Special thanks to Dr. Peter Bradley for the antibody to detect the T. gondii mitochondria. The work was supported by National Institute of Health Grants AI072028 and AI107431 to D.G.M and a generous donation to New York Medical College for the study of tropical medicine.

注：包括使用动物的所有协议均按照规定由纽约医学院的动物护理和使用委员会的指导原则和规定执行。 注：详细有限稀释38，小鼠骨髓巨噬细胞39的隔离，T.增长协议化学诱变38，寄生虫孤立弓形虫在人包皮成纤维细胞（HFF）细胞和囊肿生产的巨噬细胞和基本免疫荧光分析（IFA）32顷引用。进行，在37℃的所有细胞培养在5％CO 2在D10培养基（Dulbecco改进的高糖的Eagle培养基补充有10％胎牛血清，2mM的L-谷氨酰胺，100U / ml青霉素和100μg/ ml链霉素） 。保留所有试剂整个电池隔离和细胞培养无菌。 IFN-γ和LPS以确定精矿1.剂量滴定ations使用激活巨噬细胞感染的正向遗传学屏幕<ol><li>培养的鼠骨髓衍生巨噬细胞的O / N的8腔室玻片在3×10 5个细胞/ 250微升每室D10介质ml的浓度。从骨髓分离后使用的巨噬细胞一到两个周。 注：商会幻灯片购买具有增长提高RS洗。 </li><li>用血球计数从一个T25组织培养瓶中生长的人包皮成纤维细胞（HFF）细胞采集野生型寄生虫。重悬的寄生虫，以每毫升5×10 5个野生型寄生虫D10介质的浓度。该浓度将导致在大约1的感染复数：1作为巨噬细胞会激增O / N。使用聚苯乙烯或PET管与寄生虫寄生枝聚丙烯的所有操作。 </li><li>取出D10媒体在玻片并更换250微升的暂停野生型寄生虫。轻轻使其流下的每个腔室的内表面添加寄生虫悬浮于滑动的各腔室。不要让巨噬细胞干出在协议中的任何一点。 </li><li>在5％CO 2感染寄生虫与室滑动盖并置于培养箱中在37℃下4小时盖室载玻片，以便有时间寄生虫侵入并建立一个光伏。 </li><li>使在1ml D10媒体在无菌试管中对剂量滴定的LPS和IFN-γ的稀释液。剂量滴定保持要么LPS或IFN-γ浓度不变，改变其他刺激40。 <ol><li>例如，保持的LPS恒定在每毫升10纳克LPS和变化IFN-γ（0，1单位/ ml，10单位/ ml，100单位/ ml，1000单位/ ml）的浓度。保持的IFN-γ在100单位/ ml不变而变化的LPS（0，0.1纳克/毫升，1毫微克/毫升，10纳克/毫升，100纳克/毫升）的浓度。简要超声LPS股票没有2分钟加热的浴超声仪（不与尖端超声波仪）之前稀释。 注：超声推荐破坏了，可能无法正确​​绑定到宿主细胞LPS形成胶束聚集。 </li></ol></li><li> 4小时，在室滑动的寄生虫和粘附的巨噬细胞之间的共培养开始后，弃去D10媒体在每个室中，并将它与300微升的IFN-γ和LPS的D10介质适当稀释替换。包括只用D10媒体的控制，以评估寄生虫复制在没有巨噬细胞活化的。 </li><li>重新盖玻片与室滑动盖并置于培养箱中在37℃，在5％CO 2的24至30个额外小时。 注：孵育时间取决于野生型寄生虫菌株的范围可以从6-12小时取决于寄生虫菌株的倍增时间。一时间点的选择之前，寄生虫幼稚的巨噬细胞裂解，但足够长的时间，使至少3-4斗金光闪闪次。 </li><li>使在Dulbecco磷酸盐缓冲盐水（PBS）2.5％的甲醛溶液。丢弃介质在室中滑动，并替换为300微升2.5％的甲醛溶液。定为20分钟，在RT。 </li><li>漂洗载玻片（多个）两次，用300微升PBS每室。对于每次漂洗，弃去PBS中滑动，并轻轻添加新的PBS向下各滑动室的内面，以避免破坏附着在滑动的巨噬细胞。添加300微升PBS，每室和地点盖玻片，防止染色前晒出的幻灯片。 注：幻灯片可以染色前被放置在4℃下3天，在这一点上。 </li><li>染色协议T.