Name: forward genetics screens using macrophages to identify toxoplasma gondii genes important for resistance to ifn-γ-dependent cell autonomous immunity
Uploaded: 2015-03-12T15:00:00.000+00:00
Duration: 11 min 21 s
Description: 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

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的事实证明该协议可以专门用来识别那些在它们抵抗巨噬细胞活化的能力特别受损的突变体。使用原代骨髓源巨噬细胞的建议相对于RAW 264.7巨噬细胞系为屏幕因为寄生虫和每VACU寄生虫数目的形态OLE更容易显现于骨髓来源的巨噬细胞。 所描述的屏幕加上正向遗传学提供了一个多功能的工具来剖析寄生虫的基因和抗细胞自主免疫功能的特定介质最终的重要遗传途径。这样向前遗传学研究是重要的，以确定可以遵循像字符串开始解开用于抵抗在体外和发病机制中的特定抗微生物介质重要途径寄生虫疟原虫基因。先前的困难正向遗传学的方法是识别打乱每个突变的关键基因。粘粒库互补的T.弓形虫一直为细胞分裂突变体功能互补相当有效。然而，事实证明，野生型寄生虫复制也被抑制由IFN-γ和LPS的只是比突变体在较小程度上，使得功能互补更难塔n对于细胞分裂的突变体。全基因组突变图谱在T.化学和插入突变弓形虫最近出现作为一个生产，甚至成本效益，渠道，以确定负责正向遗传学屏幕34,36,37,44突变表型的基因。因此，使用下一代测序整个基因组的突变图谱具有增强T的化学诱变的潜在用途弓形虫的正向遗传学研究，以确定基因的具体功能很重要。如在当前的协议描述了这样一个正向遗传学方法是重要的，因为大多数T的基因组中的弓形虫仍然假设一个多数不具有同源性的其他基因或功能结构域可能在候选寄生虫基因免疫逃避重要的识别有助于预测基因的。 96孔板的IFA分析通常不被视为遇到一个高通量筛选HOD。在我们的经验是合理的一人进行染色和屏幕10的96孔板中，每天或约960的突变体。在纸张通过Skariah。等 ，约8000的突变体进行了分析和14个独立的突变体中分离，对于生存/复制的激活，但不幼稚的巨噬细胞28被显著损害。突变体的障存活率为主易感性增加一氧化氮的结果。在整个方法的限制，主要是在获得的寄生虫单个克隆，而不是由镜筛选作为在96孔板的初始画面是定性和合理地快速的水平。在96孔板的筛选中鉴定的突变体的表型的确认被随访在步骤3中通过使用在腔室玻片的巨噬细胞的定量和定性分析，并通过添加野生型或突变的寄生虫的数目相等。使用其它分析筛选方法如荧光在寄生虫的总人口的水平，而不是作为评价用显微镜个体寄生虫的水平，是有问题的，在当前的协议一样多的退化寄生虫染色用抗T的多克隆抗体弓形虫的强劲与野生型寄生虫与突变比定量的荧光强度的水平更加定性的差异。此外，IFN-γ和分离的突变体与抗活化感染的巨噬细胞的结果在野生型寄生虫抑制复制尽管在稍后的时间点的缺陷所需的LPS的剂量。因此，对于正向遗传学屏幕包括利用发光的寄生虫总数寄生虫数量的测定是有问题的。然而，这是可能的染色方案可在筛选步骤被消除，如果用于化学或插入突变的亲代克隆的寄生虫表示的组成型或诱导荧光或路西弗酶标记。