Name: streamlined single cell tcr isolation and generation of retroviral vectors for in vitro and in vivo expression of human tcrs
Uploaded: 2017-09-10T13:00:00.000+00:00
Duration: 11 min 21 s
Description: Watch this Scientific Journal Video about Streamlined Single Cell TCR Isolation and Generation of Retroviral Vectors for In Vitro and In Vivo Expression of Human TCRs at JoVE.com

这项研究由 JDRF 1-2014-243-N, ADA 7-14-JF-07, NIH 5 P30 DK079638-05 飞行员 PJ, 和罗伯特和珍妮丝捷基金会资助。
我们感谢桑德拉和 Andrene 麦当劳的耐心招聘, 塞缪尔. 布鲁姆为技术援助, Dr. 乔治 Makedonas 的礼物抗体和控制 DNA 用于 PCR 优化。

1. 确定感兴趣的 T 细胞群体. 
 注意: 抗原特异增殖与细胞分裂染料 (如羧基琥珀酰亚胺酯, CFSE) 可用于分离 T 细胞的基础上, 其增殖反应抗原刺激。如果从 PBMCs 开始, 7 天的 体外 扩展应该足以识别抗原特异的 CFSE 低种群 12 。
<ol> <li> 将 PBMCs 1:1 与 MHC 匹配的进纸单元混合, 并使用5和 #181 的标签; M CFSE 细胞分裂染料. </li>
	<li> 板 2-2. 5 x 10 5 每个井在 96-井圆底培养板中的每个单元格200和 #181; L 10% FCS 完整的 RPMI 介质. 
	注: 完整的 RPMI 含有2毫米 l-谷氨酰胺, 1 毫米丙酮酸钠, 100 和 #181; M 不必要的氨基酸, 5 毫米 HEPES, 5.5 和 #215; 10 和 #8722; 5 单位 2-基, 100 U mL 和 #8722; 1 青霉素和100和 #181; g mL 和 #8722; 1 链霉素 </li>
	<li> 用 25 #181; M 肽抗原刺激细胞. </li>
	<li> 在第四天的文化, 小心删除和更换100和 #181; L 的媒体. 
	注: 抗原特异 T 细胞识别的一种更为优化的方法是使用肽 HLA 体 13 。聚丙烯染色允许同时评估抗原特异性以及 HLA 限制. </li>
</ol> 2。设置单个单元格排序. 
<ol> <li> 执行引擎盖或指定区域中的所有过程, 没有放大的模板。分的试剂, 以避免污染, 清洁所有的工作表面, 如果可行, 设备与10% 漂白剂之前, PCR 设置。使用过滤提示, 核糖核酸和 dnasei 免费试剂和塑料在所有的 PCR 和载体克隆步骤。关键试剂可以在 材料表 中找到. 
	注意: 多重 PCR 反应是高度敏感的, 低水平的污染会导致产品的放大. </li>
	<li> 通过将 10x rt 反应缓冲、25X DNTPs、9 u rt 酶、0.01% 海卫 x、0.7 u 核糖核酸抑制剂和 383 nM 的每个 rt TCR 底漆 (列于 表 1 ) 中, 在无菌移油藏中生成反向转录主混合。对于每96个井板, 组成足够的主混合10额外的反应 (共 106), 以弥补液体损失在移。请参见 表 2 , 以了解反应组件和每井的金额. </li>
	<li> 准备一个96孔 PCR 收集板的细胞分类由移6和 #181; L 的反向转录主混合每井与多通道吸管。把盘子放在冰上或放在寒冷的街区. </li>
	<li> 将带有抗体的细胞标记为细胞表面标志物 (如 CD4 和 CD3, 或 CD8 和 CD3, 分别在2和 #181; g/毫升, 结合细胞分裂染料或聚丙烯染色). </li>
	<li> 对每个单元格中的 T 单元格进行排序, 跳过第十二行, 这将作为一个负控制. 
	注意: 排序的效率将取决于在板持有人的精确收集板对齐, 以及保持在一个较低的温度下的细胞和板块在 cDNA 反应之前。收集板应保持在冰的时刻, 除了在排序, 当他们固定在冷却板。并非所有机都装有温度调节板架;但是, 强烈建议使用一种。由于该类通常需要约 10-15 分钟每盘, 有一个增加的潜力, RNA 退化, 如果该板块不保持在低温. </li>
	<li> 作为一个正向控制, 在这个阶段用吸管将大量的细胞 (100 细胞) 手动添加到其中的一个井中。另外, 以前分离的 RNA 从汇集的细胞可以使用作为正面控制在 10 ng 每井. 
	注: 为了避免其他井的交叉污染和假阳性结果, 移液管精确、小心地纯化了 RNA. </li>
	<li> 用胶膜封板, 在 1000 x g 处旋转5分钟, 在4和 #176; C. </li>
	<li> 将 RNA 转录到25和 #176; c 为 10 min、37和 #176; c 为 45 min、85和 #176; c 为 10 min. 
	注意: 为了保证有效的 pcr 反应, 建议在同一天进行第一次 pcr 反应。另外, 转录板可以保存在-80 和 #176; C 最多2月. </li>
</ol> 3。执行多重嵌套 PCR 注意: 准备所有的主混合在一个干净的无模板区, 以避免污染. 
<ol> <li> 将每个可变区域底漆重新重建为100和 #181; M 与 dnasei 和核糖核酸自由 H 2 O。接下来, 结合20和 #181; L 每个44引物生成 V 型α入门池。生成 v 型β-beta 入门池结合20和 #181; l 每 40 v 型β底漆加上80和 #181; l dnasei 和核糖核酸自由 H 2 o. 底漆序列在 表 1 中列出。一旦汇集, 每引物的集中在混合是2.3 和 #181; m. </li>
	<li> 将常量区域底漆重建为100和 #181; m 库存解决方案与 dnasei 和核糖核酸自由 H 2 o. 将常量区域底漆稀释到10和 #181; m 工作解决方案和分使用. </li>
	<li> 准备两个单独的主混合 TCR-α和 TCR-β放大。通过合并5X 缓冲区来生成主混合80和 #181; m dNTPs, 3% 亚砜, 2 和 #181; m 底漆池 (每个底漆的最终浓度 46 nm), 200 nm TCR-恒定区域反转底漆, 1 U DNA 聚合酶, dnasei 和核糖核酸自由 H 2 O ( 表2/强 >)。为每96个井板, 构成足够的主混合为10另外的反应 (共计 106), 为了补偿液体损失在移期间. 
	注: 计算依据的是25和 #181 的最终 pcr 量; 每个井, 22.5 和 #181; i. pcr 组合与2.5 和 #181; l cDNA 模板. </li>
	<li> 在冰上或在冷块中保留该板的 cDNA。使用多通道, 以吸管22.5 和 #181; L TCR β反应到一个新的96井 PCR 板。然后使用多通道传输2.5 和 #181; L cDNA 进入 PCR 板. </li>
	<li> 使用多通道吸管22.5 和 #181; L TCR α反应直接进入含有其余的 cDNA 的板块. </li>
	<li> 用粘合剂将板材密封, 轻轻涡旋, 然后在 1000 x g 处旋转5分钟, 在4和 #176; C. </li>
	<li> 运行多重 PCR 反应与以下周期: 5 分钟在95和 #176; c 和34周期二十年代在95和 #176; c, 三十年代在54和 #176; c, 1 min 在72和 #176; c, 7 min 在72和 #176; c. </li>
