作者感谢H.艾森，M. Gubbels，光面包车Grinsven，E.吉兰，A.德雷克，五马哈詹，问:高，富有成果的讨论和试剂和G.邑。我们感谢J. Dausman，R.弗兰纳里，协助鼠标殖民地的管理和J.杰克逊。我们感谢FACSorting体育Wisniewski。电磁场是由人类前沿科学计划的支持。 RJ是由国家卫生赠款RO1的HD045022和R37 - CA084198研究所的支持。 HLP的是支持由美国国立卫生研究所资助。

使用这种方法是，在报告的<a href="http://www.sciencemag.org/cgi/content/abstract/328/5975/243">研究Kirak等 。，科学328（5975），243-248（2010</a> ） 。 1。供体细胞的分离之前特定的T细胞或B细胞可以用来作为供体细胞产生transnuclear小鼠模型，小鼠感染病原体的利益需要或利益抗原免疫。既然我们已经使用了弓形虫具体的CD8 +细胞，以下协议描述的这些细胞，并根据个人利益的需要相适应的生成和隔离。 <ol><li> BL / 6 x的BALB / C F1（B6CF1）小鼠，从而使隔离的H - 2，B和H - 2 ð限制CD8 + T细胞，腹腔内注入从小鼠脑组织匀浆（约40）派生的囊肿。 </li><li>感染小鼠的免疫反应的高峰期，安乐死，脾分离并放置到一个含有6毫升红细胞（RBC）的裂解液的培养皿。 </li><li>使用两个封面幻灯片磨砂双方，脾仔细地面，并在室温下5分钟的红细胞裂解液孵育。 </li><li>添加14毫升PBS，通过细胞过滤器（40或70微米）过滤到50毫升猎鹰管300克5分钟离心的细胞悬液。 </li><li>去除上清液，重悬细胞沉淀在1 mL PBS和转移到1.5 mL Eppendorf管。离心300克5分钟，事后去除上清。 </li><li>在50μL的PBS重悬细胞沉淀，加入荧光标记的抗体，以确定感兴趣的细胞类型（如CD3 +，B220，CD8 +和CD4），荧光标记的MHC -Ⅰ四聚体（MHC -Ⅱ类分子四聚体的CD4 T细胞）加载感兴趣的肽的细胞悬液和30分钟的孵育在4 ° C </li><li>加入1 mL PBS，300克，在3 mL PBS重悬沉淀5分钟离心，并通过细胞过滤器（40或70微米）过滤成管状的细胞悬液。 </li><li>执行严格设置大门（图1B）FACSorting。 </li></ol> 2。体细胞核移植体细胞核移植的几个协议。描述一个中期- II在被捕的卵母细胞和抑制的第二次减数分裂的细胞松弛素B。因此供体细胞的分裂是在G0/G1期。 <ol><li>卵母细胞的制备<ol><li> BDF1背景的雌性小鼠的腹腔内注射5IU上午9时至下午5时和上午10时至下午7时之间每鼠标的PMS。 </li><li>约47小时后，每只小鼠注射5IU HCG的腹腔内。 </li><li>准备注射HCG当天的卵母细胞培养皿（KSOM - AA下油滴），并把它们放入孵化器37℃，5％的CO 2。此外，准备必要的针体细胞核移植（7微米眼球摘除和4或淋巴细胞核的核移植5微米）与汞加载它们。 </li><li>安乐死小鼠和隔离有关HCG后13h的输卵管（约上午08时）。收集1-2滴的HCZB输卵管。 </li><li>一个由一搬进包含M2的瓦特/透明质酸下降输卵管。尼克仔细forcep输卵管和移动到下拉卵母细胞的卵丘复杂。经过所有的卵母细胞，卵丘复合物已被隔离，菜是培养在37 ° C。 </li><li> 2-5分钟后（确切时间取决于一批透明质酸酶）收集的卵母细胞，洗HCZB他们下降到KSOM - AA板。 </li></ol></li><li>剜除卵母细胞<ol><li>使用培养皿的盖子准备的体细胞核移植板。显微镜和显微操作。 </li><li>眼球摘除开始之前，在PVP洗净眼球摘除针（7微米）。 </li><li>眼球摘除，放入一个卵母细胞与细胞松弛素B（5微克/毫升）的HZCB下降组（10-30）。虽然孵化（至少5分钟）线的卵母细胞的水平。 </li><li>一个卵母细胞和它放置在前面控股针。拿着针头的卵母细胞的修复采用真空。