使用在96孔板中的巨噬细胞的IFA和显微镜筛选所描述的协议允许突变体在活化的巨噬细胞缺陷的存活/复制的隔离，但它也清楚地射门由于可实现使用96孔板格式作为有限微观分辨率的一些潜在的突变体在这个分辨率非晶肿泡了染色的寄生虫抗原可能被误认为是在一个正常的健康的PV复制寄生虫。 96孔盖玻璃板，代替的96孔板用光学表面的替代，有可能实现感染的巨噬细胞在96孔板中在屏幕中更大的分辨率。 用IFN-γ和LPS中所描述的协议，以激活下面寄生虫入侵的巨噬细胞所确定的突变绝大多数有一个缺陷在他们的保护自己免受一氧化氮28的能力。在这方面，虽然屏幕旨在是无偏，可富集寄生虫突变体具有增加的易感性取决于活化条件，类型和巨噬细胞的种类以及相对于巨噬细胞活化寄生虫侵入被选择用于在屏幕的定时特定抗微生物介质的隔离。从而选择用于屏幕和施加的激活刺激的先天免疫细胞是影响抗微生物介质向其中所得寄生虫突变体可能是敏感的类型的关键参数。寄生虫的基因型也是一个关键的参数作为I型寄生虫的基因型诱导响应于IFN-γ的免疫相关GTP酶（IRGs）具有相对的抗性，而II型和III基因型更容易受到26,45,46。在所描述的协议，巨噬细胞被寄生虫侵袭后激活。虽然II型基因型，Prugnaud应变，在所描述的屏幕中使用易受免疫相关GTP酶的作用下，一llowing寄生虫入侵的巨噬细胞能首先与野生型寄生虫复制全感应IRGs前。此外，它并不清楚IRGs有效对抗如果它们诱导寄生虫侵袭和复制起始随后巨噬细胞的寄生虫。 所描述的协议使用的巨噬细胞，以确定寄生虫基因抗IFN-γ依赖型的细胞中自主免疫，特别是一氧化氮介重要。共享一个明显易感性氧化氮以及一氧化氮依赖性的线粒体碎片的共享表型的突变体寄生虫的池的隔离提供了一种独特的工具集，以确定寄生虫基因和通路的抗性很重要的一氧化氮和理解一氧化氮的更广泛的抗T的动作弓形虫和其它真核pathogens.The描述协议与小的修改，可用于正向遗传学studie秒鉴别寄生虫基因抗性的细胞中自主免疫不同介质重要。潜在的修饰包括改变宿主细胞的感染的类型，激活刺激，或相对活化的定时寄生虫侵袭。例如，预活化的鼠巨噬细胞通过加入寄生虫前的IFN-γ会导致活化的免疫相关GTP酶。 T的这样的模式可以用于确定寄生虫的基因中的I型基因型弓形虫可能有助于通过ROP18和ROP5到免疫相关GTP酶24,25介导的抗性。对T.细胞自主的免疫介导弓形虫在人类先天免疫细胞没有得到很好定义为啮齿动物。因此，使用人外周血单核细胞的筛选T的化学突变弓形虫可能人类感染中先天免疫细胞，以及机制在人类FO重要期间确定既寄生虫基因生存重要以T·R抗菌药物耐药性弓形虫 。寄生虫基因抗性特定抗微生物介质可以通过分离的突变体不能存活感染的宿主细胞，以药物制​​剂的曝光，如活性氧或氮物种鉴定重要。同样地，协议可以被修改以分离寄生虫突变体具有增加的易感性的炎性活化47-49或巨噬细胞嘌呤能受体50的ATP刺激。 综合来看，这些协议描述的方法使用正向遗传学和先天免疫细胞分离寄生虫突变为T.重要弓形虫抗IFN-γ依赖型细胞中自主免疫力。重要的是，这种方法提出了具有通用性，并且可以容易地修改以分离寄生虫基因重要回避特定的抗微生物介质，寄生虫存活在特定类型的宿主细胞或抗应力条件encounte的弓形体病中的红色。此外，对于抗宿主细胞活化重要的寄生虫的基因在许多情况下，也可以直接或间接地在囊肿生产感染过程中发挥作用的体内 。

