Name: dextran enhances the lentiviral transduction efficiency of murine and human primary nk cells
Uploaded: 2018-01-15T12:30:00.000+00:00
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Description: Watch this Scientific Journal Video about Dextran Enhances the Lentiviral Transduction Efficiency of Murine and Human Primary NK Cells at JoVE.com

我们感谢露西亚 Sammarco 和她的露露的柠檬水站的灵感, 动力, 和支持。这项工作得到了部分由 NIH R01 AI102893 和 R01 CA179363 (克里希纳) 的支持;NHLBI-HL087951 (纳丹);NIH-CA151893-K08 (M.J.R.);1R01CA164225 (L. W);亚历克斯柠檬水站立基础 (克里希纳);委员会基金的 HRHM 计划 (克里希纳;纳丹;硕士 T);尼古拉斯家族基金会 (克里希纳);Gardetto 家族 (克里希纳);现代学者计划 (M.S.T.);现代希望在轮子 (纳丹);委员会基金 (M.S.T. 和克里希纳);儿童研究所, MCW (纳丹);凯西菲 Fogerty 奖 (M.J.R.)。

所有动物协议跟随了动物的人道和道德治疗并且由机构动物护理和用途委员会 (IACUC) 批准在威斯康辛医学学院的生物医学研究中心 (MCW), 密尔沃基, WI。使用人外周血单个核细胞 (PBMCs) 被批准的机构审查委员会 (IRB) 血液研究所的血液研究中心威斯康星州, 密尔沃基, WI。
1. 小鼠、细胞系和载体
<ol><li>从商业供应商那里获得 C57BL/6 的老鼠。保持在无病原体条件下的小鼠菌落, 并在6和12周的年龄段使用雌性和雄性小鼠。从 IRB 批准的来源获得去除人 PBMCs。</li>
	<li>麻醉在丙烯乙二醇 (1, 2-丙二醇, USP 等级) 中混合 20-30% v/v 异氟醚的动物, 以麻醉小鼠。将兽医软膏涂抹于眼部, 防止小鼠麻醉时干燥。</li>
	<li>对动物进行安乐, 在麻醉诱导后进行颈椎脱位。<ol>
<li>用一只手牢牢抓住尾巴的底座来抑制老鼠。在头骨的底部放置一支结实的棍子型钢笔或拇指和另一只手的手指, 放在颈部的后部。</li>
		<li>为了产生脱位, 推手抑制动物的头部向前, 并向下推, 而向后拉的手持有的尾巴基地。通过感觉宫颈组织分离来验证脱位的有效性。</li>
	</ol>
</li>
	<li>保持在室内/笼子里的动物至少5分钟, 并删除他们只有当呼吸活动是缺席或当有一个检测的心跳缺乏。</li>
	<li>从商业供应商那里获得 K562 和 YAC-1 细胞, 并在含有10% 热灭活 FBS 的 RPMI1640 培养基中维持它们。定期测试这些细胞系, 以排除支原体污染的可能性。</li>
</ol>2. 慢载体的制备和滴定
<ol><li>培养 5 x 106 293T 细胞过夜在 T75 cm2烧瓶中, 含有20毫升的 RPMI1640 培养基, 10% FBS, 100 U/毫升青霉素, 100 µg/毫升链霉素, 1 毫米丙酮酸钠, 5% 的7.5% 碳酸氢钠溶液, 0.001%β-基。将烧瓶放入37° c 的恒温箱中, 注入 5.2% CO2。</li>
	<li>用磷酸缓冲盐 (PBS) 中的胰蛋白酶 (0.025%)/EDTA (1 毫米) 获得293T 细胞。在 PBS 中加入5毫升的胰蛋白酶/EDTA, 在37° c 的孵化箱中孵育10分钟的烧瓶, 使293T 细胞脱离 T75 cm2烧瓶。<ol>
<li>收集分离的细胞, 并清洗两次在 PBS, 以消除任何痕迹的胰蛋白酶和 EDTA。用例计数单元格, 并将单元格数调整为每毫升100万单元格。</li>
	</ol>
</li>
	<li>染的293T 细胞18与3.95 µg 的 psPAX2, 1.32 µg pMD2G, 和5.26 µg pLEP-gfp 普洛 (生成 in-house 在 EF1alpha 的克隆 pWPI 启动子, 在 pLenti 的地方的巨细胞病毒的启动子,-gfp-普洛) 19.<ol>
<li>准备1毫升150毫米氯化钠加63µL 0.6 毫克/毫升亚 (PEI; 2.5万 kD, 线形)。混合好, 然后加入质粒和混合。在室温下孵育20分钟, 然后将其添加到细胞中。</li>
		<li>16 h post-transfection, 用新鲜培养基加4.5 毫米丁酸钠取代转染培养基。收获含有病毒 48 h post-transfection 的上清液, 用隔夜离心法浓缩 5000 x g。请参阅材料表。</li>
	</ol>
</li>
	<li>通过流式细胞仪 72 h post-transduction20进行串行稀释和检测, 确定使用293T 细胞的病毒滴。使用绿色荧光蛋白 (GFP) 的表达作为量化病毒滴的措施。</li>