	<li> 在25和 #181 的最终容量中进行嵌套 pcr 反应; 使用2.5 和 #181; 采用多重 pcr 混合的 l 作为模板。
	<ol> <li> 将内部和适配器的底漆重新制作为100和 #181; m 与 dnasei 和核糖核酸自由 H 2 o. 10 和 #181; 等分为工作库存. </li>
		<li> 准备两个单独的主混合 TCR-α和 TCR-β ampl分类.混合5X 缓冲器, 25X DNTP, 3% 亚砜, 200 nm 前向适配器底漆, 200 nm 反向嵌套底漆, 1 U dna 聚合酶, 和 dna 聚合酶和无核酸的 H 2 O 为最后的容量22.5 和 #181; L ( 表 2 )。对于每96个井板, 组成足够的主混合10额外的反应 (共 106), 以弥补液体损失在移. 
		注: 在第二 pcr 反应中使用 5X pcr 缓冲含有负载染料, 以方便琼脂糖凝胶负载. </li>
		<li> 使用多通道, 吸管的主混合为 TCR α和 TCR β反应在22.5 和 #181; L 每井成两个新的 96-井 PCR 板. </li>
		<li> 在2.5 和 #181 添加多重 PCR 反应模板; 每井. 
		注: 其余第一个 PCR 反应应密封, 并存储在-20 和 #176; C 作为附加模板的源, 如果需要 (请参见 步骤4.3 注意 ). </li>
		<li> 密封板, 轻轻涡, 并在 1000 x g 旋转下来5分钟, 在4和 #176; C. </li>
		<li> 运行带有以下循环的嵌套 PCR 反应: 5 分钟在95和 #176; c 和34周期二十年代在95和 #176; c, 三十年代在56和 #176; c, 1 min 在72和 #176; c, 7 min 在72和 #176; c. </li>
		<li> 运行5和 #181; L 最后的反应, 在1% 琼脂糖凝胶含有溴溴 (包括负和阳性控制井). 
		注: 预计波段应运行约 500 bp 大小。 图 2 中显示了一个示例反应. </li>
		<li> 纯化第二反应的其余部分 (20 和 #181; l) 使用96井格式 PCR 纯化试剂盒, 洗与15和 #181; l 70 和 #176; C H 2 O 每井, 并执行桑格测序。使用适当的 TCR-α或 TCR-β适配器前向引物进行测序 ( 表 1 ). 
		注意: 可以使用在线免疫软件分析序列. </li>
	</ol> </li> </ol> 4。亚克隆α和β PCR 链. 
 注: 第一反应中使用的正向引物的池, 第二个 PCR 反应中的反向引物用于合并限制部位。因此, 所获得的α和β链 PCR 产品可以按顺序 sub-cloned 到模板反转录病毒载体 ( 图 1 ). 
<ol> <li> 使用 BstbI 和 MfeI ( 表 3 ) 从 beta 嵌套反应中摘录纯化的 PCR 产品. 
	注: 请勿超过 5 #181; g 插入. </li>
	<li> 并行, 用 BstbI 和 MfeI ( 表 3 ) 来消化模板向量。使用1-2 和 #181; g 的模板向量每 TCR。在琼脂糖凝胶上运行的消化载体, 并隔离较大的波段 (〜 7500 bp)。用凝胶 DNA 纯化试剂盒净化载体. 
	注: 在限制酶消化期间, 模板小鼠 TCR-β变区被删除, 允许增加人类 TCR-β变量区域. </li>
	<li> 使用 pcr 纯化试剂盒提纯经消化的 pcr 产物. 
	注: 验证套件中所含列的 DNA 纯化能力;必要时使用多个列。成功结扎反应所需的纯化插入物的最小用量为 50 ng, 最低浓度为 10 ng/和 #181;如果纯化的插入量是不足够的成功结扎, 第二个 PCR 反应可以重复. </li>
	<li> 将模板向量与 10 U CIP 酶用于1小时的37和 #176; c 以防止 self-ligation, 其次是热失活10分钟, 在75和 #176; c. 用 PCR 纯化试剂盒提纯 DNA ( 表 3 ). </li>
	<li> 结扎 TCR-β插入和向量, 使用 DNA 连接在20和 #181; L 总体积在6摩尔, 超过30分钟的插入物在室温下. 
	注: 总的说, 结扎反应应包含约 150 ng 的 DNA ( 表 3 ). </li>
	<li> 用于转换, 混合8和 #181; 80 和 #181 的结扎反应 l; 具有化学能力的 大肠杆菌 DH5 和 #945; 细胞 (材料的 表 ) 和在冰上孵育30分钟的热休克在42和 #176; C 为四十五年代. 添加500和 #181; 我超级物抑制 (soc) 培养基的最佳肉汤, 在37和 #176 时摇动1.5 小时; C. </li>
	<li> 将250和 #181; 在含有性汤 (LB) 琼脂板的氨苄西林中培养细菌。在37和 #176 的夜间 (约16小时) 孵育盘; C. </li>
	<li> 第二天选择细菌菌落, 并在一夜之间在含有氨苄西林的3毫升培养基中生长。用 dna 质粒纯化试剂盒分离质粒 dna. </li>
	<li> 确认通过 small-scale 测试-摘要成功插入 beta 链, 并使用限制酶 BstbI 和 MfeI。在12和 #181; l 总反应量, 结合200-500 和 #181; g 质粒 DNA, 0.25 和 #181; 每种酶的 l, 和10X 酶缓冲 ( 表 3 )。运行的反应1% 琼脂糖凝胶含有溴溴, 以确认大小的插入带 (〜 500 bp). </li>
	<li> 在确认插入的存在后, 使用 IRES 反向底漆 ( 表 1 ) 序列化向量的 TCR-beta 部分. </li>
	<li> TCR-beta 插入的顺序确认后, 执行类似的过程以插入 alpha 变量区域。从α嵌套反应中提炼纯化的 PCR 产物, 以及新生成的 TCR-β插入载体, 其中含有 SnaBI 和 SacII. 
	注: 在限制酶消化期间, 模板小鼠 TCR α变区被移除, 允许增加人类的 TCR-α变量区域. </li>
	<li> 结扎在20和 #181 中使用 DNA 连接对相应的 beta 变量区域进行编码, 并将其插入到 TCR 向量中; 在室温下, 6 摩尔的总体积超过30分钟的插入量. 
	注: 总的说, 结扎反应应包含约 150 ng 的 DNA ( 表 3 ). </li>
	<li> 用于转换, 混合8和 #181; 80 和 #181 的结扎反应 l; 具有化学能力的 大肠杆菌 DH5 和 #945; 细胞 (材料的 表 ) 和在冰上孵育30分钟的热休克在42和 #176; C 四十五年代. 添加500和 #181; 我 soc在37和 #176 时为1.5 小时的介质和抖动; C. </li>
	<li> 将250和 #181; 在含有性汤 (LB) 琼脂板的氨苄西林中培养细菌。在37和 #176 的夜间 (约16小时) 孵育盘; C. </li>
	<li> 第二天选择细菌菌落, 并在一夜之间在含有氨苄西林的3毫升培养基中生长。用 dna 质粒纯化试剂盒分离质粒 dna. </li>
	<li> 确认将α链成功插入的 small-scale 测试摘要与限制酶 SnaBI 和 SacII。在12和 #181; l 总反应容量结合200-500 和 #181; g 质粒 DNA, 0.25 和 #181; 每种酶的 l, 和10x 酶缓冲 ( 表 3 )。运行的反应1% 琼脂糖凝胶含有溴溴, 以确认大小的插入带 (〜 500 bp). </li>