弓形虫检测通过免疫荧光显微镜（IFA）。 <ol><li>使在PBS中的0.2％的Triton X-100溶液（透化缓冲液）。放置在一个管中的溶液在37℃水浴中，并简短地涡旋，以确保所述的Triton X-100进入溶液。放弃的PBS在腔室玻片并用300μl的透化缓冲液的更换。留在室温30分钟。 </li><li>使10％的羊血清的PBS（封闭溶液）的溶液。丢弃在幻灯片的通透缓冲并更换300微升堵每室解决方案。留在室温30分钟。 注：胎牛血清可以取代在所有染色方案山羊血清。 </li><li>对于一步IFA染色协议，使1：1000稀释荧光标记的抗体T的弓形虫在阻断溶液中。从幻灯片除去封闭溶液，并添加150微升每室抗体溶液。取代室滑盖滑动，以防止细胞干燥。保温1小时，在室温。 注：抗体的浓度必须根据来源来凭经验确定。抗体被购买直接偶联至荧光染料或偶联至荧光染料由使用者。 <ol><li>为一个两步的IFA染色使用协议的非结合抗体T.弓形虫和荧光标记的二抗，染色载玻片150微升抗T。未偶联的第一抗体弓形虫在封闭缓冲液中进行1小时，在室温。冲洗3倍与300微升PBS加1％山羊血清（洗涤缓冲液）。 </li><li>添加150微升荧光标记的二抗的特异性起源物种的初级抗体。取代室滑盖滑动，以防止细胞干燥。保温1小时，在室温。 </li></ol></li><li>漂洗载玻片3倍，用PBS加1％山羊血清（洗涤缓冲液）。对于每次漂洗，通过倾出它的内容中删除在腔室载玻片中的溶液，并更换用300μl的洗涤缓冲液。 </li><li>漂洗幻灯片2倍用300μl的PBS的。 </li><li>从室滑动按照制造商的说明取出室中。 </li><li>摩滑动带安装含有DAPI的培养基（4&#39;6二脒基-2-苯基吲哚，dilactate）和一个22毫米×50毫米的盖玻片。 </li></ol></li><li>检查使用相衬和荧光显微镜幻灯片。通过检查至少每室100的PV以及确定每液泡寄生虫的平均数目。 IFN-γ和LPS的用于在屏幕的选择剂量需要足以抑制，但不能防止，野生型寄生虫（ 见图1）的复制。 </li></ol> 2，隔离寄生虫突变体与受感染的巨噬细胞的健身缺陷继激活注：随机T.的库弓形虫突变体是必需的正向遗传学屏幕。 T.随机诱变弓形虫可通过化学（CHS / EMS）或插入诱变27,28,38来执行。通过有限稀释和在含粘附HFF细胞在200微升D10介质32,38的体积的96孔板生长单个克隆下列诱变克隆的寄生虫。通过显微镜96孔板中的巨噬细胞的寄生虫筛选具有光学塔底使微观筛查是至关重要的。相衬和荧光显微镜对染色的筛选96孔板需要与相衬4，10，20或40倍物镜配备长工作距离的倒置荧光显微镜。一个4倍目的是看整个不错，但更好的分辨率寄生虫达到与20X目标是有用的。 <ol><li>转移50微升生长在汇合HFF细胞在96孔组织培养板中分成两速殖子的突变体复制在100μlD10介质的96孔板含鼠骨髓衍生的巨噬细胞（分离后1-2周）。 注意：请在基于寄生虫培养物体积的寄生虫的96孔板的初始屏幕，以避免必须计算每各转移以及寄生虫的数目。因此，在2.6的显微镜分析的是wheth定性基于呃寄生虫普遍复制或不可复制的。步骤3描述了使用野生型或突变的寄生虫的数目相等感染的巨噬细胞，以证实突变体在腔室玻片的表型的方法。 <ol><li>直接在每个寄生虫添加到D10媒体已经很好。感染两口井与野生型速殖子作为阳性对照寄生虫复制。 </li></ol></li><li>将感染的细胞进入培养箱（37℃和5％的CO 2）4小时，以允许寄生虫时间侵入细胞和创建他们的PV。 </li><li>倾倒板的内容从一个盘取出介质。使用手腕的单个倒装在板介质倾倒入含有洗涤剂cidal用于寄生虫丢弃盆。 <ol><li>添加100微升“巨噬细胞活化媒体”用无菌盆地媒体和多道移液器。使用的LPS的最佳浓度和IFN-γ从第1步判定为Macrophage激活介质。同样从复制控制板取出纸张，替换为100微升D10媒体。 </li></ol></li><li>将感染的细胞进入培养箱（37℃和5％CO 2）的额外24-30小时。 </li><li>染色方案对于96孔板为IFA。 <ol><li>放弃在平板媒体和更换100微升的PBS 2.5％的甲醛。孵育20分钟，在RT。使用无菌盆为甲醛和多道移液器。 </li><li>丢弃在板的甲醛溶液，并添加100微升的PBS冲洗从板的甲醛。 </li><li>丢弃在板的溶液，并添加100微升的透化缓冲液（PBS中含有0.2％的Triton X-100）到每个孔中。温育30分钟，在室温。 </li><li>丢弃在板的溶液，并添加100微升的封闭溶液（PBS中，用10％山羊血清）的每个孔中。温育30分钟，在室温。 </li><li>丢弃在p溶液晚。添加50微升/孔的荧光缀合的抗- T。弓形虫抗体稀释1：1000于PBS加1％山羊血清。保温1小时，在室温上并在摇杆板盖。 <ol><li>或者使用对T.的非结合初级抗体弓形虫随后如在1.10.3.1描述与荧光标记的二抗染色。 </li></ol></li><li>丢弃在板的抗体溶液，并替换为100μl/孔PBS中加1％山羊血清。执行此冲洗3倍后2 PBS单独额外漂洗。离开200微升PBS中每个孔，并更换盖子上的96孔平板，以防止井从干燥。 注：具有上可缠绕在封口膜和在分析前置于4℃下3天用显微镜盖的板。 </li></ol></li><li>检查使用20-40X的目标倒置荧光显微镜下的细胞。是复制在北美和选择突变＃239;已经巨噬细胞板，但主要​​是无法复制超过一个寄生虫/液泡中的“激活的巨噬细胞”或出现无定形或“活化巨噬细胞”退化。 注：每液泡野生型寄生虫的数量将取决于IFN-γ和所用的LPS的剂量，但是理想地是每液泡周围4寄生虫;一个数字，它反映的生长抑制，但尚未复制的活化刺激完全抑制。 </li></ol> 3.评估突变，以确定缺陷是寄生虫生存的水平或复制以下感染的巨噬细胞活化<ol><li>传输所选的突变体寄生虫从96孔板（的整个孔内容），以含有HFF细胞T25s，并允许复制。 </li><li>培养骨髓衍生的巨噬细胞的O / N的8室玻片在3×10 5个细胞/ 250微升D10介质ml的浓度。准备一张幻灯片比较PA在天真的巨噬细胞rasite复制和一个额外的幻灯片来比较寄生虫复制下面的激活感染的巨噬细胞。 </li><li>收获速殖子的寄生虫​​和计数的血球。添加5×10 4 T。弓形虫的寄生虫以及包含D10媒体和文化的巨噬细胞4小时一室。包括一个孔，与野生型亲本的寄生虫作为对照。接种与将不接收，以便监测在幼稚的巨噬细胞的寄生虫复制激活媒体相同的寄生虫克隆的相同复制幻灯片。 </li><li>从1复制玻片的放弃D10媒体和更换250μl的“激活媒体”。从另一个重复室幻灯片放弃D10媒体和更换250微升D10媒体。孵育无论是“激活”和“幼稚”的巨噬细胞上的腔室滑动盖滑动，在37℃和5％CO 2的一个additiOnal地区24-30小时。 </li><li>修复，通透，并为IFA染色和寄生虫分析模块幻灯片按照步骤1描述完好室进行IFA染色。 <ol><li>共染色的细胞与抗体溶酶体膜相关1（LAMP1）或Lysotracker和抗体T。弓形虫评估活化感染的巨噬细胞的是否被触发的融合突变体的PV与溶酶体（图4）。 </li><li>使用抗体对特定区室/细胞器的寄生虫/ PV内染色通过IFA来识别是很明显的早以下巨噬细胞活化28改变在寄生虫突变体。 注意：这种改变可以提供洞察underlies突变的下列巨噬细胞激活的缺陷生存/复制的机制。 </li></ol></li><li>检查采用100X相油浸物镜和荧光显微镜幻灯片。 <ol><li>通过确定评价寄生虫复制在100个随机液泡每光伏寄生虫数目。执行至少2个字的每个100液泡进行统计分析。 </li><li>评估同时使用荧光分析一般寄生虫的形态以及相差显微镜。下阶段100X客观地看待寄生虫。健康的寄生虫有寄生虫紧密内贴身虫空泡包围。该具有非晶宽敞液泡与相致密轮缘或非晶硅寄生虫寄生虫建议寄生虫死亡，而不是仅仅在复制一个不良（数字1和3）。 </li></ol></li></ol> 4.评价突变体寄生虫的感染，以巨噬细胞活化的易感是否伴有已知的抗微生物调解员<ol><li>从野生型C57 / BL6小鼠分离的骨髓衍生的巨噬细胞，iNOS的- / - ，gp91-PHOX - / -或Irgm1 / Irgm3 - / -小鼠32。 </li><li>培养各自骨髓来源MAC噬细胞的O / N的8腔室玻片在3×10 5个细胞/ 250微升D10介质ml的浓度。 </li><li>与寄生虫的挑战，IFA染色和分析进行，如步骤3所述。 </li><li>确定突变体寄生虫生存和复制以下活化感染的巨噬细胞的能力在不存在活性氧或氮物种或特异性免疫相关GTP酶28被恢复。 </li><li>使用药理学试剂，如一氧化氮供体，以评估特定抗微生物介质是否足以自行削弱在HFF细胞或幼稚的巨噬细胞的突变体的寄生虫，或者如果它们只采取一致行动，与巨噬细胞的活化28其他介质。 </li></ol> 5.评估是否缺陷的突变体寄生虫妥协慢性感染<ol><li>隔离野生型，突变或有针对性的基因删除寄生在T25s合作种植速殖子ntaining HFF细胞。寄生虫必须从HFF文化被新鲜裂解。 </li><li>计数使用血球寄生虫。重悬寄生虫在每毫升合适的浓度用于通过腹膜内（IP）注射在200μl递送的寄生虫的亚致死剂量的Hanks平衡盐溶液（HBSS）中。 </li><li>使用1毫升结核菌素注射器和25g针注入寄生虫用200μl的体积腹膜内（IP）。该剂量取决于该寄生虫的野生型菌株和鼠标的基因型。 注：寄生虫的I型基因型是致命的小鼠在感染无关寄生虫剂量的急性期，并且不适合用于慢性感染的在没有化疗为T.研究弓形虫抑制寄生虫复制。 </li><li>寄生虫激发后三十天，牺牲的小鼠吸入的CO 2从加压罐中的拥挤室（一个标准尺寸鼠笼可能含有不超过5米冰），接着通过颈脱位。 </li><li>使用既定程序41-43鼠标隔离大脑。 <ol><li>躺在鼠标在其前侧。喷雾头，用70％的乙醇消毒的区域。 </li><li>用锋利的剪刀，通过脑干削减。使用解剖剪刀做出浅切口横向地围绕右侧，然后从脑干的基部开始头骨的左侧。使用镊子轻轻剥开颅骨，露出大脑。 </li><li>轻轻提起颅骨的大脑和基极之间的脑和地点剪刀剪开嗅神经释放的大脑。提起出大脑用无菌镊子或小铲和地方装有10ml无菌PBS的细菌培养皿。 </li></ol></li><li>切开大脑中的一半与通过左右半球之间的中心的无菌手术刀。 </li><li>一半脑的添加到含1ml PBS中的小砂浆。使用研钵和研杵对使该可穿过一个大口径20-200微升移液管尖的大脑的精细组织悬浮液。 注：脑的另一半应固定在2.5％的甲醛1小时，如果需要为组织病理学组织切段。 </li><li>放置100微升脑裂解物在1.7 ml离心管。加入1毫升在PBS中的2.5％的甲醛到含有脑裂解物，以固定用于染色脑悬浮液的管中。在室温下孵育30分钟。 </li><li>离心溶胞产物以8000×g离心5分钟，在微量离心，小心弃去上清液。 </li><li>加入1毫升的透化/封闭缓冲液的溶胞产物（10％山羊血清，0.2％的Triton X-100的PBS中）。在室温下孵育30分钟。 </li><li>离心裂解液在8000 XG为5分钟，除去上清液。 </li><li>加入200μl的FITC缀合的双花扁豆凝集素（DBA）。使用1：100稀释的外源凝集素的PBS加10％山羊血清的。轻轻悬浮裂解液吹打。孵化为1小时，在室温。 注：DBA是配体结合于CST1对寄生虫囊壁。 </li><li>离心溶胞产物以8000×g离心5分钟并弃去上清。添加1ml的PBS中，以沉淀。在室温下孵育5分钟。重复PBS洗3倍。删除以下用吸管最后洗的上清。 </li><li>使用大口径20-200微升枪头制定5微升的染色脑裂解液和地点在显微镜载玻片。安装了25×25mm的盖玻片幻灯片创建脑裂解液的湿坐骑。使用5微升脑裂解物每3个重复的载玻片。 </li><li>通过使用10X物镜（图6）的荧光显微镜计数每各5微升等份囊肿的数量。 注意：有些囊肿较大（8个或更多寄生虫约），而有些是非常小的（每1-2个囊肿寄生虫）。算上每相对于大的小囊肿数量每张幻灯片，但文件编号囊肿的总数。 </li><li>加入囊肿DET数量ected在三个不同的5微升等份和乘以总稀释倍数×2（只有一半脑部被拍成裂解物）来估计每个脑总囊肿的数量。 </li></ol>

