这项工作得到 NIH 资助 ai 105813 和 ai 079705 (对 pc), 狼疮研究的目标识别联盟狼疮补助金 ALR 295955 (对 pc), 和关节炎国家研究基金会研究补助金 (HZ)。TS 由中国长沙中南大学第二湘医院儿科医学中心支持, 湘医学院圣安东尼奥医科学生访问计划的背景。

该协议遵循圣安东尼奥德克萨斯大学健康科学中心机构动物保育和使用委员会的动物保育准则. 
 1. 刺激企业社会责任、血浆细胞分化和 HDI 治疗的小鼠 B 细胞 <ol> <li> 脾细胞悬浮液的制备 注: 执行除鼠标外的层流罩中的所有步骤安乐死和解剖。
	<ol> <li> 通过 CO 2 吸入安乐特定的无病原体 C57BL/6J 小鼠 (8-12 周龄)。
		<li> 将鼠标放在解剖板上, 腹部朝上。用18口径1.5 英寸的皮下注射针头把所有四英尺的老鼠引脚. </li>
		<li> 用70% 乙醇浸泡过的湿巾对皮肤进行消毒. </li>
		<li> 使用一组蒸压的外科工具 (镊子和解剖剪刀) 切开胸腔下方的皮肤, 使脾脏可视化 (在腹部左侧, 就在肝脏下方). </li>
		<li> 使用另一种无菌钳和剪刀来提取脾脏, 它位于胃的更大的曲率之下, 通过用镊子钳夹脾并用解剖剪刀切断所有的连接组织。确保删除所有连接的组织. </li>
		<li> 将脾脏放入含有1000和 #181 的1.5 毫升离心管中; 完全 RPMI1640 培养基 (RPMI 1640 培养基补充10% 热灭活胎小牛血清 (FBS), 2 毫米 l-谷氨酰胺, 100 和 #181; g/毫升青霉素, 100 和 #181; g/毫升链霉素, 0.25 和 #181; g/毫升两性霉素 B, 和50和 #181; M 和 #946;-基). </li>
		<li> 将 70 #181; m 细胞过滤器放入一个50毫升的聚丙烯锥形离心管中, 然后将脾脏从离心管上倒入细胞滤网。使用15毫升聚丙烯锥形离心管的尖端, 通过过滤器轻轻地将脾脏捣碎。用15到20毫升的完全介质冲洗细胞过滤器. </li>
		<li> 向下旋转 350 x g 的单元格5分钟并丢弃上清. </li>
		<li> 取出脾脏细胞悬浮液中的红血球。重细胞颗粒在裂解缓冲液中 (每脾5毫升)。在室温下孵育4分钟, 偶尔摇动, 然后用25毫升完全培养基进行淬火. </li>
		<li> 将 350 x g 的单元格向下旋转5分钟, 然后丢弃上清。重1或10毫升的完整培养基中的细胞颗粒。使用例计数可用的单元格. </li>
	</ol> </li> <li> b 单元格隔离 注意: 按照制造商和 #39 的指示, 按负选择隔离 B 单元格。非 b 细胞的目标是去除化抗体针对非 b 细胞 (CD4, CD8, CD11b, CD43, CD49b, CD90.2, Ly-6C/G (Gr-1) 和 TER119) 和亲和涂层磁性粒子。
	<ol> <li> 准备0.25 和 #160; 在无菌8毫升 (5 x 12 mm) 聚苯乙烯圆底管的完整介质中, 1 x 10 75 细胞/毫升的2毫升细胞悬浮液。将正常大鼠血清 (50 和 #181; L/毫升) 添加到样品中. </li>
		<li> 添加50和 #181; 与样品的分离鸡尾酒。通过轻轻地移 上下 2-3 次并在室温下孵育10分钟, 将单元格混合在一起. </li>
		<li> 添加75和 #181; 将亲和的磁性微粒涂到样品中。混合的混合物, 轻轻移上下2-3 次和孵化2.5 分钟的室温. </li>
		<li> 添加完整的 RPMI 1640 介质。混合的细胞, 轻轻移上下 2-3 倍. </li>
		<li> 将管 (不带盖子) 放入磁体中, 在室温下孵育2.5 分钟. 将含有未接触 B 细胞的上清液倒入新的15毫升锥形管中. </li>
		<li> 使用例和台盼蓝染色对活细胞进行计数。通过流式细胞仪分析 b 细胞表面标记物, 如 B220 和 CD19, 评估 b 细胞的纯度. </li>
	</ol> </li> <li> b 细胞刺激和 HDI 治疗 <ol> <li> 重完全 RPMI 1640 培养基中的纯化 b 细胞, 最终浓度为 0.3 x 10 6 细胞/毫升. </li>
		<li> 使用48孔细胞培养板进行细胞培养。对于每个井, 添加1毫升纯化 B 细胞悬浮液 (0.3 x 10 6 细胞/毫升), LPS (3 和 #181; g/毫升最终浓度) 从 大肠杆菌 , IL-4 (5 ng/毫升最终浓度), 和0或500和 #181; M HDI (戊酸;VPA). </li>
		<li> 孵育37和 #176 的细胞; C 和 5% CO 2 60 h 用于 qRT PCR 和高通量 mRNA 和 miRNA 序列分析, 或 96 h 用于流式细胞仪分析. </li>
	</ol> </li> <li> 流式细胞仪分析 CSR 和等离子体细胞分化 <ol> 在培养了96小时后, 通过多次移单元格, 将每一个井中的细胞分离出来。将细胞悬浮液转化为1.5 毫升的离心管。用台式离心机将电池在 350 x g 上旋转5分钟, 丢弃上清液. </li>
		<li> 重100和 #181 的单元格 (HBSS), 包含 1% BSA 和 0.5 ng/ml 荧光素 ( FITC )- 共轭山羊抗 - 小鼠 IgM 抗体 (Ab) , 0.2 ng/ml 复方 (APC)-共轭鼠 anti-mouse IgG1单克隆抗体, 0.2 ng/ml 藻 (PE)-共轭大鼠 anti-mouse B220 抗体, 0.2 ng/毫升 PE-Cy7-conjugated 鼠 anti-mouse CD138 抗体, 2 ng/ml 7-aminoactinomycin D (7-反倾销). </li>
		<li> 在室温下用荧光共轭抗体 (步骤 1.4.2) 孵育细胞30分钟. </li>
		<li> 用1毫升的 HBSS 和 1% BSA 冲洗细胞. </li>
		<li> 使用台式离心机向下旋转 1500 x g 的电池5分钟, 并丢弃上清液. </li>
		<li> 重300和 #181 中的细胞, HBSS 1% BSA, 将细胞悬浮液转移到圆底的聚苯乙烯管上。用箔盖住管子以避免光线照射. </li>
		<li> 对单细胞悬浮液进行流式细胞仪分析。为每个补偿样本收集5万事件和其他示例的25万事件。使用设备软件分析数据. </li>
		<li> 通过使用脉冲几何门 (fsc-h x fsc-a 和 ssc-h x ssc a) 消除碎片和双峰。适当的门 7-反倾销的阴谋, 以排除死细胞. </li>
	</ol> </li> </ol> 2。高通量的 mRNA-Seq <ol> <li> 在60小时的区域性之后, 从 2-4 和 #215 中提取总 rna; 10 6 单元格使用总 rna 隔离套件, 它可以根据制造商和 #39 的指示恢复小 rna。包括一 dnasei I 治疗步骤. </li>
	<li> 根据制造商和 #39 的说明, 使用 bioanalyzer 验证 RNA 完整性. </li>