使用眼球摘除针，卵母细胞染色体主轴复杂（CSC，通常被称为"核心"），在3点钟位置，直到转。 </li><li>使用压电脉冲渗透不破坏卵母细胞的透明带，仔细。将针相邻中期第二主轴的开放和应用真空。 "核心"应该缓慢移动，进针，一旦它被完全去除，膜应密封本身。 </li><li>去核卵母细胞转移到KSOM - AA滴，并多次把它们洗干净。重复的电子一个新的卵母细胞的核步骤。继续，直到所有的鸡蛋都被摘除或约上午11时至。 </li></ol></li><li>核移植<ol><li>核传输，交换核转移针（​​4或5微米），用PVP的眼球摘除针（7微米）。 </li><li>放入您感兴趣的细胞（如CD8 + T细胞）与PVP下降，拌匀孵育至少10分钟。 </li><li>放入组（10-30）的去核卵母细胞与细胞松弛素B（2.5微克/毫升）的HCZB下降。虽然孵化（至少5分钟）通过核移植针行卵母细胞的水平。 </li><li>从PVP的细胞悬液，拿起一个供体细胞和吸和，直到外膜破裂（细胞膜和细胞质的片段应该是可见的的）。如果有必要压电脉冲均可适用。一旦核已被隔离，将它一点点针，并继续，直到你有10-30拿起核。 </li><li>移动包含对齐的去核卵母细胞的下降。真空应用，解决一个卵母细胞的控股针。将核移植针（原子核应远离开幕）相邻的透明带，适用于压电脉冲，通过透明带了，直到针。小心地将一个原子核针尖，针推入卵母细胞，直到针尖是2 / 3内。套用一个小的负压，使卵母细胞质膜的点点针。 </li><li>接下来的步骤是非常关键的，因为它是唯一的一次卵母细胞实际上是"开放"时，。应用压电单脉冲，推针的核应用正压，小心地拉了回来针，使针尖在卵母细胞的1 / 3左右，然后施加负压力，并继续给拉了回来针密封的卵母细胞。重复此步骤，每一个卵母细胞。 </li><li>组中的所有卵母细胞后收到一个原子核，它们洗净，培养KSOM - AA媒体。所有的卵母细胞重复此过程。 </li><li>最后一组后一直在KSOM - AA至少30分钟的文化，体细胞核移植胚胎转移到含有氯化锶钙激活媒体的下降。 </li><li>经过6个小时的激活，伪原核量（PPN）的阳性体细胞核移植胚胎是确定的。胚胎，然后洗净，在KSOM - AA培养滴，直到他们达到了胚泡的阶段，增加3.5天。 </li><li>药物或化学品的选择（如曲古菌素A）可以添加到激活和文化传媒。 </li></ol></li></ol> 3.Derivati​​on的胚胎干细胞可用于体细胞核移植的囊胚，要么转移到假孕的女性（又称直接或一步克隆）或胚胎干细胞（也称为间接或两步克隆）。因为它是更有效的产生ES细胞，我们选择了两个步骤。囊胚情况下，科学家是在直接克隆感兴趣，可以转移到假孕的女性直接在第5节描述。 有描述的胚胎干细胞衍生的许多协议。这里描述的是一个标准的技术，它采用饲养层细胞，胎牛血清。 <ol><li>核移植后的第二天，准备胚胎干细胞衍生的96 U型孔板。 </li><li>加入100μL，明胶，以及到每一个96 - U孔板孵育至少10分钟，之后将其删除。 </li><li>解冻馈线细胞和悬浮在根据"公共服务电子化"的中期量，如饲养层细胞， 相当于 25平方厘米的面积都在10毫升ESD介质和200μL的细胞悬液悬浮添加到每个明胶治疗。 </li><li>板放入孵化器和文化在37℃，5％的CO 2。 </li><li>经过3.5天，胚胎在囊胚阶段。 </li><li>电镀几滴ES细胞培养基和酸性可控硅整流准备无菌细菌培养皿。 </li><li>胚泡是首先从KSOM机管局滴转移到含有滴正常的ES细胞培养基洗涤两次。 </li><li>小心地转移到一滴酸性可控硅整流的囊胚组（5-10囊胚），并保持在那里，直到溶解透明带。 </li><li>的囊胚，然后转入下降与ES培养基，洗涤两次，然后放入馈线涂层的U - 96孔板（一到一个很好的囊胚）。 </li><li>胚胎体外培养5-7天孵化器在37℃，5％的CO 2，而不会干扰他们。这使胚胎的时间到ttach饲养层，形成一个产物。 </li><li>准备24孔板，每孔在ESD介质电镀馈线，如重新悬浮在12毫升的ESD介质相当于一个50厘米2馈线，并添加到每孔500μL的细胞悬液。 </li><li>小心地从"公共服务电子化"的媒介，与胚胎的U - 96孔板，每孔250微升HEPES（或PBS）的两倍洗，加50μL胰蛋白酶和5分钟左右在37 ° C。 </li><li>移液器上下多次，直到产物除了已经下降到一个单细胞悬液，转移到24孔板孵育37℃，5％的CO 2。 </li><li>如果ESC键推导是成功的，ESC键殖民地应该是可见的，经过7-10天。 </li></ol> 4.Blastocyst注射囊胚注射液是一种常规技术，产生任何一种转基因小鼠模型。重要的是要提的，需要定时以及囊胚注射，随后转移到假孕的女性。 <ol><li>总理与PMS和HCG的雌性小鼠在第2.1节所述。注射HCG后把雌性小鼠与雄性小鼠（一名女性和每一个笼子里的雄性小鼠）。 </li><li>第二天，检查插头的女性。这是算作0.5dpc（天性交）。 </li><li>从2.1节所述，并在KSOM - AA培养额外的3天输卵管受精胚胎，然后孤立。 </li><li>准备囊胚注射液（3.5dpc）当天胚胎干细胞。 </li><li>取出胚胎干细胞培养基，HEPES（或PBS）洗两次，加入胰蛋白酶在37℃孵育5分钟° C。 </li><li>胰酶消化细胞悬浮ES介质，在培养皿中板，孵育45-60分钟，在37 ° C，5％的CO 2减少的饲养层细胞（饲养层细胞坚持速度比ES细胞）的数量。 </li><li>成管状细胞悬液通过细胞过滤器（40或70微米）传输，并在1000RPM离心5分钟。 </li><li>删除冰上清，悬浮细胞沉淀，直到500μL，ES介质和存储需要。 </li><li>准备由电镀含滴PVP和HCZB无菌细菌培养皿的盖子。 </li><li>加载汞，放置位置和洗衣机与PVP准备15微米针头。 </li><li>放入一组囊胚（10-30囊胚）与HCZB下降，并添加到另一个HCZB下降5-50μLES细胞悬液（取决于浓度）。 </li><li>拿起ES细胞的注射针（50-100细胞）。 </li><li>移动到下拉的囊胚。水平对齐的囊胚，并修复与应用真空控股针之一。 </li><li>使用注射针转的囊胚内细胞团（ICM）"，直到在9点钟位置。 </li><li>放置在3点钟的注射针头，确保有针尖没有ES细胞，并注射针，直到通过透明带和成囊胚的滋养层穿透，适用于压电脉冲。 </li><li>发行数的ES细胞（5-15细胞），使胚胎干细胞粘到ICM。继续，直到已与ES细胞注入囊胚组中的所有。 </li><li>完成一组囊胚后，洗两次在KSOM - AA，并让他们在KSOM - AA下降，直到所有被注入囊胚。 </li></ol> 5。胚胎移植胚胎转移到假孕的女性代表一种外科手术，这需要进行非常仔细，并根据研究员的研究所的指导方针。 <ol><li>麻醉雌性受体小鼠，刮胡子右下象限，和消毒。 </li><li>用剪刀打开的皮肤，从皮肤中分离出来的腹膜。 </li><li>找到卵巢，通过腹膜（红点），打开腹膜，卵巢接近。 </li><li>使用镊子，小心地抢输卵管和子宫拉出。 </li><li>毛细管连接到口吸管，拿起一组约10囊胚。 </li><li>修正了一个钳子宫，冲了进去一个小的整体，用一个小针头，并插入的毛细血管进入子宫腔的囊胚。 </li><li>小心地释放到子宫的胚胎，并删除您的毛细血管。 </li><li>推回子宫进入腹腔。 </li><li>找到腹膜边缘，并关闭它与几针。 </li><li>几个订书钉关闭鼠标的皮肤。 </li><li>对于手术后的疼痛，管理5微克每毫克体重卡洛芬。 </li></ol><img src="/files/ftp_upload/2168/2168fig1.jpg" alt="图1" /> 体细胞核移植技术图1。B6CF1背景预先定义的T细胞的转移。 CD8 + T细胞和体细胞核移植囊胚的胚胎干细胞生成的胚胎的绝对和相对数量。 <img src="/files/ftp_upload/2168/2168fig2.jpg" alt="图2" /> 图2代表流量与体细胞核移植胚胎干细胞嵌合体小鼠注射的流式细胞仪分析。门和数量（每总CD8 + T细胞％）表明存在特异性CD8 + T细胞在嵌合体小鼠。

