这项工作由儿童癌症研究基金（CCRF）和NIH R01 AI146009到B.S..M资助。

从健康献血者那里获得的白血病样本是从当地血库获得的。此处描述的所有实验均被确定为豁免供机构审查委员会 （IRB） 研究，并经明尼苏达大学机构生物安全委员会 （IBC） 批准。
注:所有实验都是按照血液传播病原体的普遍预防措施进行的，采用无菌/无菌技术和适当的生物安全2级设备。
1. 为B细胞扩张介质准备补充剂
<ol><li>将 CpG 寡核苷酸重组为浓度为 1 毫克/mL。</li>
	<li>将 CD40 配体 （CD40L） 重组为浓度为 100μg/mL。</li>
	<li>重组重组人类IL-10（rhIL-10），浓度为50微克/mL。</li>
	<li>重组重组人类IL-15（rhIL-15），浓度为10微克/mL。 注意:将每种补充剂保持在-20°C至-80°C的小参考中长达6个月。</li>
</ol>2. 准备基础介质
<ol><li>结合B细胞基底介质与5%（v/v）介质补充体外免疫细胞扩张（如CTS免疫细胞SR）和1%（v/v）青霉素和链霉素。</li>
	<li>使用 0.22μm 滤芯适配器将基底介质过滤消毒到消毒瓶中。</li>
	<li>将基底介质保持在 4 °C，最长达 1 个月。</li>
</ol>3. 准备B细胞扩展介质
<ol><li>将基底介质所需的量转移到无菌容器中，以 5 × 105 细胞/mL 培养 B 细胞。</li>
	<li>以 1 μg/mL CpG、100 ng/mL CD40L、50 ng/mL rhIL-10 和 10 ng/mL rhIL-15 补充基础介质。</li>
	<li>使用 0.22 μm 过滤器过滤 B 细胞扩展介质。</li>
	<li>在 37 °C、5% CO2、 湿度至少 30 分钟之前，在组织培养孵化器中平衡 B 细胞膨胀介质。 注意:准备新鲜的B细胞扩展介质，使用一天。 不要 准备 B 细胞扩展介质使用多天。这种介质配方鼓励记忆B细胞的增殖，并激活原发性人类B细胞。</li>
</ol>4. 人类B细胞净化和扩张
注:按照制造商的指示，在温度控制的冷冻容器中加入 99–100% 异丙醇，并在开始第 4.1 步之前将冷冻容器冷却在 4 °C。
<ol><li>从白血病样本中分离 PBMC
	<ol>
<li>将白血病样本（约 8–10 mL）转移到无菌的 50 mL 锥形管中。</li>
		<li>将体积调高至 35 mL，无菌 1x 磷酸盐缓冲盐水 （PBS）。</li>
		<li>在 15 mL 密度梯度介质上仔细分层 35 mL leuka 磷化样本。</li>
		<li>离心机在500× 克 25分钟没有刹车，去除等离子层，而不会干扰抛光外套（PBMC层），从接口收集PCBCs，并转移到一个新的无菌50mL圆锥管。</li>
		<li>将 PBMC 与 1 倍 PBS 一起提起 50 mL。</li>
		<li>离心机在500× 克 5分钟没有刹车。在不干扰 PBMC 颗粒的情况下取出超自然，PBMC 颗粒可能呈红色。</li>
		<li>加入7mL的铵-氯化钾-溶解缓冲器，移液器3次混合良好，并在室温下（RT）孵育3分钟。</li>
		<li>将音量调高至 50 mL，并配备 1 倍 PBS。</li>
		<li>离心机在400× 克 5分钟低阻力制动器。在不干扰颗粒的情况下取出超自然。弹丸应该看起来粉红色或白色。 注意:要继续培养刚分离的 B 细胞，请在开始 B 细胞分离之前准备 B 细胞扩展介质（步骤 4.2）。</li>
	</ol></li>
	<li>使用人类初级 B 细胞负隔离套件从 PBMC 中分离 B 细胞
	<ol>
<li>在隔离缓冲区中将 PBMC 恢复为浓度为 5 × 107 个单元/mL。 注意:如果 PBMC 总数小于 5 × 107 个单元，请缩小隔离缓冲器的体积，以保持 5 × 107 个单元格/mL。细胞悬架的最小和最大体积分别为 0.25 mL 和 8 mL。</li>
		<li>将高达 8 mL（5 × 107 个电池/mL）转移到带盖子的无菌聚丙烯圆底管中。</li>
		<li>在 PBMC 中添加 50μL/mL 的鸡尾酒增强剂。</li>
		<li>在 PBMC 中加入 50μL/mL 的隔离鸡尾酒，盖上管子，然后倒置 2~3 次混合。</li>
		<li>在 RT 孵化 5 分钟;在孵化的第4 分钟，涡流磁性微珠至少30秒。</li>
		<li>每 1 mL PBMC 传输 50μL 的磁性微珠，封盖管子，并倒置 2–3 次混合。</li>
		<li>使用隔离缓冲区充值至 10 mL，并轻轻上下 2–3 次移液器。</li>
		<li>将管子放在磁站中，在 RT 孵化 3 分钟。</li>
		<li>将磁铁和管子保持在一起，在一个动作中，将磁铁和管子倒在一起，将细胞悬架倒入新管中。丢弃旧管子。</li>
		<li>重复步骤 4.2.8（将孵化时间缩短到 2 分钟），并将 B 细胞悬架倒入干净的圆锥形管中。</li>
		<li>富集的 B 细胞已准备好使用。如果立即使用细胞，请继续使用第 4.3 节（人类 B 细胞扩展）。通过流动细胞学（可选）检查分离 B 细胞的纯度。如果细胞在使用前要冻结，请继续踩踏 4.2.12。</li>
		<li>要冻结B细胞，离心机在400× 克 5分钟，并丢弃超自然分子，而不会干扰颗粒。</li>
		<li>将细胞在冷冻介质中以107 个细胞/mL进行恢复，并引用1mL/低温。</li>
		<li>将冷冻容器放在冰冷的冷冻容器中，并在 -80 °C 过夜;然后，将冷冻低温转移到液氮罐中:保持冷冻细胞长达1年。 注:分离B细胞的预期产量占全氟辛起伏碳化合物总量的2%-8%，生存能力为95%-99%。</li>
	</ol></li>
	<li>人类B细胞扩张 注意:如果使用刚分离的 B 细胞，请跳过步骤 4.3.1– 4.3.5。数数细胞并将所需细胞数转移到无菌圆锥管中，并继续执行步骤 4.3.6。<ol>
<li>在解冻B细胞之前，在水浴中预热胎儿牛血清（FBS）。准备20mL的B细胞膨胀介质，转移到T25烧瓶，并在组织培养孵化器中预平衡介质（在37°C，5%CO2，湿度），至少15分钟使用前。</li>