	<li> 使用500-1、000 ng 高质量的总 rna (rna 完整性数字和 #62; 8.0) 用于 rna 序列库的制备和商业 rna 样本 p代表工具包按照制造商和 #39 的说明操作. </li>
	<li> 根据各自的适配器的 6-bp 索引部分将单个 mRNA 序列库汇集在一起, 并按 50 bp/序列顺序排列库。根据制造商和 #39 的协议使用高通量的 DNA 系统. </li>
	<li> 顺序运行后, 解与 CASAVA 一起生成每个示例的 fastq 文件。执行读取映射和生物信息学分析, 如前面所概述的 11 . </li>
	<li> 使用 TopHat2 默认设置将所有顺序读取与其参考基因组 (UCSC 小鼠基因组构建 mm9) 对齐 14 。使用 HTSeq 来处理 bam 文件, 以获取所有样本中每一个基因的计数. </li>
</ol> 3。高通量 miRNA-seq <ol> <li> 使用 100 ng-1 和 #181; 在步骤2.1 中编写的高质量总 rna g, 使用商业小 rna-seq 套件编写小的 rna 序列库. </li>
	<li> 结扎将退化的3和 #39; 适配器放到5和 #8242 上; 开始的小 RNA 分子的末端与商业结扎试剂盒。结扎退化的5和 #39; 适配器到3和 #8242; 开始的小 RNA 分子的末端与商业结扎试剂盒。通过反向转录将 rna 转化为 cDNA, 用商用试剂盒放大 PCR 扩增小 rna 序列库. </li>
	<li> 使用6% 本机页凝胶分离最后的小 RNA 序列库。运行的凝胶与1X 的缓冲区在200伏, 直到溴蓝色跟踪染料带接近底部的凝胶 (0.5-1 厘米). </li>
	<li> 从玻璃盘中取出凝胶, 然后用溴溴化物 (0.5 和 #181; 水中的 g/毫升) 在一个干净的容器中 2-3 min. 可视化 UV transilluminator 或其他凝胶文件仪器上的凝胶带. </li>
	<li> 用干净的剃刀把 150 bp 波段剪掉, 放到1.7 毫升的管子里. </li>
	<li> 根据制造商的说明使用凝胶萃取试剂盒提取 DNA. </li>
	<li> 检查最终库的大小分布, 并使用商用的超敏 DNA 检测方法, 并按照制造商和 #39 的商业 dsDNA 检测浓度进行检测; 说明. </li>
	<li> 将库用于放大, 然后根据制造商和 #39 的协议, 使用商业高通量 DNA 测序系统进行后续排序. </li>
	<li> 解与 CASAVA 一起为每个制造商和 #39 的协议生成每个示例的 fastq 文件. </li>
	<li> 对于每个样本的小 rna-seq 分析, 根据制造商和 #39 的协议使用闪烁进行小的 rna 对准。
	<ol> <li> 删除与污染物 (如线粒体、rRNA、底漆等) 对齐的读取. </li>
		<li> 将数据与成熟的 miRNA 序列对齐. </li>
		<li> 将数据与发夹回路序列 (前体 miRNA) 对齐. </li>
		<li> 将数据与其他小 RNA 序列对齐 (使用 fRNA 数据库) 15 . </li>
	</ol> </li> <li> 在对所有示例进行量化后, 定义不同组/示例之间的差异表达 rna。预测 rna 目标选择的基因或基因, 可以使用在线 miRNA 目标预测工具 (如 TargetScan 16 、微观世界 17 和 PicTar 18) 的选择性 miRNA 来瞄准。.</li>
	<li> 如果需要功能注释或路径分析, 请将预测的基因提交到独创性路径分析 (国际音标) 或 DAVID. </li>
</ol> 4。定量 rt-pcr (qRT pcr) 的基因和 rna <ol> <li> qRT-pcr 分析 mRNA <ol> <li> 从 0.2-5 和 #215 中提取 rna; 10 6 细胞使用商业总 rna 隔离套件, 可以恢复小 rna, 跟随制造商和 #39 的指示。包括一 dnasei I 治疗步骤. </li>
		<li> 用第一链 cdna 合成系统的全 RNA cdna, 利用寡糖-dT 引物. </li>
		<li> 采用 qRT pcr 方法, 利用 2x real-time pcr 方法, 用适当的引物对 cDNA 进行定量, 并与以下协议相结合:95、#176; c 为十五年代, 40 周期94和 #176; c 为十年代, 60 和 #176; c 为三十年代, 72 和 #176; c 为三十年代. </li>
		<li> 在72和 #176 期间执行数据获取; c 扩展步骤, 并执行从72到95和 #176 的熔融曲线分析; c. </li>
	</ol> </li> <li> qRT miRNA <ol> <li> 分 RNA 的 PCR 分析从步骤4.1.1 中准备的样本中提取. </li>
		<li> 反转-转录 RNA 为 cDNA。按照制造商和 #39 的说明使用商业 rna 反转录套. </li>
		<li> 使用 SYBR 绿色 real-time pcr 大师组合与 250 nM 成熟 miRNA 前向引物与通用反向底漆的 rna 进行 real-time pcr。
		<ol> <li> 解冻 2x PCR 主混合、miRNA 特定的正向底漆和室温下的通用反向底漆。混合各个解决方案. </li>
			<li> 准备一个25和 #181 的反应组合; L 每好反应容量 (用于96井 PCR 板): 2x PCR 主混合12.5 和 #181; l miRNA 前引物 (2.5 和 #956; m) 2.5 和 #181; l 通用反向底漆 (2.5 和 #956; m) 2.5 和 #181; l RN无 water5 和 #181; l </li> <li> 添加22.5 和 #956; l 的反应混合到一个96的 PCR 板。添加2.5 和 #181; L 模板 cDNA (50 pg-3 ng) 到各自的板材井. </li>
			<li> 紧紧地封住底片。离心板1分钟, 在 1000 x g 和室温. </li>
			<li> 将盘放在 real-time 循环中, 并启动以下循环程序:95、#176; c 为5分钟, 40 周期为94和 #176; c 为十五年代, 55 和 #176; c 为三十年代, 和72和 #176; c 为三十年代. </li>
		</ol> </li> <li> 使用 and #916; #916; 用电子表格进行 qRT PCR 数据分析的 Ct 方法。将相关 rna 的表达规范化到小核/核仁 rna Rnu6/RNU61/2、Snord61/SNORD61、Snord68/SNORD68 和 Snord70/SNORD70 的表达. </li>
	</ol> </li> <li> 统计分析 <ol> <li> 执行统计分析, 通过配对和未配对的学生和 #39 来确定 p 值 t 使用电子表格进行测试, 并考虑 p 值和 #60; 0.05 作为重要. </li>
	</ol>
	</li>
</ol>