	<li> 在确认插入的存在后, 使用 MSCV2-forward 入门 ( 表 1 ) 将向量的 TCR-alpha 部分序列化. </li>
	<li> 一旦验证了 alpha 链序列, 请通过 TCR-β-反转和 IRES 反向底漆 ( 表 1 ) 的顺序验证完整的 TCR 插入. </li>
</ol> 5。验证 TCR 细胞表面的表达和特异性. 
 注意: HEK293T 单元用于测试成功的 TCR 链ng 和单元格表面表达式 ( 图 3A ) 8 。多肽 MHC 聚丙烯染色也可以对转染的 HEK293T 细胞进行测试的保留抗原特异性后 TCR 基因分离 ( 图 3B ). 
<ol> <li> 将12个组织培养皿中的 HEK293T 细胞在1毫升的 1 x 10 5 细胞中, 在5% 和 #176 的完整 DMEM 介质中包含37个 FCS 和区域性; c. 板出足够的细胞为每个新的 TCR 指定单独的井, 控制模板 TCR 矢量, 仅 CD3 向量, 控制 TCR 向量, non-transfected. 
	注: 用完整的鼠标模板 TCR 载体转染 (纶) 与 mCD3 和 #949 结合; #947; #948; #950; pMIG 向量, 单纶向量, mCD3 #949; #947; #948; #950;-pMIG 矢量单独被用作正负控制. 
	注: 完整的 DMEM 包含2毫米 l-谷氨酰胺, 1 毫米丙酮酸钠, 100 和 #181; M 非必需氨基酸, 5 毫米 HEPES 缓冲, 5.5 x 10 -5 单位 2-基, 100 U/毫升青霉素/链霉素. </li>
	<li> 第二天, 染1和 #181 的单元格; 纶向量、1和 #181; g 鼠标 CD3 和 #949; #947; #948; #950;-pMSCVII-GFP (pMIG) 载体, 和 liposome-based 转染试剂按照制造商的指示. </li>
	<li> 对于每个 TCR, 添加6和 #181; 将试剂转染为100和 #181; l 室温, 无血清 DMEM。短暂的旋涡, 并在室温下孵育15分钟. </li>
	<li> 在单独的1.5 毫升管中结合1和 #181; g TCR 纶向量与1和 #181; g 鼠标 CD3 和 #949; #947; #948; #950;-pMSCVII-GFP (pMIG) 矢量, 或1和 #181; 每个矢量的 g 分别用于控件. </li>
	<li> 滴, 将转染试剂/DMEM 溶液添加到包含矢量 DNA 的管中。室温孵育15分钟. </li>
	<li> 滴, 将转染试剂/DMEM/DNA 溶液添加到 HEK293T 细胞中。轻轻地轻敲盘子混合。孵育在37和 #176; C 为 24 h. </li>
	<li> 在24小时后替换媒体, 并将单元格保留为24小时. </li>
	<li> 通过大力移将细胞从板上移除, 并用 anti-mouse CD3 抗体 (2.5 和 #181; g/毫升) 对细胞进行染色, 以验证流式细胞术对嵌合 TCR 的表面表达. </li>
	<li> 为了比较被转染为类似水平的单元格之间的 CD3 表达式, 对表达类似高水平荧光报告分子的细胞进行分析 (GFP 为 CD3 复合体, 石为 TCR 表达载体)。优选地, 分析应该在 10 4 到 10 5 荧光强度 ( 图 3A ) 内的 GFP + 石 + 细胞上进行封闭. 
	注: 在转嵌的细胞中的 CD3 表达水平应与控制鼠标 (原始模板) 矢量 ( 图 3A ) 相媲美。生成的 TCR 反转录病毒载体与先前描述的 4 中的 在体内 表达式系统兼容。
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<ol>
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J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+naive+CD4++T+cells+from+a+diverse+repertoire+produce+different+effector+cell+types+during+infection.">Single naive CD4+ T cells from a diverse repertoire produce different effector cell types during infection.</a> Cell. 153 (4), 785-796 (2013).</li><li>Sprouse, M. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+identification+and+expression+of+human+TCRs+in+retrogenic+mice.">Rapid identification and expression of human TCRs in retrogenic mice.</a> J Immunol Methods. , (2016).</li><li>Lennon, G. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T+cell+islet+accumulation+in+type+1+diabetes+is+a+tightly+regulated,+cell-autonomous+event.">T cell islet accumulation in type 1 diabetes is a tightly regulated, cell-autonomous event.</a> Immunity. 31 (4), 643-653 (2009).</li><li>Dash, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paired+analysis+of+TCRalpha+and+TCRbeta+chains+at+the+single-cell+level+in+mice.">Paired analysis of TCRalpha and TCRbeta chains at the single-cell level in mice.</a> J Clin Invest. 121 (1), 288-295 (2011).</li><li>Kobayashi, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+cloning+and+expression+system+yields+and+validates+TCRs+from+blood+lymphocytes+of+patients+with+cancer+within+10+days.">A new cloning and expression system yields and validates TCRs from blood lymphocytes of patients with cancer within 10 days.</a> Nat Med. 19 (11), 1542-1546 (2013).</li><li>Szymczak, A. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correction+of+multi-gene+deficiency+in+vivo+using+a+single+'self-cleaving'+2A+peptide-based+retroviral+vector.">Correction of multi-gene deficiency in vivo using a single 'self-cleaving' 2A peptide-based retroviral vector.