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。

<table><tbody><tr><td>DMEM</td><td>Hyclone</td><td>SH3008101</td><td>https://www.fishersci.com/ecomm/servlet/itemdetail?storeId=10652&amp;amp;langId=-1&amp;amp;catalog&lt;br/&gt; Id=29101&amp;amp;productId=3255471&amp;amp;dis&lt;br/&gt; type=0&amp;amp;highlightProductsItemsFlag&lt;br/&gt; =Y&amp;amp;fromSearch=1&amp;amp;searchType=&lt;br/&gt; PROD&amp;amp;hasPromo=0</td></tr><tr><td>Hyclone FBS</td><td>Thermo</td><td>SH3091003</td><td>https://www.fishersci.com/ecomm/servlet/itemdetail?storeId=10652&amp;amp;langId=-1&amp;amp;catalog&lt;br/&gt; Id=29104&amp;amp;productId=11737973&amp;amp;dis&lt;br/&gt; type=0&amp;amp;highlightProductsItemsFlag&lt;br/&gt; =Y&amp;amp;fromSearch=1&amp;amp;searchType=&lt;br/&gt; PROD&amp;amp;hasPromo=0</td></tr><tr><td>Hyclone DPBS</td><td>Thermo</td><td>SH3002802&amp;nbsp;</td><td>https://www.fishersci.com/ecomm/servlet/itemdetail?storeId=10652&amp;amp;langId=-1&amp;amp;catalog&lt;br/&gt; Id=29104&amp;amp;productId=2434305&amp;amp;dis&lt;br/&gt; type=0&amp;amp;highlightProductsItemsFlag&lt;br/&gt; =Y&amp;amp;fromSearch=1&amp;amp;searchType&lt;br/&gt; =PROD&amp;amp;hasPromo=0</td></tr><tr><td>Hyclone L-glutamine</td><td>Thermo</td><td>SH3003401&amp;nbsp;</td><td>https://www.fishersci.com/ecomm/servlet/itemdetail?storeId=10652&amp;amp;langId=-1&amp;amp;catalog&lt;br/&gt; Id=29104&amp;amp;productId=3311957&amp;amp;dis&lt;br/&gt; type=0&amp;amp;highlightProductsItemsFlag&lt;br/&gt; =Y&amp;amp;fromSearch=1&amp;amp;searchType&lt;br/&gt; =PROD&amp;amp;hasPromo=0</td></tr><tr><td>Hyclone Pen strep</td><td>Thermo</td><td>SV30010&amp;nbsp;</td><td>https://www.fishersci.com/ecomm/servlet/fsproductdetail?storeId=10652&amp;amp;productId=1309668&lt;br/&gt; 6&amp;amp;catalogId=29104&amp;amp;matchedCat&lt;br/&gt; No=SV30010&amp;amp;fromSearch=1&amp;amp;&lt;br/&gt; searchKey=SV30010&amp;amp;highlightPro&lt;br/&gt; ductsItemsFlag=Y&amp;amp;endecaSearch&lt;br/&gt; Query=%23store%3DRE_SC%23nav%3D0%23rpp%3D25%23offSet%3D0%23keyWord%3DSV30010%2B%23searchType%3DPROD%23SWKeyList%3D%5B%5D&amp;amp;xrefPartType=From&amp;amp;savings= 0.0&amp;amp;xrefEvent=1407777949003_0&lt;br/&gt; &amp;amp;searchType=PROD&amp;amp;hasPromo=0</td></tr><tr><td>Hyclone Hanks BSS</td><td>Thermo</td><td>SH3003002</td><td>https://www.fishersci.com/ecomm/servlet/itemdetail?storeId=10652&amp;amp;langId=-1&amp;amp;catalog&lt;br/&gt; Id=29104&amp;amp;productId=3064595&amp;amp;dis&lt;br/&gt; type=0&amp;amp;highlightProductsItemsFlag&lt;br/&gt; =Y&amp;amp;fromSearch=1&amp;amp;searchType=&lt;br/&gt; PROD&amp;amp;hasPromo=0</td></tr><tr><td>LPS</td><td>LIST biologicals</td><td>201</td><td>http://www.listlabs.com/products-tech.php?cat_id=4&amp;amp;product_id=81&amp;amp;keywords&lt;br/&gt; =LPS_from_%3Cem%3EEscherichia_coli%3C/em%3E_O111:B4</td></tr><tr><td>IFN-&amp;#947;</td><td>Pepro