<ol>
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P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Predicting+effective+microRNA+target+sites+in+mammalian+mRNAs.">Predicting effective microRNA target sites in mammalian mRNAs.</a> Elife. 4, (2015).</li><li>Griffiths-Jones, S., Saini, H. K., van Dongen, S., Enright, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=miRBase:+tools+for+microRNA+genomics.">miRBase: tools for microRNA genomics.</a> Nucleic Acids Res. 36, D154-D158 (2008).</li><li>Anders, G., 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=doRiNA:+a+database+of+RNA+interactions+in+post-transcriptional+regulation.">doRiNA: a database of RNA interactions in post-transcriptional regulation.</a> Nucleic Acids Res. 40, D180-D186 (2012).</li></ol>

作者声明他们没有竞争的金融利益。

该协议提供了诱导 B 细胞类交换和细胞分化的综合方法;分析其对表观遗传调节剂的影响, 即 HDI;并检测人的 HDI 对这些细胞的 mRNA 和 miRNA 表达的影响。大多数这些方法也可以用来分析表观遗传因子对人 B 细胞功能和 mRNA/miRNA 表达的影响。qRT PCR 和 mRNA-seq/miRNA 的方法也可以用来分析 B 细胞分离的小鼠与后生调节剂, 如助。
表观遗传调节可能影响细胞增殖和活力等多种不同的细胞?...

表观遗传标记或因素, 如 DNA 甲基化, 组蛋白后修饰, 编码 rna (包括 rna), 通过改变基因表达1调节细胞功能。表观遗传修饰调节 b 淋巴细胞功能, 如免疫球蛋白类-开关 DNA 重组 (CSR), 体细胞变异 (SHM), 并分化为记忆 b 细胞或血浆细胞, 从而调节抗体和自我抗体响应2,3。CSR 和 SHM 的关键要求激活诱导的胞脱氨酶 (援助, 编码为Aicda), 这是高度诱导 B 细胞响应 t 依赖性和 t 独立抗原4。类交换/hypermutated b 细胞进一步分化成血浆细胞, 其中分泌大量的抗体在时尚严重依赖于 B 淋巴细胞诱导成熟蛋白 1 (Blimp1, 编码为Prdm1)5。B 细胞异常的表观遗传变化可能导致异常的抗体/自身抗体应答, 这会导致过敏反应或自身免疫1,4。了解后生因素, 如 rna, 调节 B 细胞内在基因表达不仅对疫苗的发展很重要, 而且对于揭示潜在的异常抗体/自身免疫反应的机制也是必不可少的。
组蛋白乙酰化和脱乙酰度是由组蛋白乙酰 (HAT) 和组蛋白乙酰 (HDAC) 催化的蛋白组蛋白的赖氨酸残留修饰。这些修改导致染色质的增加或减少的可及性, 并进一步允许或阻止转录因子或蛋白质与 DNA 的结合和基因表达的改变5,6,7,8. HDAC 抑制剂 (HDI) 是一类干扰 HDACs 功能的化合物。在这里, 我们使用 HDI (VPA) 来解决 HDAC 对 B 细胞固有基因表达谱的调节及其机制。
rna 是小的, 编码 rna 大约18到22核苷酸长度是通过几个阶段产生的。miRNA 寄主基因转录和形成发夹主 rna (pri-rna)。它们被出口到细胞质中, 其中 rna 被进一步加工成前体 rna (pre-miRNAs)。最后, 成熟的 rna 是通过 pre-miRNAs 的裂解形成的。rna 识别在其目标基因的 3 "未翻译区域" 中的互补序列6,7。通过后沉默, rna 调节细胞活动, 如增殖, 分化, 和凋亡10,11。由于多个 rna 可以针对同一 mRNA, 而一个单一的 miRNA 可能针对多个基因, 因此重要的是要有一个上下文视图的 miRNA 表达式配置文件, 以了解个人的价值和 rna 的集体效应。rna 已被证明参与 b 细胞的发育和外周分化, 以及 b 细胞阶段特异性分化, 抗体应答, 和自身免疫1,4,9。在 3 "UTR Aicda和Prdm1中, 有几个经过验证或预测的进化保守站点, 可通过 rna8进行攻击。
后生调制, 包括组蛋白后修饰和 rna, 显示一个细胞类型和细胞阶段特异的基因表达调控模式9。在这里, 我们描述的方法, 以确定人的 HDI 介导的 miRNA 和 mRNA 表达, 社会责任, 和血浆细胞分化。其中包括诱导 B 细胞接受 CSR 和细胞分化的协议;用 HDI 治疗 B 细胞;并通过 qRT PCR、miRNA-seq 和 mrna-seq10、11、12、813来分析 miRNA 和 mrna 的表达。