</a> Nat Biotechnol. 22 (5), 589-594 (2004).</li><li>Szymczak, A. L., Vignali, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+2A+peptide-based+strategies+in+the+design+of+multicistronic+vectors.">Development of 2A peptide-based strategies in the design of multicistronic vectors.</a> Expert Opin Biol Ther. 5 (5), 627-638 (2005).</li><li>Bettini, M. L., Bettini, M., Vignali, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T-cell+receptor+retrogenic+mice:+a+rapid,+flexible+alternative+to+T-cell+receptor+transgenic+mice.">T-cell receptor retrogenic mice: a rapid, flexible alternative to T-cell receptor transgenic mice.</a> Immunology. 136 (3), 265-272 (2012).</li><li>Holst, J., Vignali, K. M., Burton, A. R., Vignali, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+analysis+of+T-cell+selection+in+vivo+using+T+cell-receptor+retrogenic+mice.">Rapid analysis of T-cell selection in vivo using T cell-receptor retrogenic mice.</a> Nat Methods. 3 (3), 191-197 (2006).</li><li>Mannering, S. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+sensitive+method+for+detecting+proliferation+of+rare+autoantigen-specific+human+T+cells.">A sensitive method for detecting proliferation of rare autoantigen-specific human T cells.</a> J Immunol Methods. 283 (1-2), 173-183 (2003).</li><li>James, E. A., LaFond, R., Durinovic-Bello, I., Kwok, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+antigen+specific+CD4++T+cells+using+MHC+class+II+tetramers.">Visualizing antigen specific CD4+ T cells using MHC class II tetramers.</a> J Vis Exp. (25), (2009).</li><li>Corthay, A., Nandakumar, K. S., Holmdahl, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+the+percentage+of+peripheral+T+cells+with+two+different+T+cell+receptor+alpha-chains+and+of+their+potential+role+in+autoimmunity.">Evaluation of the percentage of peripheral T cells with two different T cell receptor alpha-chains and of their potential role in autoimmunity.</a> J Autoimmun. 16 (4), 423-429 (2001).</li><li>Lee, T., Shevchenko, I., Sprouse, M. L., Bettini, M., Bettini, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retroviral+Transduction+of+Bone+Marrow+Progenitor+Cells+to+Generate+T-cell+Receptor+Retrogenic+Mice.">Retroviral Transduction of Bone Marrow Progenitor Cells to Generate T-cell Receptor Retrogenic Mice.</a> J Vis Exp. (113), (2016).</li><li>Bettini, M. L., Bettini, M., Nakayama, M., Guy, C. S., Vignali, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+T+cell+receptor-retrogenic+mice:+improved+retroviral-mediated+stem+cell+gene+transfer.">Generation of T cell receptor-retrogenic mice: improved retroviral-mediated stem cell gene transfer.</a> Nat Protoc. 8 (10), 1837-1840 (2013).</li><li>Cohen, C. J., Zhao, Y., Zheng, Z., Rosenberg, S. A., Morgan, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+antitumor+activity+of+murine-human+hybrid+T-cell+receptor+(TCR)+in+human+lymphocytes+is+associated+with+improved+pairing+and+TCR/CD3+stability.">Enhanced antitumor activity of murine-human hybrid T-cell receptor (TCR) in human lymphocytes is associated with improved pairing and TCR/CD3 stability.</a> Cancer Res. 66 (17), 8878-8886 (2006).</li></ol>