Tech Inc</td><td>50-813-664</td><td>https://www.fishersci.com/ecomm/servlet/itemdetail?itemdetail=&#039;item&#039;&amp;amp;storeId=10652&amp;amp;&lt;br/&gt; productId=2988494&amp;amp;catalogId=29&lt;br/&gt; 104&amp;amp;matchedCatNo=50813664&amp;amp;&lt;br/&gt; fromSearch=1&amp;amp;searchKey=murine+ifn+pepro+tech&amp;amp;highlightProductsItemsFlag&lt;br/&gt; 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PROD&amp;amp;hasPromo=0</td></tr><tr><td>96-well tissue culture plates</td><td>Fisher</td><td>353072</td><td>https://www.fishersci.com/ecomm/servlet/itemdetail?storeId=10652&amp;amp;langId=-1&amp;amp;catalog&lt;br/&gt; Id=29104&amp;amp;productId=3158736&amp;amp;dis&lt;br/&gt; type=0&amp;amp;highlightProductsItemsFlag&lt;br/&gt; =Y&amp;amp;fromSearch=1&amp;amp;searchType=&lt;br/&gt; PROD&amp;amp;hasPromo=0</td></tr><tr><td>Tissue culture flast T25</td><td>Fisher</td><td>156367</td><td>https://www.fishersci.com/ecomm/servlet/fsproductdetail?storeId=10652&amp;amp;productId=127039&lt;br/&gt; 67&amp;amp;catalogId=29104&amp;amp;matchedCat&lt;br/&gt; No=12565351&amp;amp;fromSearch=1&amp;amp;&lt;br/&gt; searchKey=156367&amp;amp;highlightProdu&lt;br/&gt; ctsItemsFlag=Y&amp;amp;endecaSearchQu&lt;br/&gt; ery=%23store%3DRE_SC%23nav%3D0%23rpp%3D25%23offSet%3D0%23keyWord%3D156367%23searchType%3DPROD%23SWKeyList%3D%5B%5D&amp;amp;xrefPartType=From&amp;amp;savings&lt;br/&gt; =0.0&amp;amp;xrefEvent=1407778974800_&lt;br/&gt; 0&amp;amp;searchType=PROD&amp;amp;hasPromo&lt;br/&gt; =0</td></tr><tr><td>Ted Pella EM grade formaldehyde</td><td>Ted Pella</td><td>18505</td><td>http://www.tedpella.com/chemical_html/chem3.htm#anchor267712</td></tr><tr><td>Triton X-100</td><td>Fisher</td><td>BP151</td><td>https://www.fishersci.com/ecomm/servlet/itemdetail?storeId=10652&amp;amp;langId=-1&amp;amp;catalog&lt;br/&gt; Id=29104&amp;amp;productId=3425922&amp;amp;dis&lt;br/&gt; type=0&amp;amp;highlightProductsItemsFlag&lt;br/&gt; =Y&amp;amp;fromSearch=1&amp;amp;searchType=&lt;br/&gt; PROD&amp;amp;hasPromo=1</td></tr><tr><td>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><td>goat serum</td><td>MP Biomedicals</td><td>ICN19135680</td><td>https://www.fishersci.com/ecomm/servlet/itemdetail?storeId=10652&amp;amp;langId=-1&amp;amp;catalog&lt;br/&gt; Id=29104&amp;amp;productId=2133236&amp;amp;dis&lt;br/&gt; type=0&amp;amp;highlightProductsItemsFlag&lt;br/&gt; =Y&amp;amp;fromSearch=1&amp;amp;crossRefData&lt;br/&gt; =ICN19135680=2&amp;amp;searchType&lt;br/&gt; =PROD&amp;amp;hasPromo=0</td></tr><tr><td>Vectashield mounting media</td><td>Vector Labs</td><td>H1200</td><td>https://www.vectorlabs.com/catalog.aspx?prodID=428</td></tr><tr><td>FITC-conjugated