<table><tbody><tr><td>C57BL/6 mice</td><td>Jackson Labs</td><td>664</td><td></td></tr><tr><td>Corning cellgro RPMI 1640 Medium (Mod.) 1X with L-Glutamine (Size: 6 x 500mL; With L-Glutamine)</td><td>Fisher Scientific</td><td>MT 10-040-CV&amp;nbsp;</td><td></td></tr><tr><td>FBS</td><td>Hyclone</td><td>SH300</td><td></td></tr><tr><td>HyClone Antibiotic Antimycotic Solution 100 mL</td><td>Fisher Scientific - Hyclone</td><td>SV3007901&amp;nbsp;</td><td></td></tr><tr><td>&amp;beta;-Mercaptoethanol</td><td>Fisher Scientific</td><td>44-420-3250ML</td><td></td></tr><tr><td>Falcon Cell Strainers</td><td>Fisher Scientific</td><td>21008-952&amp;nbsp;&amp;nbsp;</td><td></td></tr><tr><td>Trypan Blue Stain 0.04%</td><td>GIBCO/Life Technologies/Inv</td><td>15250</td><td></td></tr><tr><td>ACK Lysis Buffer&amp;nbsp;</td><td>Fisher Scientific</td><td>BW10-548E&amp;nbsp;</td><td></td></tr><tr><td>Hausser Scientific Bright-Line Counting Chamber</td><td>Fisher Scientific</td><td>02-671-51B</td><td></td></tr><tr><td>EasySep Magnet</td><td>Stem Cell Technologies</td><td>18000</td><td></td></tr><tr><td>Falcon Round-Bottom Polystyrene Tubes with Cap</td><td>Fisher Scientific</td><td>14-959-1A</td><td></td></tr><tr><td>EasySep Mouse B cell Isolation Kit</td><td>Stem Cell Tech</td><td>19854</td><td></td></tr><tr><td>BD Needle Only 18 Gauge 1.5 inch SHORT BEVEL 100/box&amp;nbsp;</td><td>BD Biosciences</td><td>&amp;nbsp;305199</td><td></td></tr><tr><td>PE/Cy7 anti-mouse CD138 (Syndecan-1) Antibody</td><td>BioLegend&amp;nbsp;</td><td>142513 (25 ug)</td><td></td></tr><tr><td>PE-Cy7 B220 antibody</td><td>BioLegend</td><td>103222</td><td></td></tr><tr><td>7-AAD (1 mg)</td><td>Sigma Aldrich</td><td>A9400-1MG</td><td></td></tr><tr><td>APC anti-mouse/human CD45R/B220 antibody</td><td>Biolegend</td><td>103212</td><td></td></tr><tr><td>Mouse APC-IgG1 200 &amp;micro;g</td><td>Biolegend</td><td>406610</td><td></td></tr><tr><td>FITC anti-mouse IgM Antibody</td><td>Biolegend</td><td>406506</td><td></td></tr><tr><td>FITC anti-mouse/human CD45R/B220 Antibody</td><td>Biolegend</td><td>103206</td><td></td></tr><tr><td>PE Anti-Human/Mouse CD45R (B220) (RA3-6B2)</td><td>Biolegend</td><td>103208</td><td></td></tr><tr><td>HBSS 1X</td><td>Fisher Scientific</td><td>MT-21-022-CM</td><td></td></tr><tr><td>Bovine Serum Albumin, Fraction V, Heat Shock Treated&amp;nbsp;</td><td>Fisher Scientific</td><td>BP1600-100</td><td></td></tr><tr><td>LPS 25mg (Lipopolysaccharides from Escherichia coli 055:B5)</td><td>Sigma Aldrich</td><td>L2880-25MG</td><td></td></tr><tr><td>Recombinant mouse IL-4 (carrier-free)</td><td>BioLegend</td><td>574302 (size: 10 ug)</td><td></td></tr><tr><td>Valproic acid sodium salt</td><td>Sigma Aldrich</td><td>P4543</td><td></td></tr><tr><td>SterilGARD e3 Class II Type A2 Biosafety Cabinet</td><td>The Baker Company</td><td>SG404</td><td></td></tr><tr><td>Large-Capacity Reach-In CO&lt;sub&gt;2&lt;/sub&gt; Incubator&amp;nbsp;</td><td>Thermo Scientific</td><td>3950</td><td></td></tr><tr><td>Isotemp Digital-Control Water Baths: Model 205</td><td>Fisher Scientific</td><td>15-462-5Q</td><td></td></tr><tr><td>5mL Round Bottom Polystyrene Test Tube</td><td>Fisher Scientific</td><td>&amp;nbsp;14-959-5</td><td></td></tr><tr><td>Corning CentriStar 15ml Centrifuge Tubes&amp;nbsp;</td><td>Fisher Scientific&amp;nbsp;</td><td>05-538-59A</td><td></td></tr><tr><td>1.7 mL Microtube, clear</td><td>Genesee</td><td>22-282</td><td></td></tr><tr><td>Higher-Speed Easy Reader Plastic Centrifuge Tubes 50ml</td><td>Fisher Scientific</td><td>06-443-18</td><td></td></tr><tr><td>ELMI SkySpin CM-6MT</td><td>ELMI</td><td>CM-6MT</td><td></td></tr><tr><td>Rotor 6M</td><td>ELMI</td><td>6M</td><td></td></tr><tr><td>Rotor 6M.06</td><td>ELMI</td><td>6M.06</td><td></td></tr><tr><td>Drummond Portable Pipet-Aid XP Pipet Controller</td><td>Drummond Scientific</td><td>4-000-101</td><td></td></tr><tr><td>25 mL serological pipette tips</td><td>Fisher Scientific</td><td>89130-900</td><td></td></tr><tr><td>10 mL serological pipette tips</td><td>Fisher Scientific</td><td>89130-898</td><td></td></tr><tr><td>5 mL serological pipette tips</td><td>Fisher Scientific</td><td>898130-896</td><td></td></tr><tr><td>48-well plates</td><td>Fisher Scientific</td><td>07-200-86</td><td></td></tr><tr><td>Allegra 6 Benchtop Centrifuge, Non-Refrigerated</td><td>Beckman Coulter</td><td>366802</td><td></td></tr><tr><td>GH-3.8A Rotor, Horizontal, ARIES Smart Balance</td><td>Beckman Coulter</td><td>366650</td><td></td></tr><tr><td>Allegra 25R Benchtop Centrifuge, Refrigerated</td><td>Beckman Coulter</td><td>369434</td><td></td></tr><tr><td>TA-15-1.5 Rotor, Fixed Angle</td><td>Beckman Coulter</td><td>368298</td><td></td></tr><tr><td>Fisher Scientific AccuSpin Micro 17</td><td>Fisher Scientific</td><td>13-100-675</td><td></td></tr><tr><td>Fisher Scientific Analog Vortex Mixer&amp;nbsp;</td><td>Fisher Scientific</td><td>02-215-365</td><td></td></tr><tr><td>miRNeasy Mini Kit (50)</td><td>Qiagen</td><td>217004</td><td></td></tr><tr><td>Direct-zol RNA MiniPrep kit</td><td>Zymo Research</td><td>R2050</td><td></td></tr><tr><td>Chloroform (Approx. 0.75% Ethanol as Preservative/Molecular Biology)</td><td>Fisher Scientific</td><td>BP1145-1</td><td></td></tr><tr><td>Rnase-Free Dnase set (50)</td><td>QIAGEN</td><td>79254</td><td></td></tr><tr><td>NanoDrop 2000 Spectrophotometers</td><td>Thermo Scientific</td><td>ND-2000</td><td></td></tr><tr><td>Superscript III First-strand Synthesis System RT-PCR</td><td>Invitrogen</td><td>175013897</td><td></td></tr><tr><td>iTaq Universal SYBR Green Supermix</td><td>Bio-rad&amp;nbsp;</td><td>172-5121</td><td></td></tr><tr><td>Fisherbrand 96-Well Semi-Skirted PCR Plates, case of 25</td><td>Fisher</td><td>14-230-244</td><td></td></tr><tr><td>Microseal &#039;B&#039; Adhesive Seals</td><td>Bio-Rad</td><td>MSB-1001</td><td></td></tr><tr><td>MyiQ Optics Module</td><td>Bio-Rad</td><td>170-9744</td><td></td></tr><tr><td>iCycler Chassis</td><td>Bio-Rad</td><td>170-8701</td><td></td></tr><tr><td>Optical Kit</td><td>Bio-Rad</td><td>170-9752</td><td></td></tr><tr><td>BD LSR II Flow Cytometry Analyzer</td><td>BD Biosciences</td><td></td><td></td></tr><tr><td>FACSDiva software</td><td>BD Biosciences</td><td></td><td></td></tr><tr><td>FlowJo 10</td><td>BD Biosciences</td><td></td><td></td></tr><tr><td>2100 Bioanalyzer</td><td>Agilent Technologies</td><td>G2943CA</td><td></td></tr><tr><td>S200 Focused-ultrasonicator</td><td>Covaris</td><td>S200</td><td></td></tr><tr><td>SPRIworks Fragment Library System I for Illumina</td><td>Beckman Coulter</td><td>A288267</td><td></td></tr><tr><td>cBot Cluster Generation Station</td><td>illumina</td><td>SY-312-2001</td><td></td></tr><tr><td>HiSeq 2000 Genome Sequencer</td><td>Illumina</td><td>SY-401-1001</td><td></td></tr><tr><td>TruSeq RNA Library Prep Kit v2</td><td>Illumina</td><td>RS-122-2001</td><td></td></tr><tr><td>TruSeq Small RNA Library Prep Kit</td><td>Illumina</td><td>RS-200-0012</td><td></td></tr><tr><td>NEXTflex Illumina Small RNA Sequencing Kit v3</td><td>Bioo Scientific</td><td>5132-05</td><td></td></tr><tr><td>2200 TapeStation</td><td>Agilent</td><td>G2964AA</td><td></td></tr></tbody></table>