作者没有什么可透露的。

在当前的协议中, 我们描述了一个有效的方法单细胞 TCR 放大和随后的 sub-cloning 配对 TCR α和β链成模板逆转录病毒表达载体。虽然, 已经开发了几个单细胞 PCR 协议, 到目前为止没有与直接 sub-cloning 兼容到表达载体。在大多数情况下, 包含高度可变的 CDR3 区域的部分序列被放大, 具有足够的序列来推断变量区域。这种较小的 pcr 产品尺寸支持更高的 pcr 效率, 但需要de 从头TCR 基因合成功能研究?...

t 细胞受体 (TCR) 指示 t 细胞命运决定在发展期间, 稳态/稳态, 和抗原刺激的外围1,2,3。最近的深度测序技术的扩展揭示了先前未被低估的抗原特异 T 细胞反应中的 TCR 多样性。TCR 多样性表明了功能广泛的 T 细胞反应的潜力。为了将 TCR 序列剧目分析与 TCR 功能分析相结合, 测序方法应设计为与实验系统兼容, 并用于后续的功能性研究的体内模型。TCRs我们已经开发了一个有效的方法, 人类 TCR 序列隔离和简化 sub-cloning 成嵌合体的人/小鼠 TCR 模板载体与 TCR 表达的 HLA-人性化的老鼠4。heterodimeric TCRs 的相应α和β链的分离需要从单个细胞中扩增两个链。虽然, 已经开发和利用了几个单细胞的 TCR 克隆协议, 但迄今为止, 没有一个能够很容易地与高通量简化的 Vα/Vβ TCRs 的直接克隆相兼容, 成为重新体内的反转录病毒载体 4、5、6、7。以前的研究采用了两种主要方法, 要么是有选择地放大 TCR 的有限部分足以推断序列, 要么放大整个 TCR 序列4,5,6,7. 通过第一种方法获得的 TCR 序列的下游功能分析需要昂贵的在硅片中程序集和de 从头构造 TCR。虽然第二种方法提供完整的 TCR 序列, 但人的恒定区域与在鼠标模型中克隆的 TCRs 的在体内表达不兼容。我们的方法是专门设计为与在体内的功能分析 TCRs 在小鼠模型中兼容。我们开发了一个高效和简化的单细胞 pcr 协议, 允许直接 sub-cloning pcr 片段到模板表达载体。
我们的方法采用了一个高度敏感的多重嵌套 PCR 反应, 这是两个步骤执行。在第一个复用反应步骤中, 所有 v-β链的40引物的池, 以及所有 v 型α链专用的44个引物池, 用于放大 TCR-α或 TCR-beta, 而无需事先了解序列 (表 1)。前引物有一个适配器序列, 这是纳入 PCR 产品的 5 "。反向底漆基于 TCR 的恒定区域。我们已经确定了在人类变量或连接 TCR 区域中不存在的唯一限制站点, 并将它们合并到新重新设计的 TCRα和 TCRβ引物 (图 1) 中。在第二个嵌套反应中, 使用在恒定区域内的适配器序列和嵌套反向底漆的底漆, 用于进一步放大 TCR 链, 增加特异性 (表 1,图 1)。单细胞分离后, 两轮 pcr (第一反应与池的 Vα和 Vβ引物, 第二次与适配器引物) 产生的 pcr 产品, 可以直接 sub-cloned 到模板逆转录病毒载体。最后的 TCR 结构将编码, 在一个单一的开放阅读框架, 人的可变区域结合的小鼠恒定区域连接的 ' self-cleaving ' 蛋白序列, P2A (hVα-mCα-P2A-hVβ-mCβ)8。P2A 序列已在多个系统中使用, 并已被广泛测试, 专门用于表达 TCRs8、9、10、11。虽然, 在翻译以后多数 P2A 序列仍然附有由一个灵活的链接器连接的阿尔法链子的 C 末端, 而 beta 链信号序列有另外的脯氨酸, 这种修改对 TCR 作用没有有害作用。在构造中使用鼠标常量区域, 而不是人类, 以避免 re-expressed 在鼠标单元格中时与下游信号组件的潜在改变的交互。单一的, 将导致化学计量独立表达α和β链9,11。目前的协议是基于广泛可用的试剂和方法, 并且设计为以流线型和高效的方式进行。虽然我们已经明确地使用这项技术来检测 TCRs 从反应 T 细胞牵连自身免疫性糖尿病, 我们预计, 该协议可以广泛适用于识别和功能评估的人 TCRs 具体自身免疫表位, 肿瘤抗原表位, 或对病原体和疫苗的反应。