dolichos</td><td>Vector Labs</td><td>FL-1031</td><td>https://www.vectorlabs.com/catalog.aspx?prodID=188</td></tr><tr><td>Antibody to LAMP1</td><td>Developmental Studies Hybridoma Bank</td><td>http://dshb.biology.uiowa.edu/LAMP-1</td><td></td></tr><tr><td>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><td>C57BL6 mice</td><td>Jackson Laboratories</td><td>664</td><td>http://jaxmice.jax.org/strain/000664.html</td></tr><tr><td>gp91 phox knock out mice</td><td>Jackson Labaoratories</td><td>2365</td><td>http://jaxmice.jax.org/strain/002365.html</td></tr><tr><td>iNOS knock out mice</td><td>Jackson Laboratories</td><td>2609</td><td>http://jaxmice.jax.org/strain/002609.html</td></tr><tr><td>sodium nitroprusside</td><td>ACROS Organics</td><td>AC21164-0250&amp;nbsp;</td><td>https://www.fishersci.com/ecomm/servlet/itemdetail?storeId=10652&amp;amp;langId=-1&amp;amp;catalog&lt;br/&gt; Id=29104&amp;amp;productId=2627727&amp;amp;dis&lt;br/&gt; type=0&amp;amp;highlightProductsItemsFlag&lt;br/&gt; =Y&amp;amp;fromSearch=1&amp;amp;searchType=&lt;br/&gt; PROD&amp;amp;hasPromo=1</td></tr><tr><td>DETA NONOate</td><td>ACROS Organics</td><td>AC32865-0250</td><td>https://www.fishersci.com/ecomm/servlet/itemdetail?storeId=10652&amp;amp;langId=-1&amp;amp;catalog&lt;br/&gt; Id=29104&amp;amp;productId=2252389&amp;amp;dis&lt;br/&gt; type=0&amp;amp;highlightProductsItemsFlag&lt;br/&gt; =Y&amp;amp;fromSearch=1&amp;amp;searchType=&lt;br/&gt; PROD&amp;amp;hasPromo=1</td></tr><tr><td>Monoclonal mouse anti-Toxoplasma gondii Ab</td><td>Fitzgerald Industries International</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，对受感染的巨噬细胞。在这个模型中，野生型寄生虫复制减缓，但寄生虫复制仍然前进约24小时，在此期间的巨噬细胞逐渐变得在抑制寄生虫复制更擅长。该屏幕被设计为充当所需强效的巨噬细胞一时间之间的改性竞争模型ctivati​​on与其间的野生型寄生虫或寄生虫突变体可以继续其光伏内复制的时间。在屏幕上的第一个步骤是选择的LPS的剂量和IFN-γ对可用于活化感染的巨噬细胞。剂量是根据经验通过评价寄生虫复制中的巨噬细胞与任一恒定剂量IFN-γ的结合的范围内的LPS浓度的或恒定剂量的LPS与一系列浓度的IFN-（步骤1）激活测定。激活刺激的剂量需要对用于建立所述寄生虫的突变体通过化学或插入突变的亲代野生型寄生虫进行评估。理想的情况是LPS和IFN-γ的剂量将允许寄生虫复制到平均每光伏24-30小时2-8寄生虫活化后相比每光伏8-16寄生在幼稚巨噬细胞（图1和2）。 一旦LPS和IFN-的适当浓度^7;被确定时，突变体进行筛选在96孔光学底组织培养板中。在板中的光学底部是重要的，因为在塑料凹凸是用于传统的组织培养板使得难以实现适当的分辨率筛选寄生虫通过相差和荧光显微镜。在96孔板的相衬和荧光显微镜也需要与相衬4，10，20或40×物镜配备长工作距离的倒置荧光显微镜。的突变体中筛选在含有鼠骨髓衍生的巨噬细胞的复制板中。与寄生虫突变体攻击后，感染的巨噬细胞中一个板的孔中被激活用LPS和IFN-γ而含有感染的巨噬细胞的另一个板是在仅D10培养基中培养。板在37℃和5％CO 2的加入LPS和IFN-γ的后24-30小时。孵育后，细胞是音响固定的，染色的寄生虫和同时使用荧光和相差显微镜由IFA分析。突变体选择具有正常寄生虫数目和在幼稚的巨噬细胞的复制，但更少的寄生虫或更小的空泡与受感染的巨噬细胞减少寄生虫被激活。一旦突变体被选择他们应该被重新筛选在腔室玻片（步骤3）在幼稚和活化的巨噬细胞在两个独立的实验，以便对寄生虫形态分析在更高的放大倍数（100X物镜和油），并确认它们在幼稚的巨噬细胞正常复制但在复制/存活缺陷下巨噬细胞活化。 与抗活化感染的巨噬细胞缺陷的突变体的表型通常分为两大类:出现形态完整的，但都无法复制超出每光伏单一寄生虫寄生虫;并可以degrad出现寄生虫ED并且也可以是在非晶质宽敞的PV（图1和3）。形态和寄生虫的超微结构和PV上使用100X客观，油幻灯片和相差显微镜结合荧光分析的最佳效果。寄生虫为IFA染色的抗体溶酶体相关膜蛋白1（LAMP1）是非常有用的，以确定在突变的缺陷是否与光伏无力，以防止与溶酶体融合相关联。 图4所示是内吞噬溶酶体（LAMP1阳性）对寄生虫是在光伏是从该宿主细胞（LAMP1负）的内吞系统基本上隔离的寄生虫。