genome-wide analysis of hdac inhibitor-mediated modulation of micrornas and mrnas in b cells induced to undergo class-switch dna recombination and plasma cell differentiation

抗体应答是通过几个关键的 B 细胞内在的过程来完成的, 包括体细胞变异 (SHM), 类开关 DNA 重组 (CSR) 和血浆细胞分化。近年来, 表观遗传修饰或因素, 如组蛋白脱乙酰和 rna (rna), 已被证明与 B 细胞的遗传程序, 以形成抗体反应, 而表观遗传因素的功能障碍已发现导致自身抗体应答。分析 b 细胞的基因组全 miRNA 和 mRNA 表达对表观遗传调节因子的反应, 对于了解 b 细胞功能和抗体应答的表观遗传调节具有重要意义。在这里, 我们演示了一个协议诱导 b 细胞进行 CSR 和细胞分化, 治疗这些 b 细胞与组蛋白乙酰 (HDAC) 抑制剂 (助), 并分析 mRNA 和 rna 表达。在这个协议, 我们直接分析互补 DNA (cDNA) 序列使用下一代 mrna 测序 (mrna-seq) 和 miRNA 的技术, 测序读取的基因组, 和定量的反向转录 (qRT)-PCR。通过这些方法, 我们已经定义, 在 B 细胞中诱导的 csr 和细胞分化, 人类发展指数, 一个后生的调节器, 选择性地调节 miRNA 和 mRNA 表达, 改变 csr 和细胞分化。

使用我们的协议, 纯化 B 细胞放置 LPS (3 µg/毫升) 和 IL-4 (5 ng/毫升) 的96小时可以诱导30-40% 的 CSR 到 IgG1 和〜10% 的血浆细胞分化。在与 HDI (500µM VPA) 治疗后, 对 IgG1 的 CSR 降低到 10-20%, 而血浆细胞分化降低到 ~ 2% (图 1)。post-recombination Iμ-Cγ1和成熟的 VhDJh-Cγ1记录的数量的减少进一步证实了 HDI 对 csr 的抑制, 这是已完成的 csr 的标志。由 qRT-PCR 测量,...

Watch this Scientific Journal Video about Genome-wide Analysis of HDAC Inhibitor-mediated Modulation of microRNAs and mRNAs in B Cells Induced to Undergo Class-switch DNA Recombination and Plasma Cell

Genome-wide Analysis of HDAC Inhibitor-mediated Modulation of microRNAs and mRNAs in B Cells Induced to Undergo Class-switch DNA Recombination and Plasma Cell Differentiation

后生因素可以与基因程序相互作用, 调节基因表达, 调节 B 细胞功能。结合体外b 细胞刺激、qRT PCR、高通量 rna 序列和 mRNA 序列方法, 可以分析 b 细胞中 miRNA 和基因表达的表观遗传调控。

HDAC 抑制剂介导的 B 细胞 rna 和基因的基因组分析--诱导进行类开关 DNA 重组和血浆细胞分化

Antibody responses are accomplished through several critical B cell-intrinsic processes, including somatic hypermutation (SHM), class-switch DNA ...

genome-wide-analysis-hdac-inhibitor-mediated-modulation-micrornas

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

6.1K 次观看.  University of Texas Long School of Medicine at San Antonio. 后生因素可以与基因程序相互作用, 调节基因表达, 调节 B 细胞功能。结合体外b 细胞刺激、qRT PCR、高通量 rna 序列和 mRNA 序列方法, 可以分析 b 细胞中 miRNA 和基因表达的表观遗传调控。

视频: Genome-wide Analysis of HDAC Inhibitor-mediated Modulation of microRNAs and mRNAs in B Cells Induced to Undergo Class-switch DNA Recombination and Plasma Cell Differentiation