The authors have no competing financial interests.

所描述的协议提供了使用活化的鼠骨髓衍生巨噬细胞和向前遗传学分离T的非偏置的方式弓形虫突变体在自己的能力有缺陷的生存激活的巨噬细胞感染的。突变体以下的巨噬细胞的活化的表型通常分为两大类：1）的寄生虫出现完整的，但无法复制超过每光伏1寄生虫; 2）寄生虫出现退化并可能宽敞，无定形的PV。该突变体具有类似于在没有活化的幼稚的巨噬细胞的野生型寄生虫的表型?...

弓形虫（弓形虫）是一种专性细胞内，原虫病的病原体。这是弓形体病的病原体，免疫功能低下的个体健康危险。这也是其他顶复门病原体感染人类，包括隐孢子虫和模型系统。弓形体病是通过食物或水沾染寄生虫的缓殖子或卵囊阶段摄入最常获得的。一旦摄入，这些阶段转换为复制的宿主细胞内，并系统地传播寄生虫的速殖子阶段。 T细胞，IFN-γ和，在较小程度上，一氧化氮1-4，是用于控制感染重要的，但不能够消除疾病，如速殖子的比例转换为被组织囊肿内保护缓殖子阶段产生一个长寿命的慢性感染。事实上，还没有治疗剂有效对抗慢性囊肿s踏歌疾病。严重弓形体病是最常见的是由于持续感染的再活化，与寄生虫转换回的初级和急性感染迅速复制速殖子阶段特性的缓殖子阶段。 早生存于先天免疫应答的面是非常重要的，以允许寄生虫达到足够寄生虫数目，以及到达末端位置，以使建立慢性感染的T。弓形虫已经发展战略，以抵消可能的复制和传播感染的早期能力有助于宿主的防御机制。首先，T.弓形虫形成寄生虫入侵期间的唯一的PV是从该宿主细胞的相比其他细胞内病原体5-9内吞和胞吐过程基本上隔离。此外，像所有成功的细胞内病原体T.弓形虫修改它的宿主细胞创造一个宽松的环境˚F或生长。这包括重编程的宿主细胞的基因表达，通过改变宿主细胞的转录因子包括那些用于调节细胞活化10-15重要。 ROP16 16-19 GRA15 20，GRA16 21和GRA24 22都被证明在调节转录反应和细胞信号感染T.宿主细胞的级联是重要弓形虫 。使用寄生虫株不同的表型之间的遗传杂交最近的研究已经查明背后的寄生虫基因相关的性状，包括免疫相关的GTP酶（IRGs）16,19,23-26逃避寄生虫基因高产。在小鼠中，免疫相关GTP酶（IRGs）是用于第二类型的控制和III基因型寄生虫的临界而很毒的I型基因型已经进化机制以逃避鼠IRGs。然而，同样明显的是寄生虫已经进化机制以逃避抗菌媒体除了​​IRGs和一些这些机制器可跨越寄生虫基因型27,28保守的。此外，很少有人知道的细胞自主免疫力T.关键调解人人类弓形体病弓形虫期间。寄生虫基因抗性的细胞中自主免疫介重要也可以是用于生存重要期间速殖子要缓殖子转换其也可以通过宿主的免疫反应引起的。例如，一氧化氮在高的水平，可以抑制在受感染的巨噬细胞寄生虫复制，但它也可以刺激速殖子至缓殖子转变导致囊肿生产30-32。 ToxoDB是一种功能性基因组数据库T.弓形虫充当对字段中提供的序列信息用于寄生虫的基因组，并获得公布和未公布的基因组尺度的数据包括社区注解方面的重要资源，基因EXP<html> ression和蛋白质组学数据33。类似于许多原虫病原体，多数基因组由基于基因同源性为深入了解其潜在功能可以假设基因，没有信息。因此，正向遗传学是一种强大的工具，以确定新的寄生虫基因免疫逃避，囊肿转换等功能为寄生虫的发病机制，以及对不同的发育阶段之间的转换关键重要。正向遗传学的附加优点是，它可被用作一个相对无偏见的方式来询问寄生虫为那些对在发病特定的任务，包括免疫逃避和囊肿形成重要的基因。