		<li>在37°C的水浴中解冻B细胞。在等待时，将 2 毫升预热 FBS 转移到无菌的 15 mL 圆锥管中。</li>
		<li>B细胞完全解冻后，立即在样品中加入1毫升预热的FBS。在 RT 孵化 1 分钟。</li>
		<li>轻轻移液器，以补充样品，并转移整个体积，下降，到一个圆锥管含有2毫升预热的FBS。</li>
		<li>将体积调高至 15 mL，带无菌 1 倍 PBS、盖，并轻轻地将管子倒置 2–3 倍。</li>
		<li>离心机在 400 × g 5 分钟。</li>
		<li>在不干扰细胞颗粒的情况下丢弃超细剂，用1mL的预平衡B细胞膨胀介质补充细胞颗粒，并计算细胞数。总细胞数应约为107 个单元格。</li>
		<li>将细胞转移到含有 20 mL 预平衡 B 细胞膨胀介质的烧瓶中。细胞的最终浓度应约为5×105 细胞/mL。</li>
		<li>将烧瓶垂直孵化在组织培养孵化器中。</li>
		<li>通过将 B 细胞的整个体积转移到无菌圆锥管中，并重复步骤 4.3.6–4.3.9，每 2 天完全刷新一次扩展介质。 注:T25烧瓶可容纳10-20 mL的介质:T75 烧瓶可容纳高达 20–60 mL 的介质。</li>
	</ol></li>
</ol>5. 初级人类B细胞工程
<ol><li>工程师 B 细胞在扩展/激活后以 48 ± 2 h 的速度工作，以获得最佳效果。准备B细胞膨胀介质，将介质的1mL放入48井组织培养板中，并在使用前至少15分钟在组织培养孵化器中预平衡。 注意:在为感兴趣的基因设计CRISPR/Cas9 sgRNA时，请按照下面概述的步骤操作。 - 使用在线工具11设计 sgRNA 。 - 在蛋白质的所有同位素中常见的外体上设计sgRNA。 - 从信誉良好的公司订购化学改装的 sgRNA。 -sgRNA通常以嗜血的形式出现:在无菌 DNase/无鼻塞三叶草 EDTA （TE） 缓冲区中重组 sgRNA，浓度为 1 μg/μL。</li>
	<li>通过将化学改性 sgRNA 的 1 μL （1μg/μL） 与化学改性链 球菌 Cas 9 （S.p. Cas9） 核糖核酸混合，准备 CRISPR/Cas9 变种基板。要进行控制，请使用 1 μL 的 TE 缓冲区，而不是 sgRNA。 注:当 CD19 sgRNA 用于基因 KO 实验时，请参阅 图 2 了解结果。当 AAVS1 sgRNA 用于基因 KI 实验时，请参阅 图 5 了解结果。<ul>
<li>将所有组件保留在冰上。</li>
	</ul></li>
	<li>轻轻混合，将每个反应的 2.5μL CRISPR/Cas9 变速基板传输到 0.2 mL 8 管条的管中;放在RT。</li>
	<li>打开核燃机（电极器），并准备 如表1所示的转染试剂。 注意:如果必要的话，把所有试剂都放在冰上，这是暂停的好步骤。准备恢复实验时，从冰中取出所有试剂。当使用 S.p. Cas9 蛋白质时，研究者 必须 通过将 1μg sgRNA 与 5 μg S.p. Cas9 蛋白质混合，避免任何气泡，并在 RT 中孵育混合物至少 20 分钟，然后再使用以获得最佳效果。</li>
	<li>计数并转移10个6 B细胞，每个转染反应到无菌圆锥管。</li>
	<li>将体积调高至 15 mL，无菌 1 倍 PBS，离心机在 400 × g 下 5 分钟。在等待时，准备主细胞转染试剂（表2），并放在RT。</li>
	<li>丢弃超自然的，而不会干扰细胞颗粒。</li>
	<li>用 10 mL 的无菌 1 倍 PBS 和离心机在 400 × g 下重新注入细胞颗粒 5 分钟。</li>
	<li>完全丢弃超纳特，而不会干扰细胞颗粒。</li>
	<li>每106 B细胞将0.5微克化学改性GFP mRNA（作为传染报告器）转移到细胞颗粒（可选）。</li>
	<li>每10个6B 细胞用20μL原细胞传染试剂补充细胞颗粒;通过管道轻轻混合5-6次。将 20.5 μL 的每次转染反应转移到包含 CRISPR/Cas9 转染基板 2.5 μL 的 8 管条的 0.2 mL 管中。</li>
	<li>向上和向下一 次 将整个音量 （23 μL） 混合并传输到传输库维特。轻轻盖住并轻敲长凳上的绒布，以确保液体覆盖库维特的底部。</li>
	<li>在核细胞核上使用人类原生B细胞协议进行转染。 注:核效应器（电容器）可放置在组织培养罩外使用。盖上小面包，确保不育。</li>
	<li>将电波化细胞在 RT 的库维特中休息 15 分钟。</li>
	<li>将预平衡 B 细胞膨胀介质的 80 μL 从组织培养板转移到库维特的转染反应中。将小面包放在组织培养孵化器中30分钟。</li>
	<li>轻轻移液几次，将样品的整个体积从库维特混合并转移到含有 1 mL B 细胞膨胀介质的 48 井组织培养板的适当井中。细胞的最终浓度应该是106 个细胞/mL。</li>
	<li>如果进行基因 KI 实验， 将 rAAV6 向量为 500，000 多种感染的载体转移到含有电波酸细胞的适当井中（大约 45 分钟后电波）。请参阅 图 4A中的示例 rAAV6 矢量构造。 注:例如:在没有CRISPR/Cas9或rAAV6矢量的情况下，对照样品将被电波解。仅矢量样品将电波化，无需 CRISPR/Cas9，然后与 rAAV6 矢量一起转导。KI 样品将用 CRISPR/Cas9 进行电波化，然后用 rAAV6 矢量进行转导。rAAV6 必须包含 HDR 目标 DSB 站点的上下同源臂。</li>
	<li>将盘子放在湿度为 37 °C 和 5% CO2 的组织培养孵化器中。</li>
	<li>计算细胞并记录工程后第 1 天的可行性。</li>
	<li>通过计算细胞，然后将整个细胞体积转移到一个干净的 1.5 mL 微中微富格管中，每 2 天刷新一次 B 细胞扩展介质。离心机在 400 × g 5 分钟， 并丢弃超母体， 而不会干扰颗粒。用100μL的新鲜B细胞膨胀介质补充细胞，并转移到24井组织培养板的井中。提起中等体积，在 5 × 105 细胞/mL 时实现最终细胞浓度。 注:48井板可容纳1mL中/井:一个24井板可以容纳高达2 mL中/井:12井板可容纳4mL中/井，6井板可容纳8mL中/井。</li>
	<li>允许工程细胞在进行下游分析（如流细胞学分析、TIDE 分析和交界 PCR）之前至少膨胀 5 天。</li>
</ol>