</ol>3. 小鼠原代 NK 细胞的纯化和扩展
<ol><li>纯化小鼠原发性 NK 细胞21。简要地, 通过尼龙羊毛柱通过单细胞脾悬浮物, 以耗尽由 B 细胞和巨细胞组成的黏附人群。</li>
	<li>培养换药种群洗从尼龙羊毛柱, 其中含有 1000 u/毫升白细胞介素 (IL)-2 在 RPMI1640 培养基与 10% FBS, 100 U/毫升青霉素, 100 µg/毫升链霉素, 1 毫米丙酮酸钠, 5% 7.5% 碳酸氢钠溶液, 和0.001% β-基 (RPMI1640 完全介质)。</li>
	<li>改变这种培养基4天的烧瓶, 以消除换药 T 和 NKT 细胞;仍然附着在烧瓶中的细胞主要是 NK 细胞。添加20毫升新鲜 RPMI1640 完全培养基和 1000 U/毫升 IL-2, 以补充烧瓶。</li>
	<li>在7天通过流式细胞仪检测 nk 细胞培养物的纯度, 并使用具有95% 以上 CD3 -NK1.1+填充的准备工作。</li>
</ol>4. 人类原发性 NK 细胞的纯化与扩展
<ol><li>通过密度梯度分离 PBMCs, 从健康的人类志愿者的巴菲大衣。简要地, 仔细地35毫升的 half-diluted (与 HBSS) 细胞超过15毫升的密度梯度在一个50毫升规范管。离心机在 400 x g 为30分钟在20° c 在摇摆的桶转子没有闸。</li>
	<li>根据制造商的协议, 使用商用抗体阴性选择套件来纯化人类原代 NK 细胞。</li>
	<li>分离后, 通过免疫荧光分析测定 NK 细胞的纯度。用2µg/毫升 anti-CD3 和2µg/毫升 anti-CD56 抗体在4° c 下染色 nk 细胞, 20 min. 在细胞表面没有 CD3 和 CD56 存在的情况下, 用 PBS nk 细胞的数量来冲洗两次细胞。</li>
	<li>用抗体在4° c 下孵育20分钟的细胞制剂, 用 PBS 进行冲洗, 并在流式仪中进行分析。使用 NK 细胞准备与超过85% 纯度的基因转导化验20。</li>
</ol>5. 小鼠和人原发性 NK 细胞与慢的转导
<ol><li>在 0.5 x 105/毫升培养基中, 在有 GFP 慢上清的情况下, 在5处将小鼠或人类主 NK 细胞悬浮在24井板上, 10、20种感染 (莫伊) 和有 Pb (8 µg/毫升)、PS (8 µg/毫升) 或葡聚糖 (8 µg/毫升) 的存在。</li>
	<li>离心板在 1000 x g 为60分钟。</li>
	<li>没有迁上清液, 培养细胞过夜 (16-18 小时) 在37° c 孵化器注入 5.2% CO2.
</li>
	<li>在 IL-2 (300 U/毫升) 的情况下, 用10毫升 PBS 和重在2毫升完全 RPMI1640 培养基中洗涤。</li>
	<li>分别通过在不同的 effector-to-target 细胞比22中执行51铬 (Cr) 释放试验, 测试人和小鼠主要 NK 细胞对 K562 和 YAC-1 的杀伤作用。<ol>
<li>简要地, 给100万靶细胞50µCi 标记钠铬酸盐51Cr。在4小时的潜伏期, 他们吸收了51Cr 为细胞蛋白。在孵化结束时, 清洗细胞以移除任何未注册的标签。</li>
		<li>通过从目标细胞中的绝对、自发和实验释放的51Cr 的数量来计算特定肿瘤细胞的裂解。 注: pentaplicate 实验的具体溶解百分率的计算采用以下公式:% 特定的裂解 = ((51cr 平均实验释放- 51cr 平均自发释放)/(51cr 平均值最大释放- 51cr 平均自发释放)) х 100, 其中 "51cr 平均自发释放" 是51cr 释放的目标细胞在缺乏 NK 细胞和 "51cr 平均最大释放" 是51cr由2的盐酸 (HCl) 溶解后从靶细胞释放出来。</li>
	</ol>
</li>
	<li>在7天转 NK 细胞, 轻轻敲击烧瓶。<ol>
<li>通过涂层 96-好, 高蛋白吸收聚苯乙烯板, 2.5 µg/毫升浓度的 anti-NKG2D 单克隆在一夜之间激活滴定浓度的板结合 anti-NKG2D (A10) 抗体的收获细胞。</li>
		<li>在原代 NK 细胞加入前, 用100µL PBS 三次冲洗每一个井。</li>
	</ol>
</li>
	<li>收集文化清使用多通道吸管在16和 18 h 后之间定量细胞因子例如干扰素γ。使用与酶联免疫吸附试验 (ELISA) 试剂盒提供的重组细胞因子生成标准曲线。</li>
	<li>用 annexin-V/7-amino-actinomycin D (7-反倾销) 染色法测定 nk 细胞的存活率23 , 并利用流式细胞仪测定转化 nk 淋巴细胞中坏死细胞的百分比。<ol>
<li>为了进行这种化验, 在转导后四天内采集小鼠和人 NK 细胞, 用10毫升的冷 PBS 在 500 x g 分别洗涤两次, 每5分钟, 用蛋白 V (PE)/7-反倾销试剂盒孵育。</li>
	</ol>
</li>
	<li>使用适当的流式细胞仪软件对数据进行分析。选择活细胞种群并分析 GFP (FITC 通道) 的表达作为病毒转导的一种措施24。</li>
</ol>