<table><tbody><tr><td>CFSE&amp;nbsp;</td><td>eBioscience&amp;nbsp;</td><td>65-0850-84</td><td>5 mM</td></tr><tr><td>anti-human CD4 (OKT4) BV421</td><td>Biolegend&amp;nbsp;</td><td>317433</td><td>2 mg/mL</td></tr><tr><td>anti-human CD3 (OKT3) PerCP/Cy5.5</td><td>Biolegend&amp;nbsp;</td><td>317336</td><td>2 mg/mL</td></tr><tr><td>anti-mouse CD3 AF647</td><td>Biolegend&amp;nbsp;</td><td>100322</td><td>2.5 mg/mL</td></tr><tr><td>Recombinant RNAse inhibitor biotech grd 2.5KU&amp;nbsp;&amp;nbsp;</td><td>VWR</td><td>97068-158</td><td>0.7 U</td></tr><tr><td>Applied Biosystems High capaity cDNA RT kit&amp;nbsp;</td><td>Invirtogen&amp;nbsp;</td><td>4368814</td><td>9 U (RT enzyme)&amp;nbsp;</td></tr><tr><td>NF-H2O: Nuclease-free water 1000mL</td><td>Invirtogen&amp;nbsp;</td><td>AM9932</td><td></td></tr><tr><td>Gotaq DNA polymerase 2500U</td><td>VWR</td><td>PAM3008</td><td>1 U</td></tr><tr><td>96 Well Half Skirt PCR Plate</td><td>Phenix</td><td>MPX-96M2</td><td></td></tr><tr><td>MicroAmp Clear Adhesive Film&amp;nbsp;</td><td>ThermoFisher&amp;nbsp;</td><td>4306311</td><td></td></tr><tr><td>UltraPure Agarose&amp;nbsp;</td><td>Invirtogen&amp;nbsp;</td><td>15510-027</td><td></td></tr><tr><td>SnaBI</td><td>New England Biolabs&amp;nbsp;</td><td>R0130</td><td>15 U (insert) 30 U (vector)</td></tr><tr><td>SacII</td><td>New England Biolabs&amp;nbsp;</td><td>R0157</td><td>40 U (insert) 80 U (vector)</td></tr><tr><td>MfeI</td><td>New England Biolabs&amp;nbsp;</td><td>R3589S</td><td>40 U (insert) 80 U (vector)</td></tr><tr><td>BstBI</td><td>New England Biolabs&amp;nbsp;</td><td>R0519</td><td>40 U (insert) 80 U (vector)</td></tr><tr><td>Calf Intestinal Phosphatase (CIP)</td><td>New England Biolabs&amp;nbsp;</td><td>M0290S</td><td>10 U</td></tr><tr><td>Quick ligase&amp;nbsp;</td><td>New England Biolabs&amp;nbsp;</td><td>M2200S</td><td></td></tr><tr><td>Wizard Plus minipreps DNA purification systems&amp;nbsp;</td><td>Promega&amp;nbsp;</td><td>A1460</td><td></td></tr><tr><td>Wizard Plus midipreps DNA purification systems&amp;nbsp;</td><td>Promega&amp;nbsp;</td><td>A7640</td><td></td></tr><tr><td>DNA clean &amp;amp; concentrator-25 kit</td><td>Genesee Scientific&amp;nbsp;</td><td>11-304C</td><td></td></tr><tr><td>ZR-96 DNA clean &amp;amp; concentrator-5 kit</td><td>Genesee Scientific&amp;nbsp;</td><td>11-306A</td><td></td></tr><tr><td>QIAquick gel extraction kit</td><td>Qiagen</td><td>28704</td><td></td></tr><tr><td>QIAquick PCR purification kit</td><td>Qiagen</td><td>28104</td><td></td></tr><tr><td>Subcloning Efficiency DH5a Competent Cells&amp;nbsp;</td><td>ThermoFisher&amp;nbsp;</td><td>18265017</td><td></td></tr><tr><td>Transit LT-1 Reagent 1mL (transfection reagent)</td><td>Mirus&amp;nbsp;</td><td>MIR2300</td><td></td></tr><tr><td>BD FACS Aria II (sorter)</td><td>BD</td><td></td><td></td></tr><tr><td>BD Fortessa (flow cytometer)</td><td>BD&amp;nbsp;</td><td></td><td></td></tr></tbody></table>

streamlined single cell tcr isolation and generation of retroviral vectors for in vitro and in vivo expression of human tcrs

虽然, 有几种方法的排序的配对 t 细胞受体 (TCR) α和β链从单一 t 细胞已经开发, 迄今没有有利于下游体内功能分析的 TCR heterodimers。我们已经开发了一个改进的协议的基础上 two-step 多重嵌套 pcr, 这导致一个 pcr 产品横跨整个可变区域的人类 TCR α和β链。通过确定独特的限制点, 并将它们纳入 pcr 引物, 我们已经使 pcr 产品与直接 sub-cloning 与模板逆转录病毒载体相兼容。由此产生的逆转录病毒构建编码一个嵌合的人/鼠 TCR 与鼠标的细胞内域, 这是功能在鼠标细胞或在体内鼠标模型。总的来说, 这里描述的协议结合了人类单细胞配对 TCR α和β链识别与简化的逆转录病毒载体易于适应体外和体内TCR 表达。视频和伴随的材料被设计为对单细胞 PCR 的高度详细的描述, 因此关键的步可以被跟随和潜在的陷阱避免。此外, 我们还提供了生成表达载体所需的克隆步骤的详细描述。一旦掌握, 从单细胞排序到 TCR 表达的整个过程可以在短短的两周内完成。

在步骤 3.2.7 (图 2) 中, 通过在琼脂糖凝胶上5µL 第二反应的运行, 对多重嵌套 PCR 反应的效率进行了检查。平均 TCR-β放大率的效率预计在80% 左右, 而 TCR-α反应的效率通常较低, 约 50%4。只有配对的 TCR α和 TCR β链可以用于 TCR 表达;然而, 所有 PCR 产品都可以测序, 以获得 TCR 序列和剧目信息。
<p class="jove_content" fo:keep-together.within-page="1"...

Watch this Scientific Journal Video about Streamlined Single Cell TCR Isolation and Generation of Retroviral Vectors for In Vitro and In Vivo Expression of Human TCRs at JoVE.com

Streamlined Single Cell TCR Isolation and Generation of Retroviral Vectors for In Vitro and In Vivo Expression of Human TCRs

当前的协议结合了单细胞配对人类 TCR α和β链测序与与体外和体内TCR 表达相兼容的反转录病毒载体的生成。

简化的单细胞 TCR 分离和逆转录病毒载体的生成, 用于体外和体内表达人类 TCRs

Although, several methods for sequencing of paired T cell receptor (TCR) alpha and beta chains from single T cells have been developed, none so far have ...

streamlined-single-cell-tcr-isolation-generation-retroviral-vectors

Research

JoVE Journal

Immunology and Infection

Streamlined Single Cell TCR Isolation and Generation of Retroviral Vectors for In Vitro and In Vivo Expression of Human TCRs

9.4K 次观看.  Baylor College of Medicine, Texas Children's Hospital. 当前的协议结合了单细胞配对人类 TCR α和β链测序与与体外和体内TCR 表达相兼容的反转录病毒载体的生成。