Induction and Assessment of Class Switch Recombination in Purified Murine B Cells

Humoral immunity is the branch of the immune system maintained by B cells and mediated through the secretion of antibodies. Upon B cell activation, the immunoglobulin locus undergoes a series of genetic modifications to alter the binding capacity and effector function of secreted antibodies. This process is highlighted by a genomic recombination event known as class switch recombination (CSR) in which the default IgM antibody isotype is substituted for one of IgG, IgA, or IgE. Each isotype possesses distinct effector functions thereby making CSR crucial to the maintenance of immunity. Diversification of the immunoglobulin locus is mediated by the enzyme activation-induced cytidine deaminase (AID). A schematic video describing this process in detail is available online (http://video.med.utoronto.ca/videoprojects/immunology/aam.html). AID's activity and the CSR pathway are commonly studied in the assessment of B cell function and humoral immunity in mice. The protocol outlined in this report presents a method of B cell isolation from murine spleens and subsequent stimulation with bacterial lipopolysaccharide (LPS) to induce class switching to IgG3 (for other antibody isotypes see Table 1). In addition, the fluorescent cell staining dye Carboxyfluorescein succinimidyl ester (CFSE) is used to monitor cell division of stimulated cells, a process crucial to isotype switching 1, 2. The regulation of AID and the mechanism by which CSR occurs are still unclear and thus in vitro class switch assays provide a reliable method for testing these processes in various mouse models. These assays have been previously used in the context of gene deficiency using knockout mice 3. Furthermore, in vitro switching of B cells can be preceded by viral transduction to modulate gene expression by RNA knockdown or transgene expression 4-6. The data from these types of experiments have impacted our understanding of AID activity, resolution of the CSR reaction, and antibody-mediated immunity in the mouse.

Following antigen exposure, subpopulations of activated B cells undergo a process known as class switch recombination (CSR) to produce antibody isotypes with distinct effector functions. The protocol outlined in this report explains how CSR can be induced and analyzed in vitro for the purposes of studying B cell function.

纯净小鼠的B细胞的诱导和评估类开关重组

Humoral immunity is the branch of the immune system maintained by B cells and mediated through the secretion of antibodies. Upon B cell activation, the ...

科研

免疫与感染

Recombinant Retroviral Production and Infection of B Cells

The transgenic expression of genes in eukaryotic cells is a powerful reverse genetic approach in which a gene of interest is expressed under the control of a heterologous expression system to facilitate the analysis of the resulting phenotype. This approach can be used to express a gene that is not normally found in the organism, to express a mutant form of a gene product, or to over-express a dominant-negative form of the gene product. It is particularly useful in the study of the hematopoetic system, where transcriptional regulation is a major control mechanism in the development and differentiation of B cells 1, reviewed in 2-4.
Mouse genetics is a powerful tool for the study of human genes and diseases. A comparative analysis of the mouse and human genome reveals conservation of synteny in over 90% of the genome 5. Also, much of the technology used in mouse models is applicable to the study of human genes, for example, gene disruptions and allelic replacement 6. However, the creation of a transgenic mouse requires a great deal of resources of both a financial and technical nature. Several projects have begun to compile libraries of knock out mouse strains (KOMP, EUCOMM, NorCOMM) or mutagenesis induced strains (RIKEN), which require large-scale efforts and collaboration 7. Therefore, it is desirable to first study the phenotype of a desired gene in a cell culture model of primary cells before progressing to a mouse model. 
Retroviral DNA integrates into the host DNA, preferably within or near transcription units or CpG islands, resulting in stable and heritable expression of the packaged gene of interest while avoiding transcriptional silencing 8 9. The genes are then transcribed under the control of a high efficiency retroviral promoter, resulting in a high efficiency of transcription and protein production. Therefore, retroviral expression can be used with cells that are difficult to transfect, provided the cells are in an active state during mitosis. Because the structural genes of the virus are contained within the packaging cell line, the expression vectors used to clone the gene of interest contain no structural genes of the virus, which both eliminates the possibility of viral revertants and increases the safety of working with viral supernatants as no infectious virions are produced 10. 
Here we present a protocol for recombinant retroviral production and subsequent infection of splenic B cells. After isolation, the cultured splenic cells are stimulated with Th derived lymphokines and anti-CD40, which induces a burst of B cell proliferation and differentiation 11. This protocol is ideal for the study of events occurring late in B cell development and differentiation, as B cells are isolated from the spleen following initial hematopoetic events but prior to antigenic stimulation to induce plasmacytic differentiation.

An efficient system of structure and function analysis of a gene in an ex vivo culture of splenic B-lymphocytes is described. This method takes advantage of recombinant retroviral production in a helper free, ecotrophic packaging cell line. Stable, heritable expression of a gene of interest within primary lymphocytes is achieved leading to generation of surface antibodies on B cells undergoing class switch recombination.

重组逆转录病毒的生产和B细胞的感染

The transgenic expression of genes in eukaryotic cells is a powerful reverse genetic approach in which a gene of interest is expressed under the control ...

Chromatin Immunoprecipitation (ChIP) in Mouse T-cell Lines

Signaling pathways regulate gene expression programs via the modulation of the chromatin structure at different levels, such as by post-translational modifications (PTMs) of histone tails, the exchange of canonical histones with histone variants, and nucleosome eviction. Such regulation requires the binding of signal-sensitive transcription factors (TFs) that recruit chromatin-modifying enzymes at regulatory elements defined as enhancers. Understanding how signaling cascades regulate enhancer activity requires a comprehensive analysis of the binding of TFs, chromatin modifying enzymes, and the occupancy of specific histone marks and histone variants. Chromatin immunoprecipitation (ChIP) assays utilize highly specific antibodies to immunoprecipitate specific protein/DNA complexes. The subsequent analysis of the purified DNA allows for the identification the region occupied by the protein recognized by the antibody. This work describes a protocol to efficiently perform ChIP of histone proteins in a mature mouse T-cell line. The presented protocol allows for the performance of ChIP assays in a reasonable timeframe and with high reproducibility.

Chromatin Immunoprecipitation ChIP in Mouse T-cell Lines

This work describes a protocol for chromatin immunoprecipitation (ChIP) using a mature mouse T-cell line. This protocol is suitable to investigate the distribution of specific histone marks at specific promoter sites or genome-wide.

染色质免疫沉淀（ChIP）在小鼠T细胞系

Signaling pathways regulate gene expression programs via the modulation of the chromatin structure at different levels, such as by post-translational ...