吞噬溶酶体显然由LAMP1的连续固体轮缘围绕整个寄生虫使劲。与此相反，一个光伏常呈溶酶体细胞器附近，但围绕其圆周LAMP1染色没有连续边缘。使用抗体内吨定义的结构/细胞器他寄生虫和/或PV都在跟进IFA研究，以确定在激活的巨噬细胞感染相关的每一个突变异常有用的。与抗体，盘问可以提供洞察背后的复制/存活缺陷突变的机制。例如， 图5示出了T.弓形虫线粒体沾满单克隆抗体5F4抗F1 ATP酶β亚基（由彼得·布拉德利一个样的礼物）激活感染了野生型寄生虫或寄生虫巨噬细胞突变后的24小时。T.弓形虫是共同沾上单克隆抗体T.弓形虫抗体。野生型T.弓形虫具有单个线粒体周围的寄生虫的圆周上延伸。相比之下，突变体寄生虫零散线粒体以下激活的巨噬细胞感染的野生型寄生虫寄生虫的唯一线粒体是完整的。不仅在退化/非晶线粒体碎片明显寄生虫，而且在该显示形态正常作为评价用相差显微镜寄生虫。这表明，在该寄生虫突变体的缺陷可能会增加寄生虫线粒体的易感性宿主诱导巨噬细胞活化细胞介质。 当前模型使用IFN-γ和LPS的结合来激活受感染的巨噬细胞。 IFN-γ刺激转录因子STAT1。脂多糖，像的TNF-α，刺激转录因子的NF-κB。已知的IFN-γ依赖抗菌调解员对T.细胞自主免疫功能的重要弓形虫包括IFN-γ依赖型免疫相关GTP酶除了其它试剂的阵列（IRGs，Gbps）的和活性氮和氧。以确定是否在每个突变体中的缺陷进行具体的生产已知的IFN-γ诱导性抗微生物介质相关联，骨髓衍生的巨噬细胞是从分离iNOS的- / - ，GP 91 PHOX - / -或特定IRG /英镑基因缺失的小鼠，并测定抗突变寄生虫活性。 IFN-γ和LPS的结合来激活受感染的巨噬细胞优先地导致具有增强的易感性的突变体，以一氧化氮与野生型相比寄生虫28。 受感染的巨噬细胞的活化可以诱导寄生虫阶段分化速殖子至中包含的组织囊肿bradyzoites。这种囊肿是慢性感染的特征。因此，寄生虫突变体激活后感染的巨噬细胞是有缺陷的复制/生存也可能是有缺陷的体内感染过程中转换成囊肿。为了慢性感染期间评估囊肿生产，将小鼠腹膜内（IP）与非致死剂量的亲寄生虫菌株或突变体克隆的挑战。孢囊在从不到10大脑范围在尺寸_6;米，直径大于50微米， 如图6计数大型和小型囊肿通过IFA的数量，但保持的计数分开，以便确定该寄生虫突变体受损总囊肿生产或只为建立及维护的大包囊。 <img alt="图1" src="/files/ftp_upload/52556/52556fig1.jpg" /> 图1.朴素鼠骨髓衍生的巨噬细胞是在容许T的复制弓形虫 ，但激活与IFN-γ和LPS基本抑制复制。（A）的野生型T.一虫空泡（PV） 弓形虫侵入24天真的小鼠骨髓来源的巨噬细胞后小时。（B）的野生型T.一个PV 弓形虫的寄生虫包括每泡24小时4寄生虫感染激活巨噬细胞后。（C）一种突变体寄生虫无法复制，仍然在同一寄生虫/ PV活化感染的巨噬细胞的24小时后的一个例子。（D），该出现活化感染的巨噬细胞的内后的非晶质的PV 24小时退化的突变体寄生虫的实例。顶行示出了寄生虫阶段的图像和底部的行示出了荧光图像使用针对T的多克隆抗血清弓形虫 。比例尺条为5微米。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52556/52556fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/52556/52556fig2.jpg" /> 由每光伏寄生虫的数目图2.寄生虫复制评价。图为2，4，8，16和每个光伏32寄生虫HFF细胞。每一个画面是一个合并阶段形象次在展示了寄生虫红色和HFF核蓝色（DAPI）的多克隆抗体染色。比例尺条为5微米。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52556/52556fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/52556/52556fig3.jpg" /> 数字3.突变体呈现范围表型的活化后感染的巨噬细胞。左边的列是在巨噬细胞的寄生虫的相位图像，中间的列是使用抗体来T.的荧光图像弓形虫 ，而右列是相位和荧光图像的合并。寄生虫示于绿色和蓝色的巨噬细胞的细胞核中。比例尺条为5微米。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52556/52556fig3large.jpg" target="_blank">请点击此处查看大图</a>版本这个数字。 <img alt="图4" src="/files/ftp_upload/52556/52556fig4.jpg" /> 图4. LAMP1染色区分的PV来自phagolysomes含有寄生虫。寄生虫染色用多克隆抗T.弓形虫抗体（红色）和LAMP1与单抗1D4B（绿色）。一个箭头标志的寄生虫PV已经融合与溶酶体。没有箭头的寄生虫是在虫空泡（PV）已经没有熔合溶酶体。比例尺条为5微米。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52556/52556fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/52556/52556fig5.jpg" /> 图5.野生型寄生虫维持一个完整的线粒体，而mitochondri线粒体（绿色），在野生型寄生虫（顶面板）相比，三种不同的突变体T.上被降解在活化后感染的巨噬细胞的寄生虫的突变体。 弓形虫寄生在退化（底部三板）的不同阶段激活的巨噬细胞感染后24小时。比例尺条为5微米。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52556/52556fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图6" src="/files/ftp_upload/52556/52556fig6.jpg" /> 图6.检测寄生虫包囊在使用FITC缀合的双花扁豆凝集素的脑的囊壁标记的凝集素被显示为绿色。比例尺代表50微米。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52556/52556fig6large.jpg" target="_blank">请点击此处查看大图versio</a>n这个数字。