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Biol. 246, 391-411 (2004).</li><li>Sutlu, T., 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=Inhibition+of+intracellular+antiviral+defense+mechanisms+augments+lentiviral+transduction+of+human+natural+killer+cells:+implications+for+gene+therapy.">Inhibition of intracellular antiviral defense mechanisms augments lentiviral transduction of human natural killer cells: implications for gene therapy.</a> Hum. Gene Ther. 23, 1090-1100 (2012).</li><li>Cornetta, K., Anderson, W. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protamine+sulfate+as+an+effective+alternative+to+polybrene+in+retroviral-mediated+gene-transfer:+implications+for+human+gene+therapy.">Protamine sulfate as an effective alternative to polybrene in retroviral-mediated gene-transfer: implications for human gene therapy.</a> J. Virol. Methods. 23, 187-194 (1989).</li><li>Toyoshima, K., Vogt, P. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhancement+and+inhibition+of+avian+sarcoma+viruses+by+polycations+and+polyanions.">Enhancement and inhibition of avian sarcoma viruses by polycations and polyanions.</a> Virology. 38, 414-426 (1969).</li><li>Jensen, M., 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=The+bi-specific+CD3+x+NCAM+antibody:+a+model+to+preactivate+T+cells+prior+to+tumour+cell+lysis.">The bi-specific CD3 x NCAM antibody: a model to preactivate T cells prior to tumour cell lysis.</a> Clin. Exp. Immunol. 134, 253-263 (2003).</li></ol>

提交人声称没有任何经济利益冲突。

本研究表明, 用葡聚糖作为阳离子聚合物剂, 可提高小鼠和人原代 NK 细胞的慢转导效率。此外, 其他阳离子剂, 如 Pb 或 PS, 对病毒载体向人类原发性 NK 细胞的传递无明显影响。在此之前, 人们已经证明, Pb 可以增强人类 T 细胞的基因转导17。然而, 这些结果表明, 无论是 Pb 还是 PS 在人类原代 NK 细胞上都没有类似的效率。在本研究中, Pb 对小鼠 NK 细胞的转导效果仅有轻度改善。结果?...