在新一代测序的突变分析近期的改进，使它的首选识别从正向遗传学研究的负责疟原虫基因用化学方法和插入突变34-37的方法。 ntent“&gt;重要的是要确定在弓形虫漏洞可以被利用以提高的细胞中自主免疫机制对寄生虫特别是那些也可以有效对抗耐药囊肿阶段。为了实现这一目的， 在体外鼠的效力是很重要的巨噬细胞的感染和激活模型的开发是为了确定特异性削弱弓形虫健身以下活化感染的巨噬细胞，但不是在幼稚的巨噬细胞的寄生虫的突变。该巨噬细胞的屏幕被用于询问弓形虫插入突变体文库，以最终识别弓形虫基因抗性很重要的一氧化氮27,28。 弓形虫突变体活化感染的巨噬细胞，特别是一个显着的敏感性，一氧化氮的受损性的面板的分离，证实在屏幕的效用，以确定寄生虫基因性重要到的细胞中自主免疫比鼠IRGs 28中描述的抗性机制的其他介质。插入突变有超过化学诱变优点在产生的随机突变在每个寄生虫克隆和，在理论上，便于识别突变位点的数量有限的条款。然而，确定质粒插入的T.的基因组位点弓形虫插入突变体，在实践中，已经令人惊奇地难以在许多情况下，37。一个质粒导入的基因的插入也有可能打乱一个基因的功能在对比化学诱变通常导致单核苷酸变化。然而，化学诱变是N-乙基-N-亚硝基脲（ENU）或甲磺酸乙酯（EMS），可以提供分析寄生虫基因组的较大部分增加的能力，相对于插入诱变，因为它创建多个单核苷酸多态性（每突变体34估计为10 -100）38。此外，在全基因组谱的最新进展使得有可能使用下一代测序，以确定负责突变寄生虫34,38的所识别的表型是最有可能的候选基因。不管诱变方法，确认该寄生虫基因抗性巨噬细胞活化中的作用的最终需要的基因缺失和互补履行分子柯赫氏法则。 解剖的基因通过两个寄生虫和巨噬细胞的遗传操作的功能的能力是重要的，因为许多经由正向遗传学T中鉴定的基因的弓形虫，以及其它病原体，仍然表征为假想的基因几乎没有序列同源性的其他蛋白与已知功能。当前文件概述了可被用于识别在一个突变体被破坏的基因是否是用于抵抗重要到一个已知的或一般的做法未知调解员的细胞自主的免疫力。通过评估野生型和从野生型小鼠与那些与诱导型一氧化氮合酶（iNOS），GP-91 PHOX（NADPH氧化酶）特异性基因缺失突变体的巨噬细胞的寄生虫的存活进行的宿主抗微生物因素最初的分析，并特异性免疫相关的GTP酶（IRGs）。这将确定是否所识别的寄生虫基因是抗一氧化氮，活性氧中间体或分别或者如果未知免疫机制参与免疫相关GTP酶28重要。受感染的巨噬细胞与这两种IFN-γ和LPS，在当前协议中描述的活化，导致主要在寄生虫基因抗性很重要的一氧化氮28的隔离。采用药理学试剂诱导的一氧化氮在没有巨噬细胞活化的（一氧化氮供体）证实，大多数鉴定的基因的人重新重要sistance一氧化氮，而不是一氧化氮的音乐会与巨噬细胞活化28相关的其他调解员。 步骤1和2描述了一个向前遗传学屏幕旨在隔离寄生虫突变体与健身缺陷以下体外活化感染的骨髓衍生巨噬细胞。步骤1描述了一个剂量滴定分析来凭经验确定IFN-γ和LPS的剂量用于巨噬细胞活化，减少寄生虫复制，但不能完全抑制野生型T的复制弓形虫父母菌株用于创建寄 ​​生虫突变体库。步骤2描述了突变体克隆在96孔板的巨噬细胞的前向遗传屏幕。步骤3概述的方法来确认鉴定在96孔板的屏幕，并评估在每个突变体中的缺陷是否影响寄生虫存活，复制每个突变体的表型，或囊肿生产响应于巨噬细胞的活化。步骤4描述了使用骨髓衍生的巨噬细胞的小鼠与特定抗微生物途径缺失来识别免疫介质到的寄生虫突变体是特别敏感的。步骤5概述的方法，以确定是否一个寄生虫突变体也遭到了破坏用于体内发病如在感染小鼠的脑中评价由囊肿的生产。