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O., Porteus, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CRISPR-mediated+integration+of+large+gene+cassettes+using+AAV+donor+vectors.">CRISPR-mediated integration of large gene cassettes using AAV donor vectors.</a> Cell Reports. 20 (3), 750-756 (2017).</li><li>Shirley, J. L., de Jong, Y. P., Terhorst, C., Herzog, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immune+responses+to+viral+gene+therapy+vectors.">Immune responses to viral gene therapy vectors.</a> Molecular Therapy. 28 (3), 709-722 (2020).</li><li>Martino, A. T., Markusic, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immune+response+mechanisms+against+AAV+vectors+in+animal+models.">Immune response mechanisms against AAV vectors in animal models.</a> Molecular Therapy - Methods and Clinical Development. 17, 198-208 (2020).</li></ol>

以M.J.J、K.L.和B..M S.为发明人，对制造和使用基因组编辑的B细胞的方法提出了专利。B.S..M是伊姆穆索夫特公司的顾问，并拥有该公司的股票。伊姆穆索夫特公司赞助了B.S..M实验室的研究。

直到最近9月10日，人类B细胞的精确基因组工程一直具有挑战性。我们以前曾发布过使用CRISPR/Cas9来设计原始人类B细胞9的协议。在这里，我们概述了改进的B细胞隔离，扩展和工程协议，以允许高效的KO CD19或敲击EGFP。
在这里，我们证明，我们的扩展协议允许在培养中快速扩展B细胞，在7天内扩展高达44倍（<st...

