium pH in fibroblast cells.

弓形虫囊肿壁形成激活骨髓衍生的巨噬细胞和Bradyzoite条件

Toxoplasma gondii is an obligate intracellular parasite that can invade any nucleated cell of warm-blooded animals. During infection, T. gondii ...

Transnuclear Mice with Pre-defined T Cell Receptor Specificities Against Toxoplasma gondii Obtained Via SCNT

Lymphocytes, such as T cells, undergo genetic V(D)J recombination, to generate a receptor with a certain specificity1. Mice transgenic for a rearranged antigen-specific T cell receptor (TCR) have been an indispensable tool to study T cell development and function. However, such TCRs are usually isolated from the relevant T cells after long-term culture often following repeated antigen stimulation, which unavoidably selects for T cells with high affinity. Random genomic integration of the TCR &alpha;- and &beta;-chain and expression from non-endogenous promoters can lead to variations in expression level and kinetics.
Epigenetic reprogramming via somatic cell nuclear transfer provides a tool to generate embryonic stem cells and mice from any cell of interest. Consequently, when SCNT is applied to T cells of known specificity, these genetic V(D)J rearrangements are transferred to the SCNT-embryonic stem cells (ESCs) and the mice derived from them, while epigenetic marks are reset. We have demonstrated that T cells with pre-defined specificities against Toxoplasma gondii can be used to generate mouse models that express the specific TCR from their endogenous loci, without experimentally introduced genetic modification. The relative ease and speed with which such transnuclear models can be obtained holds promise for the construction of other disease models.

Transnuclear Mice with Pre-defined T Cell Receptor Specificities Against Toxoplasma gondii Obtained Via SCNT

We demonstrate here that epigenetic reprogramming via Somatic Cell Nuclear Transfer (SCNT) can be used as a tool to generate mouse models with pre-defined T cell receptor (TCR) specificities. These transnuclear mice express the corresponding TCR from their endogenous locus under the control of the endogenous promoter.

与预先定义的T细胞受体特异性Transnuclear小鼠对弓形虫获得通过体细胞核移植

Lymphocytes, such as T cells, undergo genetic V(D)J recombination, to generate a receptor with a certain specificity1. Mice transgenic for a rearranged ...

QTL Mapping and CRISPR/Cas9 Editing to Identify a Drug Resistance Gene in Toxoplasma gondii

Scientific knowledge is intrinsically linked to available technologies and methods. This article will present two methods that allowed for the identification and verification of a drug resistance gene in the Apicomplexan parasite Toxoplasma gondii, the method of Quantitative Trait Locus (QTL) mapping using a Whole Genome Sequence (WGS) -based genetic map and the method of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 -based gene editing. The approach of QTL mapping allows one to test if there is a correlation between a genomic region(s) and a phenotype. Two datasets are required to run a QTL scan, a genetic map based on the progeny of a recombinant cross and a quantifiable phenotype assessed in each of the progeny of that cross. These datasets are then formatted to be compatible with R/qtl software that generates a QTL scan to identify significant loci correlated with the phenotype. Although this can greatly narrow the search window of possible candidates, QTLs span regions containing a number of genes from which the causal gene needs to be identified. Having WGS of the progeny was critical to identify the causal drug resistance mutation at the gene level. Once identified, the candidate mutation can be verified by genetic manipulation of drug sensitive parasites. The most facile and efficient method to genetically modify T. gondii is the CRISPR/Cas9 system. This system comprised of just 2 components both encoded on a single plasmid, a single guide RNA (gRNA) containing a 20 bp sequence complementary to the genomic target and the Cas9 endonuclease that generates a double-strand DNA break (DSB) at the target, repair of which allows for insertion or deletion of sequences around the break site. This article provides detailed protocols to use CRISPR/Cas9 based genome editing tools to verify the gene responsible for sinefungin resistance and to construct transgenic parasites.

QTL Mapping and CRISPR/Cas9 Editing to Identify a Drug Resistance Gene in Toxoplasma gondii

Details are presented on how QTL mapping with a whole genome sequence based genetic map can be used to identify a drug resistance gene in Toxoplasma gondii and how this can be verified with the CRISPR/Cas9 system that efficiently edits a genomic target, in this case the drug resistance gene.