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			<td>Thermo</td>
			<td>SH3002802&#160;</td>
			<td>https://www.fishersci.com/ecomm/servlet/itemdetail?storeId=10652&#38;langId=-1&#38;catalog 
			Id=29104&#38;productId=2434305&#38;dis 
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		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hyclone L-glutamine</td>
			<td>Thermo</td>
			<td>SH3003401&#160;</td>
			<td>https://www.fishersci.com/ecomm/servlet/itemdetail?storeId=10652&#38;langId=-1&#38;catalog 
			Id=29104&#38;productId=3311957&#38;dis 
			type=0&#38;highlightProductsItemsFlag 
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		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hyclone Pen strep</td>
			<td>Thermo</td>
			<td>SV30010&#160;</td>
			<td>https://www.fishersci.com/ecomm/servlet/fsproductdetail?storeId=10652&#38;productId=1309668 
			6&#38;catalogId=29104&#38;matchedCat 
			No=SV30010&#38;fromSearch=1&#38; 
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			&#38;searchType=PROD&#38;hasPromo=0</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hyclone Hanks BSS</td>
			<td>Thermo</td>
			<td>SH3003002</td>
			<td>https://www.fishersci.com/ecomm/servlet/itemdetail?storeId=10652&#38;langId=-1&#38;catalog 
			Id=29104&#38;productId=3064595&#38;dis 
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		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LPS</td>
			<td>LIST biologicals</td>
			<td align="right">201</td>
			<td>http://www.listlabs.com/products-tech.php?cat_id=4&#38;product_id=81&#38;keywords 
			=LPS_from_%3Cem%3EEscherichia_coli%3C/em%3E_O111:B4</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">IFN-g</td>
			<td>Pepro Tech Inc</td>
			<td>50-813-664</td>
			<td>https://www.fishersci.com/ecomm/servlet/itemdetail?itemdetail=&#39;item&#39;&#38;storeId=10652&#38; 
			productId=2988494&#38;catalogId=29 
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		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Chamber slides</td>
			<td>Thermo</td>
			<td align="right">177402</td>
			<td>https://www.fishersci.com/ecomm/servlet/itemdetail?storeId=10652&#38;langId=-1&#38;catalog 
			Id=29104&#38;productId=2164545&#38;dis 
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		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">96-well optical plates</td>
			<td>Thermo</td>
			<td align="right">165306</td>
			<td>https://www.fishersci.com/ecomm/servlet/itemdetail?storeId=10652&#38;langId=-1&#38;catalog 
			Id=29104&#38;productId=3010670&#38;dis 
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		</tr>
		<tr height="21">
			<td colspan="2" height="21" style="height:21px;">96-well tissue culture plates</td>
			<td></td>
			<td align="right">353072</td>
			<td>https://www.fishersci.com/ecomm/servlet/itemdetail?storeId=10652&#38;langId=-1&#38;catalog 
			Id=29104&#38;productId=3158736&#38;dis 
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		</tr>
		<tr height="21">
			<td colspan="2" height="21" style="height:21px;">Tissue culture flast T25</td>
			<td></td>
			<td align="right">156367</td>
			<td>https://www.fishersci.com/ecomm/servlet/fsproductdetail?storeId=10652&#38;productId=127039 
			67&#38;catalogId=29104&#38;matchedCat 
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		</tr>
		<tr height="21">
			<td colspan="2" height="21" style="height:21px;">Ted Pella EM grade formaldehyde</td>
			<td></td>
			<td align="right">18505</td>
			<td>http://www.tedpella.com/chemical_html/chem3.htm#anchor267712</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Triton X-100</td>
			<td>Fisher</td>
			<td>BP151</td>
			<td>https://www.fishersci.com/ecomm/servlet/itemdetail?storeId=10652&#38;langId=-1&#38;catalog 
			Id=29104&#38;productId=3425922&#38;dis 
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			PROD&#38;hasPromo=1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa 488 - protein conjugation kit</td>
			<td>Life Technologies</td>
			<td>A20181</td>
			<td>http://www.lifetechnologies.com/order/catalog/product/A10235</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">goat serum</td>
			<td>MP Biomedicals</td>
			<td>ICN19135680</td>
			<td>https://www.fishersci.com/ecomm/servlet/itemdetail?storeId=10652&#38;langId=-1&#38;catalog 
			Id=29104&#38;productId=2133236&#38;dis 
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			=PROD&#38;hasPromo=0</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vectashield mounting media</td>
			<td>Vector Labs</td>
			<td>H1200</td>
			<td>https://www.vectorlabs.com/catalog.aspx?prodID=428</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FITC-conjugated dolichos</td>
			<td>Vector Labs</td>
			<td>FL-1031</td>
			<td>https://www.vectorlabs.com/catalog.aspx?prodID=188</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Antibody to LAMP1</td>
			<td colspan="2">Developmental Studies Hybridoma Bank</td>
			<td>http://dshb.biology.uiowa.edu/LAMP-1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LysoTracker</td>
			<td>Life Technologies</td>
			<td>L-7526</td>
			<td>https://www.lifetechnologies.com/order/catalog/product/L7526?ICID=search-product</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">C57BL6 mice</td>
			<td>Jackson Laboratories</td>
			<td align="right">664</td>
			<td>http://jaxmice.jax.org/strain/000664.html</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">gp91 phox knock out mice</td>
			<td>Jackson Labaoratories</td>
			<td align="right">2365</td>
			<td>http://jaxmice.jax.org/strain/002365.html</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">iNOS knock out mice</td>
			<td>Jackson Laboratories</td>
			<td align="right">2609</td>
			<td>http://jaxmice.jax.org/strain/002609.html</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">sodium nitroprusside</td>
			<td>ACROS Organics</td>
			<td>AC21164-0250&#160;</td>
			<td>https://www.fishersci.com/ecomm/servlet/itemdetail?storeId=10652&#38;langId=-1&#38;catalog 
			Id=29104&#38;productId=2627727&#38;dis 
			type=0&#38;highlightProductsItemsFlag 
			=Y&#38;fromSearch=1&#38;searchType= 
			PROD&#38;hasPromo=1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DETA NONOate</td>
			<td>ACROS Organics</td>
			<td>AC32865-0250</td>
			<td>https://www.fishersci.com/ecomm/servlet/itemdetail?storeId=10652&#38;langId=-1&#38;catalog 
			Id=29104&#38;productId=2252389&#38;dis 
			type=0&#38;highlightProductsItemsFlag 
			=Y&#38;fromSearch=1&#38;searchType= 
			PROD&#38;hasPromo=1</td>
		</tr>
		<tr height="21">
			<td colspan="2" height="21" style="height:21px;">Monoclonal mouse anti-Toxoplasma gondii Ab</td>
			<td></td>
			<td>10T19A</td>
			<td>http://1degreebio.org/reagents/product/1069274/?qid=652947</td>
		</tr>
	</tbody>
</table>