B细胞是来自造血干细胞的淋巴细胞系的子群。他们在适应性幽默免疫系统中发挥关键作用，产生大量的抗体来应对免疫挑战1。B细胞也是记忆B细胞和最终分化的长寿血浆细胞的前体，从而提供持久的幽默免疫力2。特别是等离子体细胞，在免疫细胞中独一无二，它们能够产生大量的特定抗体，同时存活多年或几十年。此外，从外周血液隔离的便利性使B细胞血统成为新细胞疗法4的极好候选者。
以前，随机整合方法，如那些使用扁桃体载体或睡美人转子，已经被用来工程B细胞的转基因传递和表达5，6，7，8。然而，这些方法的非特异性使得研究B细胞中特定基因的生物功能变得困难，并具有在治疗环境中插入突变和可变转基因表达和/或沉默的固有风险。
CRISPR/Cas9系统是一个强大的基因组工程工具，使研究人员能够精确编辑许多物种中各种细胞的基因组。最近，包括我们自己的两组已经成功地开发出了原始人类B细胞9、10的外活扩张和靶向基因组工程方法。我们将描述从白血病样本中净化原人类B细胞的过程。之后，我们将描述我们更新的 B 细胞扩展和激活孤立 B 细胞的协议。然后，我们将描述一个通过电波将CRISPR/Cas9 mRNA与CD19 sgRNA一起引入激活的B细胞，从而敲除分化19（CD19）的簇、特定的B细胞受体和B细胞的标志的过程。
Cas9 mRNA 被翻译并绑定到 CD19 sgRNA，以形成 CRISPR/Cas9-sgRNA 核糖核蛋白复合物 （RNP）。随后，复合物中的sgRNA导致Cas9在基因exon2上的目标序列中产生双链断裂（DSB）。细胞将通过引入或删除核苷酸来修复DSB，导致帧移位突变并导致基因被消灭。然后，我们将用流细胞测量测量CD19的损失，并通过分解（TIDE）分析对英德尔的形成进行跟踪分析。
然后，我们将描述使用CRISPR/Cas9与重组AAV6载体（rAAV6，同源定向修复（HDR）的捐赠模板）一起，以调解在腺相关病毒集成站点1（AAVS1）基因中插入增强型绿色荧光蛋白（EGFP） 的现场特定插入过程。AAVS1基因是一种没有已知生物功能的活性球体，也是人类基因组上的AAV病毒整合站点:因此，它被认为是基因组工程的"安全港"。在这里，我们报告说，B细胞的膨胀和激活允许在7天的培养中扩大44倍（图1）。B细胞的电扩散显示，在24小时后，整体细胞健康（图2A）略有下降。对 CD19 标记（图 2B）的分散图分析显示编辑的单元块（图 2C）减少高达83%。
TIDE对色谱仪（图3A）的分析显示，英德尔的百分比与流动细胞仪（图3B）所导致的蛋白质损失相似。KI实验的流动细胞学分析表明，接受AAV载体（图4）的细胞与RNP一起表示高达64%的EGFP阳性细胞（图5A），随后通过结点聚合酶链反应（PCR）（图5B）显示成功整合。细胞计数显示，所有样本在工程后3天内迅速恢复（图5C）。

<table>
	<tbody>
		<tr>
			<td>Alt-R S.p. Cas9 Nuclease V3 protein, 500 ug</td>
			<td>IDT</td>
			<td>1081059</td>
			<td>smaller size is also available</td>
		</tr>
		<tr>
			<td>Amaxa P3 primary cell 4D- Nucleofector X Kit S (32 RCT)</td>
			<td>Lonza</td>
			<td>V4XP-3032</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium-chloride-potassium (ACK) lysing buffer</td>
			<td>Quality Biological</td>
			<td>118-156-101</td>
			<td></td>
		</tr>
		<tr>
			<td>CleanCap chemically modified Cas9 mRNA</td>
			<td>Trilink Biotechnology</td>
			<td>L-7206-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>CpG ODN 2006 (ODN 7909) 5 mg</td>
			<td>Invivogen</td>
			<td>TLRL-2006-5</td>
			<td>different sizes available</td>
		</tr>
		<tr>
			<td>Cryostor CS10, 100 mL</td>
			<td>STEMCELL Technology</td>
			<td>7930</td>
			<td></td>
		</tr>
		<tr>
			<td>CTS Immune Cell SR</td>
			<td>Thermo Fisher Scientific</td>
			<td>A2596101</td>
			<td></td>
		</tr>
		<tr>
			<td>EasySep human B cells isolation kit</td>
			<td>STEMCELL Technology</td>
			<td>17954</td>
			<td></td>
		</tr>
		<tr>
			<td>eBioscience fixable viability dye eFlour 780</td>
			<td>eBiosciences</td>
			<td>65-0865-14</td>
			<td></td>
		</tr>
		<tr>
			<td>Excellerate B cell media, Xeno-free</td>
			<td>R&#38;D Systems</td>
			<td>CCM031</td>
			<td>B-cell basal medium</td>
		</tr>
		<tr>
			<td>Falcon 14 mL Polypropylene Round-bottom Tube</td>
			<td>Corning</td>
			<td>352059</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>R&#38;D Systems</td>
			<td>S11550</td>
			<td>For thawing B cells only</td>
		</tr>
		<tr>
			<td>Ficoll-Paque Plus</td>
			<td>GE Healthcare</td>
			<td>17-1440-03</td>
			<td></td>
		</tr>
		<tr>
			<td>GeneMate SnapStrip&#174; 8-Strip 0.2 mL PCR Tubes with Individual Attached Dome Caps</td>
			<td>BioExpress</td>
			<td>T-3035-1 / 490003-692</td>
			<td></td>
		</tr>
		<tr>
			<td>Hyclone 0.0067M PBS (No Ca, No Mg) or 1x PBS</td>
			<td>GE lifesciences</td>
			<td>SH30256.01</td>
			<td></td>
		</tr>
		<tr>
			<td>Lonza 4D Nucleofector core unit</td>
			<td>Lonza</td>
			<td>AAF-1002B</td>
			<td></td>
		</tr>
		<tr>
			<td>Lonza 4D Nucleofector X unit</td>
			<td>Lonza</td>
			<td>AAF-1002X</td>
			<td></td>
		</tr>
		<tr>
			<td>Mega CD40 Ligand</td>
			<td>Enzo Life Sciences</td>
			<td>ALX-522-110-C010</td>
			<td></td>
		</tr>
		<tr>
			<td>Mr. Frosty</td>
			<td>Sigma-Aldrich</td>
			<td>C1562-1EA</td>
			<td>For freezing cells</td>
		</tr>
		<tr>
			<td>Pen/Strep 100X</td>
			<td>Sigma-Aldrich</td>
			<td>TMS-AB2-C</td>
			<td></td>
		</tr>
		<tr>
			<td>PerCP anti-human CD19 clone HIB19</td>
			<td>biolegend</td>
			<td>302228</td>
			<td>smaller size is also available</td>
		</tr>
		<tr>
			<td>rAAV6 SA-GFP pakaging ( with our SA-GFP cassette see Figure 4.)</td>
			<td>Vigene Biosciences</td>
			<td>N/A</td>
			<td>large scale packaging, 1e13 GC/mL, 500 mL</td>
		</tr>
		<tr>
			<td>Recombinant human IL-10 protein 250 ug</td>
			<td>R&#38;D Systems</td>
			<td>217-IL-250</td>
			<td>different sizes available</td>
		</tr>
		<tr>
			<td>Recombinant human IL-15 protein 25 ug</td>
			<td>R&#38;D Systems</td>
			<td>247-ILB-025</td>
			<td>different sizes available</td>
		</tr>
		<tr>
			<td>The Big Easy EasySep Magnet</td>
			<td>STEMCELL Technology</td>
			<td>18001</td>
			<td>different sizes available</td>
		</tr>
		<tr>
			<td>Tris-EDTA (TE) buffer</td>
			<td>Fisher Scientific</td>
			<td>BP2476100</td>
			<td></td>
		</tr>
	</tbody>
</table>