<ol>
	<li>Schatz, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=V(D)J+recombination.">V(D)J recombination.</a> Immunol. Rev. 200, 5-5 (2004).</li><li>Barnden, M. J., Allison, J., Heath, W. R., Carbone, F. R., R, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defective+TCR+expression+in+transgenic+mice+constructed+using+cDNA-based+alpha-+and+beta-chain+genes+under+the+control+of+heterologous+regulatory+elements.">Defective TCR expression in transgenic mice constructed using cDNA-based alpha- and beta-chain genes under the control of heterologous regulatory elements.</a> Immunol. Cell Biol. 76, 34-34 (1998).</li><li>Eggan, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hybrid+vigor,+fetal+overgrowth,+and+viability+of+mice+derived+by+nuclear+cloning+and+tetraploid+embryo+complementation.">Hybrid vigor, fetal overgrowth, and viability of mice derived by nuclear cloning and tetraploid embryo complementation.</a> Proc Natl Acad Sci U S A. 98, 6209-6209 (2001).</li><li>Kishigami, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Significant+improvement+of+mouse+cloning+technique+by+treatment+with+trichostatin+A+after+somatic+nuclear+transfer.">Significant improvement of mouse cloning technique by treatment with trichostatin A after somatic nuclear transfer.</a> Biochem Biophys Res Commun. 340, 183-183 (2006).</li><li>Wakayama, T., Yanagimachi, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+cloning+with+nucleus+donor+cells+of+different+age+and+type.">Mouse cloning with nucleus donor cells of different age and type.</a> Mol Reprod Dev. 58, 376-376 (2001).</li></ol>