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

使用巨噬细胞进行正向遗传学筛选，鉴定对抵抗 IFN-γ 依赖性细胞自主免疫很重要的弓形虫基因免疫学和感染|视频

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 ...

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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-γ-Dependent Cell Autonomous Immunity

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 ...

科研

免疫与感染

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 ...

A Parasite Rescue and Transformation Assay for Antileishmanial Screening Against Intracellular Leishmania donovani Amastigotes in THP1 Human Acute Monocytic Leukemia Cell Line

Leishmaniasis is one of the world's most neglected diseases, largely affecting the poorest of the poor, mainly in developing countries. Over 350 million people are considered at risk of contracting leishmaniasis, and approximately 2 million new cases occur yearly1. Leishmania donovani is the causative agent for visceral leishmaniasis (VL), the most fatal form of the disease. The choice of drugs available to treat leishmaniasis is limited 2;current treatments provide limited efficacy and many are toxic at therapeutic doses. In addition, most of the first line treatment drugs have already lost their utility due to increasing multiple drug resistance 3. The current pipeline of anti-leishmanial drugs is also severely depleted. Sustained efforts are needed to enrich a new anti-leishmanial drug discovery pipeline, and this endeavor relies on the availability of suitable in vitro screening models.
In vitro promastigotes 4 and axenic amastigotes assays5 are primarily used for anti-leishmanial drug screening however, may not be appropriate due to significant cellular, physiological, biochemical and molecular differences in comparison to intracellular amastigotes. Assays with macrophage-amastigotes models are considered closest to the pathophysiological conditions of leishmaniasis, and are therefore the most appropriate for in vitro screening. Differentiated, non-dividing human acute monocytic leukemia cells (THP1) (make an attractive) alternative to isolated primary macrophages and can be used for assaying anti-leishmanial activity of different compounds against intracellular amastigotes.
Here, we present a parasite-rescue and transformation assay with differentiated THP1 cells infected in vitro with Leishmania donovani for screening pure compounds and natural products extracts and determining the efficacy against the intracellular Leishmania amastigotes. The assay involves the following steps: (1) differentiation of THP1 cells to non-dividing macrophages, (2) infection of macrophages with L. donovani metacyclic promastigotes, (3) treatment of infected cells with test drugs, (4) controlled lysis of infected macrophages, (5) release/rescue of amastigotes and (6) transformation of live amastigotes to promastigotes. The assay was optimized using detergent treatment for controlled lysis of Leishmania-infected THP1 cells to achieve almost complete rescue of viable intracellular amastigotes with minimal effect on their ability to transform to promastigotes. Different macrophage:promastigotes ratios were tested to achieve maximum infection. Quantification of the infection was performed through transformation of live, rescued Leishmania amastigotes to promastigotes and evaluation of their growth by an alamarBlue fluorometric assay in 96-well microplates. This assay is comparable to the currently-used microscopic, transgenic reporter gene and digital-image analysis assays. This assay is robust and measures only the live intracellular amastigotes compared to reporter gene and image analysis assays, which may not differentiate between live and dead amastigotes. Also, the assay has been validated with a current panel of anti-leishmanial drugs and has been successfully applied to large-scale screening of pure compounds and a library of natural products fractions (Tekwani et al. unpublished).

A Parasite Rescue and Transformation Assay for Antileishmanial Screening Against Intracellular Leishmania donovani Amastigotes in THP1 Human Acute Monocytic Leukemia Cell Line

A parasite-rescue and transformation assay with THP1 cells infected in vitro with Leishmania donovani has been optimized for anti-leishmanial drug screening. The assay involves differentiation of THP1 cells, infection with promastigotes, treatment with test drugs, controlled lysis of the infected macrophages, rescue of amastigotes, transformation to promastigotes and monitoring promastigote growth and proliferation with a fluorometric assay.