QTL定位和CRISPR / Cas9编辑识别抗药性基因<em&gt;弓形虫</em

Scientific knowledge is intrinsically linked to available technologies and methods. This article will present two methods that allowed for the ...

遗传学

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 ...

Forward Genetic Approaches in Chlamydia trachomatis

Chlamydia trachomatis, the etiological agent of sexually transmitted diseases and ocular infections, remains poorly characterized due to its intractability to experimental transformation with recombinant DNA. We developed an approach to perform genetic analysis in C. trachomatis&#160;despite the lack of molecular genetic tools. Our method involves: i.) chemical mutagenesis to rapidly generate comprehensive libraries of genetically-defined mutants with distinct phenotypes; ii.) whole-genome sequencing (WGS) to map the underlying genetic lesions and to find associations between mutated gene(s) and a common phenotype; iii.) generation of recombinant strains through co-infection of mammalian cells with mutant and wild type bacteria. Accordingly, we were able to establish causal relationships between genotypes and phenotypes. The coupling of chemically-induced gene variation and WGS to establish correlative genotype&#8211;phenotype associations should be broadly applicable to the large list of medically and environmentally important microorganisms currently intractable to genetic analysis.

Forward Genetic Approaches in Chlamydia trachomatis

We describe a methodology to perform genetic analysis in Chlamydia based on chemical mutagenesis and whole genome sequencing. In addition, a system for DNA exchange within infected cells is described that can be used for genetic mapping. This method may be broadly applicable to microbial systems lacking transformation systems and molecular genetic tools.

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 ...

Following in Real Time the Impact of Pneumococcal Virulence Factors in an Acute Mouse Pneumonia Model Using Bioluminescent Bacteria

Pneumonia is one of the major health care problems in developing and industrialized countries and is associated with considerable morbidity and mortality. Despite advances in knowledge of this illness, the availability of intensive care units (ICU), and the use of potent antimicrobial agents and effective vaccines, the mortality rates remain high1. Streptococcus pneumoniae is the leading pathogen of community-acquired pneumonia (CAP) and one of the most common causes of bacteremia in humans. This pathogen is equipped with an armamentarium of surface-exposed adhesins and virulence factors contributing to pneumonia and invasive pneumococcal disease (IPD). The assessment of the in vivo role of bacterial fitness or virulence factors is of utmost importance to unravel S. pneumoniae pathogenicity mechanisms. Murine models of pneumonia, bacteremia, and meningitis are being used to determine the impact of pneumococcal factors at different stages of the infection. Here we describe a protocol to monitor in real-time pneumococcal dissemination in mice after intranasal or intraperitoneal infections with bioluminescent bacteria. The results show the multiplication and dissemination of pneumococci in the lower respiratory tract and blood, which can be visualized and evaluated using an imaging system and the accompanying analysis software.

Streptococcus pneumoniae is the leading pathogen causing severe community-acquired pneumonia and responsible for over 2&#160;million deaths worldwide. The impact of bacterial factors implicated in fitness or virulence can be monitored in real-time in an acute mouse pneumonia or bacteremia model using bioluminescent bacteria.

在实时以下的肺炎球菌毒力因子在小鼠急性肺炎模型的影响采用发光细菌

Pneumonia is one of the major health care problems in developing and industrialized countries and is associated with considerable morbidity and mortality. ...

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-&#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 ...

3-D Imaging and Analysis of Neurons Infected In Vivo with Toxoplasma gondii

Toxoplasma gondii is an obligate, intracellular parasite with a broad host range, including humans and rodents. In both humans and rodents, Toxoplasma establishes a lifelong persistent infection in the brain. While this brain infection is asymptomatic in most immunocompetent people, in the developing fetus or immunocompromised individuals such as acquired immune deficiency syndrome (AIDS) patients, this predilection for and persistence in the brain can lead to devastating neurologic disease. Thus, it is clear that the brain-Toxoplasma interaction is critical to the symptomatic disease produced by Toxoplasma, yet we have little understanding of the cellular or molecular interaction between cells of the central nervous system (CNS) and the parasite. In the mouse model of CNS toxoplasmosis it has been known for over 30 years that neurons are the cells in which the parasite persists, but little information is available about which part of the neuron is generally infected (soma, dendrite, axon) and if this cellular relationship changes between strains. In part, this lack is secondary to the difficulty of imaging and visualizing whole infected neurons from an animal. Such images would typically require serial sectioning and stitching of tissue imaged by electron microscopy or confocal microscopy after immunostaining. By combining several techniques, the method described here enables the use of thick sections (160 &#181;m) to identify and image whole cells that contain cysts, allowing three-dimensional visualization and analysis of individual, chronically infected neurons without the need for immunostaining, electron microscopy, or serial sectioning and stitching. Using this technique, we can begin to understand the cellular relationship between the parasite and the infected neuron.