自然杀手 (NK) 细胞是先天免疫系统的主要淋巴细胞种群1。NK 细胞作为宿主免疫应答的一线防御者, 抗肿瘤和感染2,3,4。NK 细胞通过分泌强有力的细胞因子和趋化因子5在耐受性发展中起着重要作用。由于其靶向和消除肿瘤细胞的强大能力, 目前正在进行多项临床试验, 以评估供者派生的人类 NK 细胞作为癌症的过继免疫治疗6,7。与 T 细胞相比, NK 细胞的发育生物学尚未良好8。这种缺乏知识的部分原因是缺少有效的技术来提供老鼠或人类原发性 NK 细胞感兴趣的基因。由于这些原因, 大多数 NK 细胞的研究都是在细胞系中进行的, 而不是在原代细胞中进行。因此, 需要一个可靠和有效的协议, 传感器原代 NK 细胞与感兴趣的基因是至关重要的。
这项研究的总目标是制定一个一致的和可靠的方法, 其中主要人或小鼠 NK 细胞可以转与 lenti 或病毒。
早先的研究试图解决这个问题, 主要是利用了原代 NK 细胞的瞬时转化。这包括质粒转染9,10, eb 病毒-巴尔毒 (eb)/逆转录酶杂交载体11, 痘苗向量12,13, 和 Ad5/F35 嵌合体腺病毒载体14。尽管这些技术的效率很高, 但转导的瞬态性使得它们不适合长期利用转基因 NK 细胞。最近的一些研究已经使用逆转录病毒载体传感器 NK 细胞, 需要多个感染周期来达到一个可接受的基因表达水平11,15。与逆转录病毒载体不同, 慢载体可以利用宿主细胞核进口机械转移病毒 pre-integration 复合体进入细胞核。这是在 non-dividing 细胞 (包括原代 NK 细胞) 中复制病毒的一个主要限制因素。
不同的细胞表面受体和病毒粒子之间的相互作用允许病毒对细胞的吸收。病毒包膜蛋白及其同源宿主受体之间的初始接触可能会受到限制, 因为这两者之间存在潜在的负电荷。许多转导技术的基本原理是, 添加阳离子聚合物, 如凝聚 (Pb), 硫酸精蛋白 (PS), 或葡聚糖, 可以给细胞表面受体积极的电荷, 从而增强病毒包络的结合蛋白质.这将提高融合效率和吸收的病毒粒子的细胞16。虽然据报道, Pb 或 PS 可以改善 T 细胞的基因转移17, 他们的应用没有任何影响的转导效率的原代 NK 细胞。此外, 还没有对这些试剂使用原代 NK 细胞进行比较分析。本研究对三阳离子聚合物的转导效率进行了比较。结果表明, 在这三阳离子聚合物中, 只有葡聚糖能显著提高小鼠和人原代 NK 细胞的有效病毒转导。

<table><tbody><tr><td>Dextran</td><td>Sigma-Aldrich</td><td>90-64-91-9</td><td></td></tr><tr><td>polybrene (Pb)</td><td>Sigma-Aldrich</td><td>TR-1003</td><td></td></tr><tr><td>protamine sulfate (PS)</td><td>Sigma-Aldrich</td><td>p3369</td><td></td></tr><tr><td>Trypsin</td><td>Corning</td><td>25-052-CI</td><td></td></tr><tr><td>RPMI1640</td><td>Corning</td><td>10-040-CV</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>ATALANTA</td><td>S11150</td><td></td></tr><tr><td>Penicillin</td><td>Corning</td><td>30-001-CI</td><td></td></tr><tr><td>B-mercaptoethanol</td><td>SIGMA</td><td>M3148</td><td></td></tr><tr><td>sodium pyruvate</td><td>Corning</td><td>MT25000CI</td><td></td></tr><tr><td>Interferon gamma (IFN-&amp;gamma; )</td><td>eBioscience</td><td>14-7311-85</td><td></td></tr><tr><td>Propidium lodding staining solution</td><td>BD</td><td>51-66211E</td><td></td></tr><tr><td>Lipofectamine 3000</td><td>Thermo Fisher</td><td>L3000015</td><td></td></tr><tr><td>Isoflurane</td><td>PHOENIX</td><td>NDC 57319-559-05</td><td></td></tr><tr><td>NK cell negative selection kit</td><td>Stem Cell</td><td>19855</td><td></td></tr><tr><td>Yac-1</td><td>ATCC</td><td>TIB-160</td><td></td></tr><tr><td>K562</td><td>ATCC</td><td>CCL-243</td><td></td></tr><tr><td>Mice</td><td>Jakson</td><td>664</td><td></td></tr><tr><td>293T cells</td><td>ATCC</td><td>CRL-3216</td><td></td></tr><tr><td>T75 flasks</td><td>Cornnig</td><td>430641U</td><td></td></tr><tr><td>antibody-based negative selection kits</td><td>Stem Cell</td><td>19055</td><td></td></tr><tr><td>&lt;sup&gt;51&lt;/sup&gt;Chromium (Cr)-release assays</td><td>perkin elmer&#039;s</td><td>NEZ030</td><td></td></tr><tr><td>ELISA kits</td><td>Ebioscience</td><td>00-4201-56</td><td></td></tr><tr><td>Sodium Butyrate</td><td>Sigma</td><td>5887-5G</td><td></td></tr><tr><td>Linear polyethylenimine</td><td>polysciences</td><td>23966-2</td><td></td></tr><tr><td>Ficoll</td><td>GE Life Science</td><td>17-1440-03</td><td></td></tr><tr><td>HBSS</td><td>Corning</td><td>21-022-CV</td><td></td></tr></tbody></table>