Antileishmanial筛选对细胞内的寄生虫救援和转化实验<em&gt;杜氏利什曼原虫</em无鞭毛体在THP1人急性单核细胞白血病细胞株

Leishmaniasis is  one of the world's most neglected diseases, largely affecting the  poorest of the poor, mainly in developing countries. Over 350 million ...

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).

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 ...

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 ...

Functional Characterization of Regulatory Macrophages That Inhibit Graft-reactive Immunity

Macrophage accumulation in transplanted organs has long been recognized as a feature of allograft rejection1. Immunogenic monocytes infiltrate the allograft early after transplantation, mount a graft reactive response against the transplanted organ, and initiate organ rejection2. Recent data suggest that suppressive macrophages facilitate successful long-term transplantation3 and are required for the induction of transplantation tolerance4. This suggests a multidimensional concept of macrophage ontogeny, activation, and function, which demands a new roadmap for the isolation and analysis of macrophage function5. Due to the plasticity of macrophages, it is necessary to provide a methodology to isolate and characterize macrophages, depending on the tissue environment, and to define their functions according to different scenarios. Here, we describe a protocol for immune characterization of graft-infiltrating macrophages and the methods we used to functionally evaluate their capacity to inhibit CD8+ T proliferation and to promote CD4+Foxp3+ Treg expansion in vitro.

Macrophages are plastic cells of the hematopoietic system that have a crucial role in protective immunity and homeostasis. In this report, we describe optimized in vitro techniques to phenotypically and functionally characterize graft-infiltrating regulatory macrophages that accumulate in the transplanted organ under tolerogenic conditions.

抑制接枝反应性免疫的调节性巨噬细胞的功能表征

Macrophage accumulation in transplanted organs has long been recognized as a feature of allograft rejection1. Immunogenic monocytes infiltrate the ...

Screening Bioactive Nanoparticles in Phagocytic Immune Cells for Inhibitors of Toll-like Receptor Signaling

Pharmacological regulation of Toll-like receptor (TLR) responses holds great promise in the treatment of many inflammatory diseases. However, there have been limited compounds available so far to attenuate TLR signaling and there have been no clinically approved TLR inhibitors (except the anti-malarial drug hydroxychloroquine) in clinical use. In light of rapid advances in nanotechnology, manipulation of immune responsiveness using nano-devices may provide a new strategy to treat these diseases. Herein, we present a high throughput screening method for quickly identifying novel bioactive nanoparticles that inhibit TLR signaling in phagocytic immune cells. This screening platform is built on THP-1 cell-based reporter cells with colorimetric and luciferase assays. The reporter cells are engineered from the human THP-1 monocytic cell line by stable integration of two inducible reporter constructs. One expresses a secreted embryonic alkaline phosphatase (SEAP) gene under the control of a promoter inducible by the transcription factors NF-&#954;B and AP-1, and the other expresses a secreted luciferase reporter gene under the control of promoters inducible by interferon regulatory factors (IRFs).Upon TLR stimulation, the reporter cells activate transcription factors and subsequently produce SEAP and/or luciferase, which can be detected using their corresponding substrate reagents. Using a library of peptide-gold nanoparticle (GNP) hybrids established in our previous studies as an example, we identified one peptide-GNP hybrid that could effectively inhibit the two arms of TLR4 signaling cascade triggered by its prototypical ligand, lipopolysaccharide (LPS). The findings were validated by standard biochemical techniques including immunoblotting. Further analysis established that this lead hybrid had a broad inhibitory spectrum, acting on multiple TLR pathways, including TLR2, 3, 4, and 5. This experimental approach allows a rapid assessment of whether a nanoparticle (or other therapeutic compounds) can modulate specific TLR signaling in phagocytic immune cells.

Toll-like receptor (TLR) signaling plays an important role in the pathophysiology of many human inflammatory diseases, and regulating TLR responses by bioactive nanoparticles is anticipated to be beneficial in many inflammatory conditions. THP-1 cell-based reporter cells provide a versatile and robust screening platform for identifying novel inhibitors of TLR signaling.

筛选生物活性纳米颗粒在噬菌体免疫细胞中的Toll样受体信号传导抑制剂

Pharmacological regulation of Toll-like receptor (TLR) responses holds great promise in the treatment of many inflammatory diseases. However, there have ...

医学

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 ...

正向遗传学屏风使用巨噬细胞识别

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