3-D Imaging and Analysis of Neurons Infected In Vivo with Toxoplasma gondii

Using this protocol, we were able to image 160 &#181;m thick brain sections from mice infected with the parasite Toxoplasma gondii, which enables visualization and analysis of the spatial relationship between the encysting parasite and the infected neuron.

神经元的3-D成像和分析感染<em&gt;在体内</em&gt;与<em&gt;弓形虫</em

Toxoplasma gondii is an obligate, intracellular parasite with a broad host range, including humans and rodents. In both humans and rodents, Toxoplasma ...

神经科学

Determination of Chemical Inhibitor Efficiency against Intracellular Toxoplasma Gondii Growth Using a Luciferase-Based Growth Assay

Toxoplasma gondii is a protozoan pathogen that widely affects the human population. The current antibiotics used for treating clinical toxoplasmosis are limited. In addition, they exhibit adverse side effects in certain groups of people. Therefore, discovery of novel therapeutics for clinical toxoplasmosis is imperative. The first step of novel antibiotic development is to identify chemical compounds showing high efficacy in inhibition of parasite growth using a high throughput screening strategy. As an obligate intracellular pathogen, Toxoplasma can only replicate within host cells, which prohibits the use of optical absorbance measurements as a quick indicator of growth. Presented here is a detailed protocol for a luciferase-based growth assay. As an example, this method is used to calculate the doubling time of wild-type Toxoplasma parasites and measure the efficacy of morpholinurea-leucyl-homophenyl-vinyl sulfone phenyl (LHVS, a cysteine protease-targeting compound) regarding inhibition of parasite intracellular growth. Also described, is a CRISPR-Cas9-based gene deletion protocol in Toxoplasma using 50 bp homologous regions for homology-dependent recombination (HDR). By quantifying the inhibition efficacies of LHVS in wild-type and TgCPL (Toxoplasma cathepsin L-like protease)-deficient parasites, it is shown that LHVS inhibits wild-type parasite growth more efficiently than &#916;cpl growth, suggesting that TgCPL is a target that LHVS binds to in Toxoplasma. The high sensitivity and easy operation of this luciferase-based growth assay make it suitable for monitoring Toxoplasma proliferation and evaluating drug efficacy in a high throughput manner.

Determination of Chemical Inhibitor Efficiency against Intracellular Toxoplasma Gondii Growth Using a Luciferase-Based Growth Assay

Presented here is a protocol to evaluate the inhibition efficacy of chemical compounds against in vitro intracellular growth of Toxoplasma gondii using a luciferase-based growth assay. The technique is used to confirm inhibition specificity by genetic deletion of the corresponding target gene. The inhibition of LHVS against TgCPL protease is evaluated as an example.

使用基于路西法抹去的生长测定，确定化学抑制剂对细胞内弓形体贡迪生长的抑制效率

Toxoplasma gondii is a protozoan pathogen that widely affects the human population. The current antibiotics used for treating clinical toxoplasmosis are ...

遗传操纵

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取得高纯度弓形虫卵囊

弓形虫囊肿壁形成激活骨髓衍生的巨噬细胞和Bradyzoite条件

与预先定义的T细胞受体特异性Transnuclear小鼠对弓形虫获得通过体细胞核移植

QTL定位和CRISPR / Cas9编辑识别抗药性基因

遗传筛选隔离弓形虫宿主细胞的出口突变

正向遗传途径

在实时以下的肺炎球菌毒力因子在小鼠急性肺炎模型的影响采用发光细菌

正向遗传学屏风使用巨噬细胞识别

神经元的3-D成像和分析感染

使用基于路西法抹去的生长测定，确定化学抑制剂对细胞内弓形体贡迪生长的抑制效率