我们已经表明，可用于生成与预先定义的特异性T细胞的transnuclear小鼠体细胞核移植。虽然这里没有显示，该技术也应该可用与预先定义的特异性的B细胞产生的transnuclear小鼠。 一个重要的事情要考虑的是应变和性别的鼠标，这是感染或接种疫苗和用于隔离T或B细胞的兴趣。由于T细胞和B细胞有一个非常低的重编程效率一般，我们建议使用所需的单体型F1杂种。由于供体细胞也决定了性，我们建议使用从雄性小鼠的T或B细胞。这意味着，只有男性的嵌合体小鼠传输通过生殖细胞的TCR或BCR与雄性小鼠更容易和更快繁殖，。 两者合计，我们认为，通过体细胞核移植后生重新编程产生的小鼠模型的一种新型免疫学领域的巨大优势，这将是一个有力的工具。

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		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Red Blood Cell lysis Buffer</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>R7757</td>
<td>&nbsp;</td>
<tr>
<td>Cell strainer</td>
<td>&nbsp;</td>
<td>BD Falcon</td>
<td>REF 352340</td>
<td>&nbsp;</td>
<tr>
<td>Pregnant Mare Serum (PMS)</td>
<td>&nbsp;</td>
<td>Calbiochem</td>
<td>367222</td>
<td>&nbsp;</td>
<tr>
<td>Human Chorionic Gonadotropin (HCG)</td>
<td>&nbsp;</td>
<td>Calbiochem</td>
<td>230734</td>
<td>&nbsp;</td>
<tr>
<td>Hyaluronidase</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>H4272</td>
<td>Type IV-S</td>
</tr>
<tr>
<td>KSOM-AA</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>MR-106-D</td>
<td>&nbsp;</td>
<tr>
<td>HCZB</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>MR-173-D</td>
<td>Customized medium</td>
</tr>
<tr>
<td>Ca-free medium for activation</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>MR-174-D</td>
<td>Customized medium</td>
</tr>
<tr>
<td>SrCl2</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>255521</td>
<td>100mM = 10x</td>
</tr>
<tr>
<td>AlbuMAX I</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>11020-021</td>
<td>&nbsp;</td>
<tr>
<td>PVP</td>
<td>&nbsp;</td>
<td>MP Biotech</td>
<td>102787</td>
<td>Make 10% solution using HCZB, and add 0.1% AlbuMAX I</td>
</tr>
<tr>
<td>Cytochalasin B</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>C6762</td>
<td>500&mu;g/ml = 100x</td>
</tr>
<tr>
<td>Trichostatin A</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>T8552</td>
<td>5mM = 1000x</td>
</tr>
<tr>
<td>Mineral Oil</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>M5310</td>
<td>&nbsp;</td>
<tr>
<td>Holding needle</td>
<td>&nbsp;</td>
<td>Humagen</td>
<td>MPH-SM-30</td>
<td>&nbsp;</td>
<tr>
<td>Injection and transfer needles</td>
<td>&nbsp;</td>
<td>Humagen</td>
<td>4-15, 7-15, 15-15</td>
<td>&nbsp;</td>
<tr>
<td>Mouth pipette</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A5177</td>
<td>&nbsp;</td>
<tr>
<td>Scissors</td>
<td>&nbsp;</td>
<td>Fine Science Instruments</td>
<td>14088-10</td>
<td>or something similar</td>
</tr>
<tr>
<td>Forceps</td>
<td>&nbsp;</td>
<td>Fine Science Instruments</td>
<td>11251-10</td>
<td>or something similar</td>
</tr>
<tr>
<td>Wound Clip Applicator</td>
<td>&nbsp;</td>
<td>BD</td>
<td>427630</td>
<td>&nbsp;</td>
<tr>
<td>Wound Clips</td>
<td>&nbsp;</td>
<td>BD</td>
<td>427630</td>
<td>&nbsp;</td>
<tr>
<td>Suture</td>
<td>&nbsp;</td>
<td>Ethicon</td>
<td>K871H</td>
<td>&nbsp;</td>
<tr>
<td>DMEM</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>D6429</td>
<td>&nbsp;</td>
<tr>
<td>FBS</td>
<td>&nbsp;</td>
<td>Hyclone</td>
<td>SH30071.03</td>
<td>15%</td>
</tr>
<tr>
<td>Penicillin/Streptomycin</td>
<td>&nbsp;</td>
<td>Lonza</td>
<td>17-602E</td>
<td>100x</td>
</tr>
<tr>
<td>Glutamin</td>
<td>&nbsp;</td>
<td>MP Biomedicals</td>
<td>101806</td>
<td>200mM = 100x</td>
</tr>
<tr>
<td>Non-essential Aminoacids</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>11140050</td>
<td>100x</td>
</tr>
<tr>
<td>&beta;-Mercaptoethanol</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>21985-023</td>
<td>5&mu;l in 500ml</td>
</tr>
<tr>
<td>LIF</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>ESG1106</td>
<td>1000x</td>
</tr>
<tr>
<td>MEK inhibitor</td>
<td>&nbsp;</td>
<td>Cell Signaling</td>
<td>9900L</td>
<td>For ES derivation only, add to ES medium (final concentration 50&mu;M)</td>
</tr>		</tbody>
		</table>
		</div>

transnuclear mice with pre-defined t cell receptor specificities against toxoplasma gondii obtained via scnt

淋巴细胞，如T细胞，进行基因V（D）J重组，生成具有一定的特异性受体1。小鼠基因为一个重新安排的抗原特异性T细胞受体（TCR）已研究T细胞的发育和功能的一个不可缺少的工具。然而，这样的电阻温度系数有关T细胞长期培养后，经常反复的抗原刺激，不可避免地选择具有高亲和力的T细胞通常是孤立的。随机的TCR基因组融合α-和β链和非内源性促销员表达可导致表达水平和动力学的变化。 通过体细胞核移植后生重新编程，提供了一个工具来生成胚胎干细胞和小鼠细胞不涉及任何利益。因此，体细胞核移植是应用已知的特异性T细胞时，这些遗传V（D）J重排的体细胞核移植胚胎干细胞（ESC）和从中获得的小鼠，而表观遗传标记被复位。我们已经证明，与预先定义的特点对弓形虫 T细胞，可用于生成表示，从他们的内源性基因位点的具体TCR无基因改造实验介绍，小鼠模型。的速度，这样的transnuclear模型可以得到相对缓和和持有其他疾病模型建设的承诺。

Watch this Scientific Journal Video about Transnuclear Mice with Pre-defined T Cell Receptor Specificities Against Toxoplasma gondii Obtained Via SCNT at JoVE.com