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.

弓形虫自由复制在幼稚的巨噬细胞，并具有6-12小时取决于该寄生虫的应变之间的倍增时间。 图1示出了在相对 ​​于幼稚激活骨髓衍生的巨噬细胞代表的寄生虫。 图2示出了寄生在HFF的一般形态宿主细胞在2，4，8，16和32个寄生虫/ PV。在当前的协议，该寄生虫可以侵入幼稚的巨噬细胞，并建立一个新生虫空泡（PV）前的强效激活刺激，LPS和IFN-的输送，对受感染的巨...

Watch this Scientific Journal Video about Forward Genetics Screens Using Macrophages to Identify Toxoplasma gondii Genes Important for Resistance to IFN-Î³-Dependent Cell Autonomous Immunity at JoVE

Forward Genetics Screens Using Macrophages to Identify Toxoplasma gondii Genes Important for Resistance to IFN-γ Dependent Cell Autonomous Immunity | Immunology and Infection | Video

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 ...

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

Unit 2

Population Genetics

Analyzing Gene Expression and Function

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11.0K 次观看.  New York Medical College. 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. 

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

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 ...

Selection of Plasmodium falciparum Parasites for Cytoadhesion to Human Brain Endothelial Cells

Most human malaria deaths are caused by blood-stage Plasmodium falciparum parasites. Cerebral malaria, the most life-threatening complication of the disease, is characterised by an accumulation of Plasmodium falciparum infected red blood cells (iRBC) at pigmented trophozoite stage in the microvasculature of the brain2-4. This microvessel obstruction (sequestration) leads to acidosis, hypoxia and harmful inflammatory cytokines (reviewed in 5). Sequestration is also found in most microvascular tissues of the human body2, 3. The mechanism by which iRBC attach to the blood vessel walls is still poorly understood.
The immortalized Human Brain microvascular Endothelial Cell line (HBEC-5i) has been used as an in vitro model of the blood-brain barrier6. However, Plasmodium falciparum iRBC attach only poorly to HBEC-5i in vitro, unlike the dense sequestration that occurs in cerebral malaria cases. We therefore developed a panning assay to select (enrich) various P. falciparum strains for adhesion to HBEC-5i in order to obtain populations of high-binding parasites, more representative of what occurs in vivo.
A sample of a parasite culture (mixture of iRBC and uninfected RBC) at the pigmented trophozoite stage is washed and incubated on a layer of HBEC-5i grown on a Petri dish. After incubation, the dish is gently washed free from uRBC and unbound iRBC. Fresh uRBC are added to the few iRBC attached to HBEC-5i and incubated overnight. As schizont stage parasites burst, merozoites reinvade RBC and these ring stage parasites are harvested the following day. Parasites are cultured until enough material is obtained (typically 2 to 4 weeks) and a new round of selection can be performed. Depending on the P. falciparum strain, 4 to 7 rounds of selection are needed in order to get a population where most parasites bind to HBEC-5i. The binding phenotype is progressively lost after a few weeks, indicating a switch in variant surface antigen gene expression, thus regular selection on HBEC-5i is required to maintain the phenotype.
In summary, we developed a selection assay rendering P. falciparum parasites a more "cerebral malaria adhesive" phenotype. We were able to select 3 out of 4 P. falciparum strains on HBEC-5i. This assay has also successfully been used to select parasites for binding to human dermal and pulmonary endothelial cells. Importantly, this method can be used to select tissue-specific parasite populations in order to identify candidate parasite ligands for binding to brain endothelium. Moreover, this assay can be used to screen for putative anti-sequestration drugs7.

Selection of Plasmodium falciparum Parasites for Cytoadhesion to Human Brain Endothelial Cells

An in vitro model for cerebral malaria sequestration is described1. Plasmodium falciparum infected red blood cells are selected for binding to immortalized human brain microvascular endothelial cells. The selected parasites show a distinct phenotype. The selection process can be applied using various P. falciparum strains and endothelial cell lines.

选择恶性疟原虫</emCytoadhesion>寄生虫对人脑血管内皮细胞

Most human malaria deaths are caused by blood-stage Plasmodium falciparum parasites. Cerebral malaria, the most life-threatening complication of the ...

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 ...

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 ...

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 ...

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 ...

Using RNA-interference to Investigate the Innate Immune Response in Mouse Macrophages

Macrophages are key phagocytic innate immune cells. When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the pathogen, produce various cytokines and chemokines to recruit and stimulate other immune cells, and present antigens to stimulate the adaptive immune response. Thus, being able to efficiently manipulate macrophages with techniques such as RNA-interference (RNAi) is critical to our ability to investigate this important innate immune cell. However, macrophages can be technically challenging to transfect and can exhibit inefficient RNAi-induced gene knockdown. In this protocol, we describe methods to efficiently transfect two mouse macrophage cell lines (RAW264.7 and J774A.1) with siRNA using the Amaxa Nucleofector 96-well Shuttle System and describe procedures to maximize the effect of siRNA on gene knockdown. Moreover, the described methods are adapted to work in 96-well format, allowing for medium and high-throughput studies. To demonstrate the utility of this approach, we describe experiments that utilize RNAi to inhibit genes that regulate lipopolysaccharide (LPS)-induced cytokine production.

In this protocol, we describe methods to efficiently transfect murine macrophage cell lines with siRNAs using the Amaxa Nucleofector 96-well Shuttle System, stimulate the macrophages with lipopolysaccharide, and monitor the effects on inflammatory cytokine production.

利用RNA干扰调查的先天免疫反应的小鼠巨噬细胞

Macrophages are key phagocytic innate immune cells.  When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the ...

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of inflammation. They are key players in the innate immune system and play major roles in cancer biology. Neutrophils have been proposed as key mediators of malignant transformation, tumor progression, angiogenesis and in the modulation of the antitumor immunity; through their release of soluble factors or their interaction with tumor cells. To characterize the specific functions of neutrophils, a fast and reliable method is coveted for in vitro isolation of neutrophils from human blood. Here, a density gradient separation method is demonstrated to isolate neutrophils as well as mononuclear cells from the blood. The procedure consists of layering the density gradient solution such as Ficoll carefully above the diluted blood obtained from patients diagnosed with chronic lymphocytic leukemia (CLL), followed by centrifugation, isolation of mononuclear layer, separation of neutrophils from RBCsby dextran then lysis of residual erythrocytes. This method has been shown to isolate neutrophils &#8805; 90 % pure. To mimic the tumor microenvironment, 3-dimensional (3D) experiments were performed using basement membrane matrix such as Matrigel. Given the short half-life of neutrophils in vitro, 3D experiments with fresh human neutrophils cannot be performed. For this reason promyelocytic HL60 cells are differentiated along the granulocytic pathway using the differentiation inducers dimethyl sulfoxide (DMSO) and retinoic acid (RA). The aim of our experiments is to study the role of neutrophils on the sensitivity of lymphoma cells to anti-lymphoma agents. However these methods can be generalized to study the interactions of neutrophils or neutrophil-like cells with a large range of cell types in different situations.

Neutrophils are the most abundant type of white blood cells. The isolation of neutrophils from human blood by density gradient separation method and the differentiation of human promyelocytic (HL60) cells along the granulocytic pathway are described here; to test their role on sensitivity of lymphoma cells to anti-lymphoma agents.