genome engineering of primary human b cells using crispr/cas9

B细胞是从造血干细胞中提取的淋巴细胞，是适应性免疫系统幽默手臂的关键组成部分。他们之所以成为细胞疗法的候选者，是因为它们容易与周围血液隔离，体外扩张能力，以及体内寿命。此外，它们的正常生物功能——产生大量的抗体——可以用来表达大量的治疗性蛋白质，如抗感染的重组抗体，或用于治疗酶病变的酶。在这里，我们提供详细的方法，将原人类B细胞从外周血单核细胞（PBMCs）中分离，并在体外激活/扩大分离的B细胞。然后，我们演示了使用CRISPR/Cas9系统在B细胞中针对内源基因的部位特定KO所涉及的步骤。这种方法可以有效地利用各种基因的KO，用于研究感兴趣的基因的生物功能。然后，我们演示了使用CRISPR/Cas9系统以及重组的、与腺相关的病毒（rAAV）载体的步骤，以有效实现B细胞中转基因表达盒的现场特定集成。该协议共同提供了一个分步工程平台，可用于人类B细胞的初级研究基因的生物功能以及B细胞治疗的开发。

更新的扩展和激活协议使B细胞在7天内迅速膨胀到44倍（图1;n=3捐赠者）。在KO实验中，使用Trypan蓝色染色进行的B细胞计数显示，在24小时后的控制和CD19 KO样本中，超过80%的可行细胞的细胞恢复略有减少（图2A:p≥0.05，n = 3捐赠者）。这一结果表明，电波轻微影响整体B细胞健康。B细胞在输血后的第5天收集，用于流动细胞学和潮汐分析。控制和KO样本的代?...

Watch this Scientific Journal Video about Genome Engineering of Primary Human B Cells Using CRISPR/Cas9 at JoVE.com

Genome Engineering of Primary Human B Cells Using CRISPR/Cas9

在这里，我们为CRISPR/Cas9基础基因组工程提供详细的分步协议，用于基因敲除（KO）和敲击（KI），以研究B细胞中基因的生物功能和B细胞治疗的开发。

使用CRISPR/Cas9的人类B细胞基因组工程

B cells are lymphocytes derived from hematopoietic stem cells and are a key component of the humoral arm of the adaptive immune system. They make ...

genome-engineering-of-primary-human-b-cells-using-crisprcas9

Research

JoVE Journal

Biology

6.1K 次观看.  University of Minnesota. 在这里，我们为CRISPR/Cas9基础基因组工程提供详细的分步协议，用于基因敲除（KO）和敲击（KI），以研究B细胞中基因的生物功能和B细胞治疗的开发。

视频: Genome Engineering of Primary Human B Cells Using CRISPR/Cas9

Gross and Fine Dissection of Inner Ear Sensory Epithelia in Adult Zebrafish (Danio rerio)

Neurosensory epithelia in the inner ear are the crucial structures for hearing and balance functions. Therefore, it is important to understand the cellular and molecular features of the epithelia, which are mainly composed of two types of cells: hair cells (HCs) and supporting cells (SCs). Here we choose to study the inner ear sensory epithelia in adult zebrafish not only because the epithelial structures are highly conserved in all vertebrates studied, but also because the adult zebrafish is able to regenerate HCs, an ability that mammals lose shortly after birth. We use the inner ear of adult zebrafish as a model system to study the mechanisms of inner ear HC regeneration in adult vertebrates that could be helpful for clinical therapy of hearing/balance deficits in human as a result of HC loss.
Here we demonstrate how to do gross and fine dissections of inner ear sensory epithelia in adult zebrafish. The gross dissection removes the tissues surrounding the inner ear and is helpful for preparing tissue sections, which allows us to examine the detailed structure of the sensory epithelia. The fine dissection cleans up the non-sensory-epithelial tissues of each individual epithelium and enables us to examine the heterogeneity of the whole epithelium easily in whole-mount epithelial samples.

Gross and Fine Dissection of Inner Ear Sensory Epithelia in Adult Zebrafish Danio rerio

The inner ear sensory epithelium of adult zebrafish is a good model system for understanding the mechanisms of hair cell regeneration in adult vertebrates. This protocol demonstrates the fine dissection of the epithelia, through which we can get tissue samples for studying the regenerative events at cellular and subcellular levels.