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

我们证明，在这里，通过体细胞核移植（SCNT）后生重新编程可以作为一个工具来生成与预先定义的T细胞受体（TCR）特异性的小鼠模型使用。这些transnuclear小鼠表达​​内源性启动子的控制下的内源性轨迹相应的TCR。

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 ...

transnuclear-mice-with-pre-defined-t-cell-receptor-specificities

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

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

14.1K 次观看.  Whitehead Institute for Biomedical Research. 我们证明，在这里，通过体细胞核移植（SCNT）后生重新编程可以作为一个工具来生成与预先定义的T细胞受体（TCR）特异性的小鼠模型使用。这些transnuclear小鼠表达​​内源性启动子的控制下的内源性轨迹相应的TCR。

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

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 ...

Derivation of Thymic Lymphoma T-cell Lines from Atm-/- and p53-/- Mice

Established cell lines are a critical research tool that can reduce the use of laboratory animals in research. Certain strains of genetically modified mice, such as Atm-/- and p53-/- consistently develop thymic lymphoma early in life 1,2, and thus, can serve as a reliable source for derivation of murine T-cell lines. Here we present a detailed protocol for the development of established murine thymic lymphoma T-cell lines without the need to add interleukins as described in previous protocols 1,3. Tumors were harvested from mice aged three to six months, at the earliest indication of visible tumors based on the observation of hunched posture, labored breathing, poor grooming and wasting in a susceptible strain 1,4. We have successfully established several T-cell lines using this protocol and inbred strains ofAtm-/- [FVB/N-Atmtm1Led/J] 2 and p53-/- [129/S6-Trp53tm1Tyj/J] 5 mice. We further demonstrate that more than 90% of the established T-cell population expresses CD3, CD4 and CD8. Consistent with stably established cell lines, the T-cells generated by using the present protocol have been passaged for over a year.

Derivation of Thymic Lymphoma T-cell Lines from Atm-/- and p53-/- Mice

In this video we demonstrate a protocol to establish mouse thymic lymphoma cell lines. By following this protocol, we have successfully established several T-cell lines from Atm-/- and p53-/- mice with thymic lymphoma.

胸腺淋巴瘤的T细胞系的推导 ATM - / -</sup P53 - / -</sup鼠标

Established cell lines are a critical research tool that can reduce the use of laboratory animals in research. Certain strains of genetically modified ...

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).

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

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

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

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

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

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

Retroviral Transduction of Bone Marrow Progenitor Cells to Generate T-cell Receptor Retrogenic Mice

T cell receptor (TCR) signaling is essential in the development and differentiation of T cells in the thymus and periphery, respectively. The vast array of TCRs proves studying a specific antigenic response difficult. Therefore, TCR transgenic mice were made to study positive and negative selection in the thymus as well as peripheral T cell activation, proliferation and tolerance. However, relatively few TCR transgenic mice have been generated specific to any given antigen. Thus, studies involving TCRs of varying affinities for the same antigenic peptide have been lacking. The generation of a new TCR transgenic line can take six or more months. Additionally, any specific backcrosses can take an additional six months. In order to allow faster generation and screening of multiple TCRs, a protocol for retroviral transduction of bone marrow was established with stoichiometric expression of the TCR&#945; and TCR&#946; chains and the generation of retrogenic mice. Each retrogenic mouse is essentially a founder, virtually negating a founder effect, while the length of time to generate a TCR retrogenic is cut from six months to approximately six weeks. Here we present a rapid and flexible alternative to TCR transgenic mice that can be expressed on any chosen background with any particular TCR.

We present a rapid and flexible protocol for a single T cell receptor (TCR) retroviral-based in vivo expression system. Retroviral vectors are used to transduce bone marrow progenitor cells to study T cell development and function of a single TCR in vivo as an alternative to TCR transgenic mice.

骨髓祖细胞的逆转录病毒转导到生成T细胞受体Retrogenic小鼠

T cell receptor (TCR) signaling is essential in the development and differentiation of T cells in the thymus and periphery, respectively.  The vast array ...