视频: Streamlined Single Cell TCR Isolation and Generation of Retroviral Vectors for In Vitro and In Vivo Expression of Human TCRs

Retroviral Transduction of T-cell Receptors in Mouse T-cells

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen presenting cells (APCs)1. This recognition leads to the activation of T-cells and a series of functional outcomes (e.g. cytokine production, killing of the target cells). Understanding the functional role of TCRs is critical to harness the power of the immune system to treat a variety of immunology related diseases (e.g. cancer or autoimmunity).
It is convenient to study TCRs in mouse models, which can be accomplished in several ways. Making TCR transgenic mouse models is costly and time-consuming and currently there are only a limited number of them available2-4. Alternatively, mice with antigen-specific T-cells can be generated by bone marrow chimera. This method also takes several weeks and requires expertise5. Retroviral transduction of TCRs into in vitro activated mouse T-cells is a quick and relatively easy method to obtain T-cells of desired peptide-MHC specificity. Antigen-specific T-cells can be generated in one week and used in any downstream applications. Studying transduced T-cells also has direct application to human immunotherapy, as adoptive transfer of human T-cells transduced with antigen-specific TCRs is an emerging strategy for cancer treatment6.
Here we present a protocol to retrovirally transduce TCRs into in vitro activated mouse T-cells. Both human and mouse TCR genes can be used. Retroviruses carrying specific TCR genes are generated and used to infect mouse T-cells activated with anti-CD3 and anti-CD28 antibodies. After in vitro expansion, transduced T-cells are analyzed by flow cytometry.

We present a protocol to produce antigen-specific mouse T-cells using retroviral transduction

T细胞受体在小鼠T细胞的逆转录病毒转导

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen ...

科研

免疫与感染

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 ...

Generating De Novo Antigen-specific Human T Cell Receptors by Retroviral Transduction of Centric Hemichain

T cell receptors (TCRs) are used clinically to direct the specificity of T cells to target tumors as a promising modality of immunotherapy. Therefore, cloning TCRs specific for various tumor-associated antigens has been the goal of many studies. To elicit an effective T cell response, the TCR must recognize the target antigen with optimal affinity. However, cloning such TCRs has been a challenge and many available TCRs possess sub-optimal affinity for the cognate antigen. In this protocol, we describe a method of cloning de novo high affinity antigen-specific TCRs using existing TCRs by exploiting hemichain centricity. It is known that for some TCRs, each TCR&#945; or TCR&#946; hemichain do not contribute equally to antigen recognition, and the dominant hemichain is referred to as the centric hemichain. We have shown that by pairing the centric hemichain with counter-chains differing from the original counter-chain, we are able to maintain the antigen specificity, while modulating its interaction strength for the cognate antigen. Thus, the therapeutic potential of a given TCR can be improved by optimizing the pairing between the centric and counter hemichains.

Generating De Novo Antigen-specific Human T Cell Receptors by Retroviral Transduction of Centric Hemichain

Herein we describe a novel method to generate antigen-specific T cell receptors (TCRs) by pairing the TCR&#945; or TCR&#946; of an existing TCR, possessing the antigen-specificity of interest, with complementary hemichain of the peripheral T cell receptor repertoire. The de novo generated TCRs retain antigen-specificity with varying affinity.

发电<em&gt;德诺</em&gt;抗原特异性人T细胞受体通过中心的Hemichain的逆转录病毒转导

T cell receptors (TCRs) are used clinically to direct the specificity of T cells to target tumors as a promising modality of immunotherapy. Therefore, ...

Retroviral Transduction of Helper T Cells as a Genetic Approach to Study Mechanisms Controlling their Differentiation and Function

Helper T cell development and function must be tightly regulated to induce an appropriate immune response that eliminates specific pathogens yet prevents autoimmunity. Many approaches involving different model organisms have been utilized to understand the mechanisms controlling helper T cell development and function. However, studies using mouse models have proven to be highly informative due to the availability of genetic, cellular, and biochemical systems. One genetic approach in mice used by many labs involves retroviral transduction of primary helper T cells. This is a powerful approach due to its relative ease, making it accessible to almost any laboratory with basic skills in molecular biology and immunology. Therefore, multiple genes in wild type or mutant forms can readily be tested for function in helper T cells to understand their importance and mechanisms of action. We have optimized this approach and describe here the protocols for production of high titer retroviruses, isolation of primary murine helper T cells, and their transduction by retroviruses and differentiation toward the different helper subsets. Finally, the use of this approach is described in uncovering mechanisms utilized by microRNAs (miRNAs) to regulate pathways controlling helper T cell development and function.

Many experimental systems have been utilized to understand the mechanisms regulating T cell development and function in an immune response. Here a genetic approach using retroviral transduction is described, which is economic, time efficient, and most importantly, highly informative in identifying regulatory pathways.

辅助性T细胞作为一种遗传方法来研究逆转录病毒转导机制控制其分化和功能

Helper T cell development and function must be tightly regulated to induce an appropriate immune response that eliminates specific pathogens yet prevents ...

Generation of Human Regulatory T Cell Clones

Human regulatory T cells (Treg) are notoriously difficult to isolate in high purity given the current methods of Treg enrichment. These methods are based on the identification of Treg through several activation-dependent cellular surface markers with varying expression levels in different physiologic and pathologic conditions. Populations isolated as &#8220;Treg&#8221; therefore often contain considerable numbers of non-Treg effector cells (i.e., Teff) which hamper the precise phenotypic and functional characterization of these cells, their genomic and proteomic characterization, their reliable enumeration in different states of health and disease, as well as their isolation and expansion for therapeutic purposes. The latter, in particular, remains a major hurdle, as the inadvertent expansion of effector cells homing in Treg-relevant cellular compartments (e.g., CD4+CD25+ T cells) may render Treg-based immunotherapy ineffective, or even harmful. This work presents a method that circumvents the problems associated with population-based isolation and expansion of Treg and shows that the generation of Treg candidate clones with the subsequent selection, culture, and expansion of only carefully vetted, monoclonal cells, enables the generation of an ultrapure Treg cell product that can be kept in culture for many months, enabling downstream investigation of these cells, including for possible therapeutic applications.