格罗斯和精细解剖内耳感觉上皮细胞在成年斑马鱼（斑马鱼）

Neurosensory epithelia in the inner ear are the crucial structures for hearing and balance functions. Therefore, it is important to understand the ...

科研

生物学

Generation of Human CD40-activated B cells

CD40-activated B cells (CD40-B cells) have been identified as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cancer immunotherapy 1-3. Compared to Dendritic cells (DCs), the best characterized APC, CD40-B cells have several distinct biological and technical properties. Similar to DCs, B cells show an increased expression of MHC and co-stimulatory molecules (Fig.1b), exhibit a strong migratory capacity and present antigen presentation efficiently to T cells, after stimulation with interleukin-4 and CD40 ligand (CD40L). However, in contrast to immature or mature DCs, CD40-B cells express the full lymph node homing triad consisting of CD62L, CCR7/CXCR4, and leukocyte function antigen-1 (LFA1, CD11a/CD18), necessary for homing to secondary lymphoid organs (Fig.1a) 3. CD40-B cells can be generated without difficulties from very small amounts of peripheral blood which can be further expanded in vitro to very large amounts of highly-pure CD40-B cells (&gt;109 cells per patient) from healthy donors as well as cancer patients (Fig.1c,d) 1,4. 
In this protocol we demonstrate how to obtain fully activated CD40-B cells from human PBMC. Key molecules for the cell culture are CD40 ligand, interleukin-4 (IL-4) and cyclosporin A (CsA), which are replenished in a 3-4 day culture cycle. For laboratory purposes CD40-stimulation is provided by NIH/3T3 cells expressing recombinant human CD40 ligand (tCD40L NIH/3T3) 5. To avoid contamination with non-transfected cells, expression of the human CD40 ligand on the transfectants has to be checked regularly (Fig.2). 
After 14 days CD40-B cell cultures consist of more than 95% pure B cells and an expansion of CD40-B cells over 65 days is frequently possible without any loss of function 1, 4. CD40-B cells efficiently take up, process and present antigens to T cells 6. They do not only prime na&#970;ve, but also expand memory T cells 7,8. CD40-activated B cells can be used to study B-cell activation, differentiation and function. Moreover, they represent a promising tool for therapeutic or preventive vaccination against tumors 9.

In this video we present the ex vivo generation and expansion of human CD40-activated B cells (CD40-B) from peripheral blood mononuclear cells (PBMC) by stimulation with CD40 ligand and interleukin-4.

人CD40激活的B细胞的生成

CD40-activated B cells (CD40-B cells) have been identified as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cancer ...

Na&iuml;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

Over the last decade, several adult stem cell populations have been identified in human skin 1-4. The isolation of multipotent adult dermal precursors was first reported by Miller F. D laboratory 5, 6. These early studies described a multipotent precursor cell population from adult mammalian dermis 5. These cells--termed SKPs, for skin-derived precursors-- were isolated and expanded from rodent and human skin and differentiated into both neural and mesodermal progeny, including cell types never found in skin, such as neurons 5. Immunocytochemical studies on cultured SKPs revealed that cells expressed vimentin and nestin, an intermediate filament protein expressed in neural and skeletal muscle precursors, in addition to fibronectin and multipotent stem cell markers 6. Until now, the adult stem cells population SKPs have been isolated from freshly collected mammalian skin biopsies.
Recently, we have established and reported that a population of skin derived precursor cells could remain present in primary fibroblast cultures established from skin biopsies 7. The assumption that a few somatic stem cells might reside in primary fibroblast cultures at early population doublings was based upon the following observations: (1) SKPs and primary fibroblast cultures are derived from the dermis, and therefore a small number of SKP cells could remain present in primary dermal fibroblast cultures and (2) primary fibroblast cultures grown from frozen aliquots that have been subjected to unfavorable temperature during storage or transfer contained a small number of cells that remained viable 7. These rare cells were able to expand and could be passaged several times. This observation suggested that a small number of cells with high proliferation potency and resistance to stress were present in human fibroblast cultures 7.
We took advantage of these findings to establish a protocol for rapid isolation of adult stem cells from primary fibroblast cultures that are readily available from tissue banks around the world (Figure 1). This method has important significance as it allows the isolation of precursor cells when skin samples are not accessible while fibroblast cultures may be available from tissue banks, thus, opening new opportunities to dissect the molecular mechanisms underlying rare genetic diseases as well as modeling diseases in a dish.

Na&#239;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

We report a method to isolate na&#239;ve multipotent skin-derived precursor (SKP) cells from primary human fibroblast cultures. We show that these SKPs derived from fibroblast cultures share similar stem cell properties to the ones derived directly from human skin biopsies. These cells express the neural crest marker, nestin, in addition to the multipotent markers such as OCT4 and Nanog.

天真成人干细胞的分离从初级人类成纤维细胞培养

Over the last decade, several adult stem cell  populations have been identified in human skin 1-4.  The isolation of multipotent adult dermal precursors ...