遗传学

Assessing Somatic Hypermutation in Ramos B Cells after Overexpression or Knockdown of Specific Genes

B cells start their life with low affinity antibodies generated by V(D)J recombination. However, upon detecting a pathogen, the variable (V) region of an immunoglobulin (Ig) gene is mutated approximately 100,000-fold more than the rest of the genome through somatic hypermutation (SHM), resulting in high affinity antibodies1,2. In addition, class switch recombination (CSR) produces antibodies with different effector functions depending on the kind of immune response that is needed for a particular pathogen. Both CSR and SHM are initiated by activation-induced cytidine deaminase (AID), which deaminates cytosine residues in DNA to produce uracils. These uracils are processed by error-prone forms of repair pathways, eventually leading to mutations and recombination1-3.
Our current understanding of the molecular details of SHM and CSR come from a combination of studies in mice, primary cells, cell lines, and cell-free experiments. Mouse models remain the gold standard with genetic knockouts showing critical roles for many repair factors (e.g. Ung, Msh2, Msh6, Exo1, and polymerase &eta;)4-10. However, not all genes are amenable for knockout studies. For example, knockouts of several double-strand break repair proteins are embryonically lethal or impair B-cell development11-14. Moreover, sometimes the specific function of a protein in SHM or CSR may be masked by more global defects caused by the knockout. In addition, since experiments in mice can be lengthy, altering expression of individual genes in cell lines has become an increasingly popular first step to identifying and characterizing candidate genes15-18.
Ramos &#x2013; a Burkitt lymphoma cell line that constitutively undergoes SHM &#x2013; has been a popular cell-line model to study SHM18-24. One advantage of Ramos cells is that they have a built-in convenient semi-quantitative measure of SHM. Wild type cells express IgM and, as they pick up mutations, some of the mutations knock out IgM expression. Therefore, assaying IgM loss by fluorescence-activated cell scanning (FACS) provides a quick read-out for the level of SHM. A more quantitative measurement of SHM can be obtained by directly sequencing the antibody genes. 
Since Ramos cells are difficult to transfect, we produce stable derivatives that have increased or lowered expression of an individual gene by infecting cells with retroviral or lentiviral constructs that contain either an overexpression cassette or a short hairpin RNA (shRNA), respectively. Here, we describe how we infect Ramos cells and then use these cells to investigate the role of specific genes on SHM (Figure 1).

We describe how to perform retroviral or lentiviral infections of overexpression or shRNA-containing constructs in the human Ramos B-cell line and how to measure somatic hypermutation in these cells.

评估拉莫斯B细胞的特定基因的过表达或击倒后的体细胞超突变

B cells start their life with low affinity antibodies generated by V(D)J recombination.  However, upon detecting a pathogen, the variable (V) region of an ...

Simultaneous Measurement of HDAC1 and HDAC6 Activity in HeLa Cells Using UHPLC-MS

The search for new histone deacetylase (HDAC) inhibitors is of increasing interest in drug discovery. Isoform selectivity has been in the spotlight since the approval of romidepsin, a class I HDAC inhibitor for cancer therapy, and the clinical investigation of HDAC6-specific inhibitors for multiple myeloma. The present method is used to determine the inhibitory activity of test compounds on HDAC1 and HDAC6 in cells. The isoform activity is measured using the ultra-high-performance liquid chromatography &#8211; mass spectrometry (UHPLC-MS) analysis of specific substrates incubated with treated and untreated HeLa cells. The method has the advantage of reflecting the endogenous HDAC activity within the cell environment, in contrast to cell-free biochemical assays conducted on isolated isoforms. Moreover, because it is based on the quantification of synthetic substrates, the method does not require the antibody recognition of endogenous acetylated proteins. It is easily adaptable to several cell lines and an automated process. The method has already proved useful in finding HDAC6-selective compounds in neuroblasts. Representative results are shown here with the standard HDAC inhibitors trichostatin A (non-specific), MS275 (HDAC1-specific), and tubastatin A (HDAC6-specific) using HeLa cells.

The present method serves to identify isoform-specific inhibitors of histone deacetylases (HDAC) in HeLa cells by the UHPLC-MS analysis of multiple substrates. This is an antibody-free method developed to reflect HDAC1 and HDAC6 activity in the living cell environment, in contrast to single-isoform cell-free assays.

同时测量 HDAC1 和 HDAC6 活动在 HeLa 细胞使用 UHPLC 女士

The search for new histone deacetylase (HDAC) inhibitors is of increasing interest in drug discovery. Isoform selectivity has been in the spotlight since ...

化学

Chromatin Immunoprecipitation (ChIP) to Assay Dynamic Histone Modification in Activated Gene Expression in Human Cells

In response to a variety of extracellular ligands, the STAT (signal transducer and activator of transcription) transcription factors are rapidly recruited from their latent state in the cytoplasm to cell surface receptors where they are activated by phosphorylation at a single tyrosine residue1. They then dimerize and translocate to the nucleus to drive the transcription of target genes, affecting growth, differentiation, homeostasis and the immune response. Not surprisingly, given their widespread involvement in normal cell processes, dysregulation of STAT function contributes to human disease, particularly to cancers2 and autoimmune diseases3. It is well established that transcription is regulated by alterations to the chromatin template4,5. These alterations include the activities of ATP-dependent complexes, as well as covalent histone modifications and DNA methylation6. Because STAT activation of gene expression is both rapid and transient, it requires specific mechanisms for modulating the chromatin template at STAT-dependent gene loci. To define these mechanisms, we characterize the histone modifications and the enzymatic activities that generate them at gene loci that respond to STAT signaling. This protocol describes chromatin immunoprecipitation, a method that is valuable for the study of STAT signaling to chromatin in activated gene expression.

Chromatin Immunoprecipitation ChIP to Assay Dynamic Histone Modification in Activated Gene Expression in Human Cells

This protocol describes how chromatin immunoprecipitation (ChIP) is used to study the dynamic alterations to the chromatin template that regulate transcription induced by a signal transduction pathway.

在激活人类细胞中的基因表达的动态检测组蛋白修饰染色质免疫沉淀（ChIP）

In response to a variety of extracellular ligands, the STAT (signal transducer and activator of transcription) transcription factors are rapidly recruited ...