This protocol describes the cloning and expansion of human regulatory T cells for the generation of ultra-high purity viable human Treg with stable demethylation at the Treg-specific demethylated region (TSDR) and Treg-specific phenotypic features.

Human regulatory T cells (Treg) are notoriously difficult to isolate in high purity given the current methods of Treg enrichment. These methods are based ...

Production of Human CRISPR-Engineered CAR-T Cells

Adoptive cell therapies using chimeric antigen receptor T cells (CAR-T cells) have demonstrated remarkable clinical efficacy in patients with hematological malignancies and are currently being investigated for various solid tumors. CAR-T cells are generated by removing T cells from a patient's blood and engineering them to express a synthetic immune receptor that redirects the T-cells to recognize and eliminate target tumor cells. Gene editing of CAR-T cells has the potential to improve safety of current CAR-T cell therapies and further increase the efficacy of CAR-T cells. Here, we describe methods for the activation, expansion, and characterization of human CRISPR-engineered CD19 directed CAR-T cells. This comprises transduction of the CAR lentiviral vector and use of single guide RNA (sgRNA) and Cas9 endonuclease to target genes of interest in T cells. The methods described in this protocol can be universally applied to other CAR constructs and target genes beyond the ones used for this study. Furthermore, this protocol discusses strategies for gRNA design, lead gRNA selection and target gene knockout validation to reproducibly achieve high-efficiency, multiplex CRISPR-Cas9 engineering of clinical grade human T cells.

Here, we present a protocol for gene editing in primary human T cells using CRISPR Cas Technology to modify CAR-T cells.

生产人类CRISPR工程的CAR-T细胞

Adoptive cell therapies using chimeric antigen receptor T cells (CAR-T cells) have demonstrated remarkable clinical efficacy in patients with ...

在同基因模型中生成小鼠 CAR-T 细胞的简化方案

Efficient Generation of Murine Chimeric Antigen Receptor (CAR)-T Cells

Engineered cell therapies utilizing chimeric antigen receptor (CAR)-T cells have achieved remarkable effectiveness in individuals with hematological malignancies and are presently undergoing development for the treatment of diverse solid tumors. So far, the preliminary evaluation of novel CAR-T cell products has predominantly taken place in xenograft tumor models using immunodeficient mice. This approach is chosen to facilitate the successful engraftment of human CAR-T cells in the experimental setting. However, syngeneic mouse models, in which tumors and CAR-T cells are derived from the same mouse strain, allow evaluation of new CAR technologies in the context of a functional immune system and comprehensive tumor microenvironment (TME). The protocol described here aims to streamline the process of mouse CAR-T cell generation by presenting standardized methods for retroviral transduction and ex vivo T cell culture. The methods described in this protocol can be applied to other CAR constructs beyond the ones used in this study to enable routine evaluation of new CAR technologies in immune-competent systems.

作者聚焦:增强同基因肿瘤模型中的 CAR-T 细胞功能 

This protocol streamlines retroviral vector production and murine T cell transduction, facilitating the efficient generation of mouse CAR-T cells.

小鼠嵌合抗原受体（CAR）-T细胞的高效生成

Engineered cell therapies utilizing chimeric antigen receptor (CAR)-T cells have achieved remarkable effectiveness in individuals with hematological ...

生物工程

<meta charset="utf8"></head><body>向导 RNA 的逆转录病毒转导用于 Th17 分化中的基因编辑

Retroviral CRISPR/Cas9-Mediated Gene Targeting for the Study of Th17 Differentiation in Vitro

T helper cells that produce IL-17A, known as Th17 cells, play a critical role in immune defense and are implicated in autoimmune disorders. CD4 T cells can be stimulated with antigens and well-defined cytokine cocktails in vitro to mimic Th17 cell differentiation in vivo. Research has been conducted extensively on the Th17 differentiation regulation mechanisms using the in vitro Th17 polarization assay.
Conventional Th17 polarization methods typically involve obtaining na&#239;ve CD4 T cells from genetically modified mice to study the effects of specific genes on Th17 differentiation and function. These methods can be time-consuming and costly and may be influenced by cell-extrinsic factors from the knockout animals. Thus, a protocol using retroviral transduction of guide RNA to introduce gene knockout in CRISPR/Cas9 knockin primary mouse T cells serves as a very useful alternative approach. This paper presents a protocol to differentiate na&#239;ve primary T cells into Th17 cells following retroviral-mediated gene targeting, as well as the subsequent flow cytometry analysis methods for assaying infection and differentiation efficiency.

<meta charset="utf8"></head><body>逆转录病毒 CRISPR/Cas9 介导的基因靶向在体外研究 Th17 分化

We present a protocol for retroviral transduction of guide RNA into primary T cells from Cas9 transgenic mice, providing an efficient alternative for gene editing in studying Th17 differentiation.

逆转录病毒 CRISPR/Cas9 介导的基因靶向在体外研究 Th17 分化

T helper cells that produce IL-17A, known as Th17 cells, play a critical role in immune defense and are implicated in autoimmune disorders. CD4 T cells ...

简化的单细胞 TCR 分离和逆转录病毒载体的生成, 用于体外和体内表达人类 TCRs

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T细胞受体在小鼠T细胞的逆转录病毒转导

骨髓祖细胞的逆转录病毒转导到生成T细胞受体Retrogenic小鼠

发电

辅助性T细胞作为一种遗传方法来研究逆转录病毒转导机制控制其分化和功能

Generation of Human Regulatory T Cell Clones

生产人类CRISPR工程的CAR-T细胞

小鼠嵌合抗原受体（CAR）-T细胞的高效生成

小鼠嵌合抗原受体（CAR）-T细胞的高效生成

小鼠嵌合抗原受体（CAR）-T细胞的高效生成

逆转录病毒 CRISPR/Cas9 介导的基因靶向在体外研究 Th17 分化