In Vivo Modeling of the Morbid Human Genome using Danio rerio

Here, we present methods for the development of assays to query potentially clinically significant nonsynonymous changes using in vivo complementation in zebrafish. Zebrafish (Danio rerio) are a useful animal system due to their experimental tractability; embryos are transparent to enable facile viewing, undergo rapid development ex vivo, and can be genetically manipulated.1 These aspects have allowed for significant advances in the analysis of embryogenesis, molecular processes, and morphogenetic signaling. Taken together, the advantages of this vertebrate model make zebrafish highly amenable to modeling the developmental defects in pediatric disease, and in some cases, adult-onset disorders. Because the zebrafish genome is highly conserved with that of humans (~70% orthologous), it is possible to recapitulate human disease states in zebrafish. This is accomplished either through the injection of mutant human mRNA to induce dominant negative or gain of function alleles, or utilization of morpholino (MO) antisense oligonucleotides to suppress genes to mimic loss of function variants. Through complementation of MO-induced phenotypes with capped human mRNA, our approach enables the interpretation of the deleterious effect of mutations on human protein sequence based on the ability of mutant mRNA to rescue a measurable, physiologically relevant phenotype. Modeling of the human disease alleles occurs through microinjection of zebrafish embryos with MO and/or human mRNA at the 1-4 cell stage, and phenotyping up to seven days post fertilization (dpf). This general strategy can be extended to a wide range of disease phenotypes, as demonstrated in the following protocol. We present our established models for morphogenetic signaling, craniofacial, cardiac, vascular integrity, renal function, and skeletal muscle disorder phenotypes, as well as others.

In Vivo Modeling of the Morbid Human Genome using Danio rerio

Here, we present a systematic approach for developing physiologically relevant, sensitive and specific in vivo assays for interpreting variation in human pathology. Transient genetic manipulation via microinjection of WT and mutant human mRNA and morpholino (MO) antisense oligonucleotides harness the tractability of the developing zebrafish embryo to rapidly assay pathogenic mutations, especially, but not exclusively, in the context of human developmental disorders.

在体内病态的人类基因组建模使用斑马鱼

Here,  we present methods for the development of assays to query potentially clinically  significant nonsynonymous changes using in  vivo complementation ...

Mouse Genome Engineering Using Designer Nucleases

Transgenic mice carrying site-specific genome modifications (knockout, knock-in) are of vital importance for dissecting complex biological systems as well as for modeling human diseases and testing therapeutic strategies. Recent advances in the use of designer nucleases such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 system for site-specific genome engineering open the possibility to perform rapid targeted genome modification in virtually any laboratory species without the need to rely on embryonic stem (ES) cell technology. A genome editing experiment typically starts with identification of designer nuclease target sites within a gene of interest followed by construction of custom DNA-binding domains to direct nuclease activity to the investigator-defined genomic locus. Designer nuclease plasmids are in vitro transcribed to generate mRNA for microinjection of fertilized mouse oocytes. Here, we provide a protocol for achieving targeted genome modification by direct injection of TALEN mRNA into fertilized mouse oocytes.

Designer nucleases such as zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) can be used to modify the genome of mouse preimplantation embryos by triggering both the nonhomologous end joining (NHEJ) and homologous recombination (HR) pathways. These advances enable the rapid generation of mice with precise genetic modifications.

小鼠基因组工程使用Designer核酸酶

Transgenic mice carrying site-specific genome modifications (knockout, knock-in) are of vital importance for dissecting complex biological systems as well ...

Whole Cell Electrophysiology of Primary Cultured Murine Enterochromaffin Cells

Enterochromaffin (EC) cells in the gastrointestinal (GI) epithelium constitute the largest subpopulation of enteroendocrine cells. As specialized sensory cells, EC cells sense luminal stimuli and convert them into serotonin (5-hyroxytryptamine, 5-HT) release events. However, the electrophysiology of these cells is poorly understood because they are difficult to culture and to identify. The method presented in this paper outlines primary EC cell cultures optimized for single cell electrophysiology. This protocol utilizes a transgenic cyan fluorescent protein (CFP) reporter to identify mouse EC cells in mixed primary cultures, advancing the approach to obtaining high-quality recordings of whole cell electrophysiology in voltage- and current-clamp modes.

Enterochromaffin (EC) cells comprise a small subset of gastrointestinal epithelial cells. EC cells are electrically excitable and release serotonin, yet difficulties in culturing and identifying EC cells have limited physiological studies. The method presented here establishes a primary culture model amenable to examination of single EC cells by electrophysiology.

原代培养小鼠肠嗜铬细胞的全细胞电生理

Enterochromaffin (EC) cells in the gastrointestinal (GI) epithelium constitute the largest subpopulation of enteroendocrine cells. As specialized sensory ...

In Situ Monitoring of Transiently Formed Molecular Chaperone Assemblies in Bacteria, Yeast, and Human Cells

J-domain proteins (JDPs) form the largest and the most diverse co-chaperone family in eukaryotic cells. Recent findings show that specific members of the JDP family could form transient heterocomplexes in eukaryotes to fine-tune substrate selection for the 70 kDa heat shock protein (Hsp70) chaperone-based protein disaggregases. The JDP complexes target acute/chronic stress induced aggregated proteins and presumably help assemble the disaggregases by recruiting multiple Hsp70s to the surface of protein aggregates. The extent of the protein quality control (PQC) network formed by these physically interacting JDPs remains largely uncharacterized in vivo. Here, we describe a microscopy-based in situ protein interaction assay named the proximity ligation assay (PLA), which is able to robustly capture&#160;these transiently formed chaperone complexes in distinct cellular compartments of eukaryotic cells. Our work expands the employment of PLA from human cells to yeast (Saccharomyces cerevisiae) and bacteria (Escherichia coli), thus rendering an important tool to monitor the dynamics of transiently formed protein assemblies in both prokaryotic and eukaryotic cells.

Cognate J-domain proteins cooperate with the Hsp70 chaperone to assist in a myriad of biological processes ranging from protein folding to degradation. Here, we describe an in situ proximity ligation assay, which allows the monitoring of these transiently formed chaperone machineries in bacterial, yeast and human cells.