Development of Stem Cell-derived Antigen-specific Regulatory T Cells Against Autoimmunity

Autoimmune diseases arise due to the loss of immunological self-tolerance. Regulatory T cells (Tregs) are important mediators of immunologic self-tolerance. Tregs represent about 5 - 10% of the mature CD4+ T cell subpopulation in mice and humans, with about 1 - 2% of those Tregs circulating in the peripheral blood. Induced pluripotent stem cells (iPSCs) can be differentiated into functional Tregs, which have a potential to be used for cell-based therapies of autoimmune diseases. Here, we present a method to develop antigen (Ag)-specific Tregs from iPSCs (i.e., iPSC-Tregs). The method is based on incorporating the transcription factor FoxP3 and an Ag-specific T cell receptor (TCR) into iPSCs and then differentiating on OP9 stromal cells expressing Notch ligands delta-like (DL) 1 and DL4. Following in vitro differentiation, the iPSC-Tregs express CD4, CD8, CD3, CD25, FoxP3, and Ag-specific TCR and are able to respond to Ag stimulation. This method has been successfully applied to cell-based therapy of autoimmune arthritis in a murine model. Adoptive transfer of these Ag-specific iPSC-Tregs into Ag-induced arthritis (AIA)-bearing mice has the ability to reduce joint inflammation and swelling and to prevent bone loss.

We present here a method to develop functional antigen (Ag)-specific regulatory T cells (Tregs) from induced pluripotent stem cells (iPSCs) for immunotherapy of autoimmune arthritis in a murine model.

干细胞衍生的抗原特异性调节性T细胞对自身免疫的发展

Autoimmune diseases arise due to the loss of immunological self-tolerance. Regulatory T cells (Tregs) are important mediators of immunologic ...

Identification of Mediators of T-cell Receptor Signaling via the Screening of Chemical Inhibitor Libraries

The T-cell receptor (TCR) signaling pathway comprises a multitude of mediators that transmit signals upon the activation of the TCR. Different strategies have been proposed and implemented for the identification of new mediators of TCR signaling, which would improve the understanding of T-cell processes, including activation and thymic selection. We describe a screening assay that enables the identification of molecules that influence TCR signaling based on the activation of developing thymocytes. Strong TCR signals cause developing thymocytes to activate apoptotic machinery in a process known as negative selection. Through the application of kinase inhibitors, those with targets that affect TCR signaling are able to override the process of negative selection. The method detailed in this paper can be used to identify inhibitors of canonical kinases with established roles in the TCR signaling pathways and also inhibitors of new kinases yet to be established in the TCR signaling pathways. The screening strategy here can be applied to screens of higher throughput for the identification of novel druggable targets in TCR signaling.

Identification of Mediators of T-cell Receptor Signaling via the Screening of Chemical Inhibitor Libraries

This paper uses a flow-cytometry-based assay to screen libraries of chemical inhibitors for the identification of inhibitors and their targets that influence T-cell receptor signaling. The methods described here can also be expanded for high-throughput screenings.

化学抑制剂库筛选 t 细胞受体信号的中介识别

The T-cell receptor (TCR) signaling pathway comprises a multitude of mediators that transmit signals upon the activation of the TCR. Different strategies ...

Single-cell Screening Method for the Selection and Recovery of Antibodies with Desired Specificities from Enriched Human Memory B Cell Populations

The human antibody repertoire represents a largely untapped source of potential therapeutic antibodies and useful biomarkers. While current computational methods, such as next generation sequencing (NGS), yield enormous sets of data on the antibody repertoire at the sequence level, functional data is required to identify which sequences are relevant for a particular antigen or set of antigens. Here, we describe a method to identify and recover individual antigen-specific antibodies from peripheral blood mononuclear cells (PBMCs) from a human blood donor. This method utilizes an initial enrichment of mature B cells and requires a combination of phenotypic cell markers and fluorescently-labeled protein to isolate IgG memory B cells via flow cytometry. The heavy and light chain variable regions are then cloned and re-screened. Although limited to the memory B cell compartment, this method takes advantage of flow cytometry to interrogate millions of B cells and returns paired heavy and light chain sequences from a single cell in a format ready for expression and confirmation of specificity. Antibodies recovered with this method can be considered for therapeutic potential, but can also link specificity and function with bioinformatic approaches to assess the B cell repertoire within individuals.

The BSelex method to identify and recover individual antigen-specific antibodies from human peripheral blood mononuclear cells combines flow cytometry with single cell PCR and cloning.

从富足人类记忆B细胞群中选择和恢复具有所需特异性的抗体的单细胞筛选方法

The human antibody repertoire represents a largely untapped source of potential therapeutic antibodies and useful biomarkers. While current computational ...