生物学

Genome-wide Analysis using ChIP to Identify Isoform-specific Gene Targets

Recruitment of transcriptional and epigenetic factors to their targets is a key step in their regulation. Prominently featured in recruitment are the protein domains that bind to specific histone modifications. One such domain is the plant homeodomain (PHD), found in several chromatin-binding proteins. The epigenetic factor RBP2 has multiple PHD domains, however, they have different functions (Figure 4). In particular, the C-terminal PHD domain, found in a RBP2 oncogenic fusion in human leukemia, binds to trimethylated lysine 4 in histone H3 (H3K4me3)1. The transcript corresponding to the RBP2 isoform containing the C-terminal PHD accumulates during differentiation of promonocytic, lymphoma-derived, U937 cells into monocytes2. Consistent with both sets of data, genome-wide analysis showed that in differentiated U937 cells, the RBP2 protein gets localized to genomic regions highly enriched for H3K4me33. Localization of RBP2 to its targets correlates with a decrease in H3K4me3 due to RBP2 histone demethylase activity and a decrease in transcriptional activity. In contrast, two other PHDs of RBP2 are unable to bind H3K4me3. Notably, the C-terminal domain PHD of RBP2 is absent in the smaller RBP2 isoform4. It is conceivable that the small isoform of RBP2, which lacks interaction with H3K4me3, differs from the larger isoform in genomic location. The difference in genomic location of RBP2 isoforms may account for the observed diversity in RBP2 function. Specifically, RBP2 is a critical player in cellular differentiation mediated by the retinoblastoma protein (pRB). Consistent with these data, previous genome-wide analysis, without distinction between isoforms, identified two distinct groups of RBP2 target genes: 1) genes bound by RBP2 in a manner that is independent of differentiation; 2) genes bound by RBP2 in a differentiation-dependent manner.
To identify differences in localization between the isoforms we performed genome-wide location analysis by ChIP-Seq. Using antibodies that detect both RBP2 isoforms we have located all RBP2 targets. Additionally we have antibodies that only bind large, and not small RBP2 isoform (Figure 4). After identifying the large isoform targets, one can then subtract them from all RBP2 targets to reveal the targets of small isoform. These data show the contribution of chromatin-interacting domain in protein recruitment to its binding sites in the genome.

Here we are presenting a chromatin immunoprecipitation (ChIP) procedure for genome-wide location analysis of protein isoforms that differ in a histone-binding domain. We are applying it to ChIP-Seq analysis to identify the targets of the KDM5A/JARID1A/RBP2 histone demethylase.

采用芯片识别亚型特定的靶基因的全基因组分析

Recruitment of transcriptional and epigenetic factors to their targets is a key step in their regulation. Prominently featured in recruitment are the ...

Repressing Gene Transcription by Redirecting Cellular Machinery with Chemical Epigenetic Modifiers

Regulation of chromatin compaction is an important process that governs gene expression in higher eukaryotes. Although chromatin compaction and gene expression regulation are commonly disrupted in many diseases, a locus-specific, endogenous, and reversible method to study and control these mechanisms of action has been lacking. To address this issue, we have developed and characterized novel gene-regulating bifunctional molecules. One component of the bifunctional molecule binds to a DNA-protein anchor so that it will be recruited to an allele-specific locus. The other component engages endogenous cellular chromatin-modifying machinery, recruiting these proteins to a gene of interest. These small molecules, called chemical epigenetic modifiers (CEMs), are capable of controlling gene expression and the chromatin environment in a dose-dependent and reversible manner. Here, we detail a CEM approach and its application to decrease gene expression and histone tail acetylation at a Green Fluorescent Protein (GFP) reporter located at the Oct4 locus in mouse embryonic stem cells (mESCs). We characterize the lead CEM (CEM23) using fluorescent microscopy, flow cytometry, and chromatin immunoprecipitation (ChIP), followed by a quantitative polymerase chain reaction (qPCR). While the power of this system is demonstrated at the Oct4 locus, conceptually, the CEM technology is modular and can be applied in other cell types and at other genomic loci.

Regulation of the chromatin environment is an essential process required for proper gene expression. Here, we describe a method for controlling gene expression through the recruitment of chromatin-modifying machinery in a gene-specific and reversible manner.

用化学表观修饰因子重定向细胞机械抑制基因转录

Regulation of chromatin compaction is an important process that governs gene expression in higher eukaryotes. Although chromatin compaction and gene ...

生物工程

In Vitro Differentiation Model of Human Normal Memory B Cells to Long-lived Plasma Cells

Plasma cells (PCs) secrete large amounts of antibodies and develop from B cells that have been activated. PCs are rare cells located in the bone marrow or mucosa and ensure humoral immunity. Due to their low frequency and location, the study of PCs is difficult in human. We reported a B to PC in vitro differentiation model using selected combinations of cytokines and activation molecules that allow to reproduce the sequential cell differentiation occurring in vivo. In this in vitro model, memory B cells (MBCs) will differentiate into pre-plasmablasts (prePBs), plasmablasts (PBs), early PCs and finally, into long-lived PCs, with a phenotype close to their counterparts in healthy individuals.&#160;We also built an open access bioinformatics tools to analyze the most prominent information from GEP data related to PC differentiation. These resources can be used to study human B to PC differentiation and in the current study, we investigated the gene expression regulation of epigenetic factors during human B to PC differentiation.

Using multi-step culture systems, we report an in vitro B cell to plasma cell differentiation model.

人正常记忆 b 细胞与长寿浆细胞体外分化模型

Plasma cells (PCs) secrete large amounts of antibodies and develop from B cells that have been activated. PCs are rare cells located in the bone marrow or ...

Study of Dendritic Cell Development by Short Hairpin RNA-Mediated Gene Knockdown in a Hematopoietic Stem and Progenitor Cell Line In vitro

Dendritic cells (DCs) are important antigen-presenting cells that connect innate and adaptive immune responses. DCs are heterogeneous and can be divided into conventional DCs (cDCs) and plasmacytoid DCs (pDCs). cDCs specializes in presenting antigens to and activate na&#239;ve T cells. On the other hand, pDCs can produce large quantities of type I interferons (IFN-I) during viral infection. The specification of DCs occurs at an early stage of DC progenitors in the bone marrow (BM) and is defined by a network of transcription factors (TFs). For example, cDCs highly express ID2, while pDCs highly express E2-2. Since more and more subsets of DCs are being identified, there is a growing interest in understanding specific TFs controlling DC development. Here, we establish a method to screen TFs critical for DCs differentiation in vitro by delivering lentivirus carrying short hairpin RNA (shRNA) into an immortalized hematopoietic stem and progenitor cell (iHSPCs) line. After the selection and in vitro differentiation, cDC and pDC potential of the stable knockdown cell lines are analyzed by flow cytometry. This approach provides a platform to identify genes potentially governing DC fates from progenitors in vitro.