细菌、酵母和人类细胞中瞬态形成的分子茶龙组件的原位监测

J-domain proteins (JDPs) form the largest and the most diverse co-chaperone family in eukaryotic cells. Recent findings show that specific members of the ...

Silencing the Spark: CRISPR/Cas9 Genome Editing in Weakly Electric Fish

Electroreception and electrogenesis have changed in the evolutionary history of vertebrates. There is a striking degree of convergence in these independently derived phenotypes, which share a common genetic architecture. This is perhaps best exemplified by the numerous convergent features of gymnotiforms and mormyrids, two species-rich teleost clades that produce and detect weak electric fields and are called weakly electric fish. In the 50 years since the discovery that weakly electric fish use electricity to sense their surroundings and communicate, a growing community of scientists has gained tremendous insights into evolution of development, systems and circuits neuroscience, cellular physiology, ecology, evolutionary biology, and behavior. More recently, there has been a proliferation of genomic resources for electric fish. Use of these resources has already facilitated important insights with regards to the connection between genotype and phenotype in these species. A major obstacle to integrating genomics data with phenotypic data of weakly electric fish is a present lack of functional genomics tools. We report here a full protocol for performing CRISPR/Cas9 mutagenesis that utilizes endogenous DNA repair mechanisms in weakly electric fish. We demonstrate that this protocol is equally effective in both the mormyrid species Brienomyrus brachyistius and the gymnotiform Brachyhypopomus gauderio by using CRISPR/Cas9 to target indels and point mutations in the first exon of the sodium channel gene scn4aa. Using this protocol, embryos from both species were obtained and genotyped to confirm that the predicted mutations in the first exon of the sodium channel scn4aa were present. The knock-out success phenotype was confirmed with recordings showing reduced electric organ discharge amplitudes when compared to uninjected size-matched controls.

Here, a protocol is presented to produce and rear CRISPR/Cas9 genome knockout electric fish. Outlined in detail are the required molecular biology, breeding, and husbandry requirements for both a gymnotiform and a mormyrid, and injection techniques to produce Cas9-induced indel F0 larvae.

沉默的火花:CRISPR/Cas9基因组编辑在弱电鱼

Electroreception and electrogenesis have changed in the evolutionary history of vertebrates. There is a striking degree of convergence in these ...

Preparing and Injecting Embryos of Culex Mosquitoes to Generate Null Mutations using CRISPR/Cas9

Culex mosquitoes are the major vectors of several diseases that negatively impact human and animal health including West Nile virus and diseases caused by filarial nematodes such as canine heartworm and elephantasis. Recently, CRISPR/Cas9 genome editing has been used to induce site-directed mutations by injecting a Cas9 protein that has been complexed with a guide RNA (gRNA) into freshly laid embryos of several insect species, including mosquitoes that belong to the genera Anopheles and Aedes. Manipulating and injecting Culex mosquitoes is slightly more difficult as these mosquitoes lay their eggs upright in rafts rather than individually like other species of mosquitoes. Here we describe how to design gRNAs, complex them with Cas9 protein, induce female mosquitoes of Culex pipiens to lay eggs, and how to prepare and inject newly laid embryos for microinjection with Cas9/gRNA. We also describe how to rear and screen injected mosquitoes for the desired mutation. The representative results demonstrate that this technique can be used to induce site-directed mutations in the genome of Culex mosquitoes and, with slight modifications, can be used to generate null-mutants in other mosquito species as well.&#160;

Preparing and Injecting Embryos of Culex Mosquitoes to Generate Null Mutations using CRISPR/Cas9

CRISPR/Cas9 is increasingly used to characterize gene function in non-model organisms. This protocol describes how to generate knock-out lines of Culex pipiens, from preparing injection mixes, to obtaining and injecting mosquito embryos, as well as how to rear, cross, and screen injected mosquitoes and their progeny for desired mutations.

准备和注射 Culex 蚊子的胚胎，使用CRISPR/Cas9产生空突变

Culex mosquitoes are the major vectors of several diseases that negatively impact human and animal health including West Nile virus and diseases caused by ...

Construction of Homozygous Mutants of Migratory Locust Using CRISPR/Cas9 Technology

The migratory locust, Locusta migratoria, is not only one of the worldwide plague locusts that caused huge economic losses to human beings but also an important research model for insect metamorphosis. The CRISPR/Cas9 system can accurately locate at a specific DNA locus and cleave within the target site, efficiently introducing double-strand breaks to induce target gene knockout or integrate new gene fragments into the specific locus. CRISPR/Cas9-mediated genome editing is a powerful tool for addressing questions encountered in locust research as well as a promising technology for locust control. This study provides a systematic protocol for CRISPR/Cas9-mediated gene knockout with the complex of Cas9 protein and single guide RNAs (sgRNAs) in migratory locusts. The selection of target sites and design of sgRNA are described in detail, followed by in vitro synthesis and verification of the sgRNAs. Subsequent procedures include egg raft collection and tanned-egg separation to achieve successful microinjection with low mortality rate, egg culture,&#160;preliminary estimation of the mutation rate, locust breeding as well as detection, preservation, and passage of the mutants to ensure population stability of the edited locusts. This method can be used as a reference for CRISPR/Cas9 based gene editing applications in migratory locusts as well as in other insects.

This study provides a systematically optimized procedure of CRISPR/Cas9 ribonuclease-based construction of homozygous locust mutants as well as a detailed method for cryopreservation and resuscitation of the locust eggs.

基于CRISPR/Cas9技术构建迁徙蝗虫纯合突变体

The migratory locust, Locusta migratoria, is not only one of the worldwide plague locusts that caused huge economic losses to human beings but also an ...