dextran enhances the lentiviral transduction efficiency of murine and human primary nk cells

将特定基因的有效转导转化为自然杀伤细胞是一个主要的挑战。成功的 transductions 是关键的定义的作用基因的兴趣在发展, 分化和功能的 NK 细胞。最近的进展与嵌合抗原受体 (汽车) 的癌症免疫治疗强调需要一个有效的方法提供外源基因的效应淋巴细胞。慢介导的基因 transductions 对原人或小鼠 NK 细胞的效率仍然很低, 这是一个主要的限制因素。最近的进展, 使用阳离子聚合物, 如凝聚, 显示了改善基因转导效率的 T 细胞。然而, 这些产品未能改善 NK 细胞的转导效率。这项研究表明, 葡聚糖是一种支链多糖, 能显著提高人和小鼠原代 NK 细胞的转导效率。这种高度可再生的转导方法为换人类原发性 nk 细胞提供了一个有效的工具, 它可以极大地改善临床基因的传递应用, 从而使 nk 干细胞免疫治疗.

葡聚糖诱导人和小鼠 NK 细胞慢载体的高效基因转移
人类 NK 细胞从 PBMC 分离纯化 (纯度超过 85%), 并在夜间与 rIL-2 300 U/毫升孵育。这些主要 NK 细胞, 然后转与 GFP 慢在不同的多重感染 (莫伊; 3, 10, 20 每细胞) 24-井板存在8µg/毫升铅, PS, 或葡聚糖。细胞被离心在 1000 x g 为60分钟和培养 (在病毒面前) 过夜在37° c 在 CO2孵化器。细?...

Watch this Scientific Journal Video about Dextran Enhances the Lentiviral Transduction Efficiency of Murine and Human Primary NK Cells at JoVE.com

Dextran Enhances the Lentiviral Transduction Efficiency of Murine and Human Primary NK Cells

这项研究的目的是制定技术, 允许成功的基因转导在原自然杀手 (NK) 细胞。葡聚糖介导的人或小鼠原代 NK 细胞的慢转导可导致较高的基因表达效率。这种基因转导方法将极大地改善 NK 细胞的遗传操作。

葡聚糖增强小鼠和人原发性 NK 细胞的慢转导效率

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining ...

dextran-enhances-lentiviral-transduction-efficiency-murine-human

Research

JoVE Journal

Immunology and Infection

11.0K 次观看.  The Blood Center of Wisconsin. 这项研究的目的是制定技术, 允许成功的基因转导在原自然杀手 (NK) 细胞。葡聚糖介导的人或小鼠原代 NK 细胞的慢转导可导致较高的基因表达效率。这种基因转导方法将极大地改善 NK 细胞的遗传操作。

视频: Dextran Enhances the Lentiviral Transduction Efficiency of Murine and Human Primary NK Cells

Development, Expansion, and In vivo Monitoring of Human NK Cells from Human Embryonic Stem Cells (hESCs) and Induced Pluripotent Stem Cells (iPSCs)

We present a method for deriving natural killer (NK) cells from undifferentiated hESCs and iPSCs using a feeder-free approach. This method gives rise to high levels of NK cells after 4 weeks culture and can undergo further 2-log expansion with artificial antigen presenting cells. hESC- and iPSC-derived NK cells developed in this system have a mature phenotype and function. The production of large numbers of genetically modifiable NK cells is applicable for both basic mechanistic as well as anti-tumor studies. Expression of firefly luciferase in hESC-derived NK cells allows a non-invasive approach to follow NK cell engraftment, distribution, and function. We also describe a dual-imaging scheme that allows separate monitoring of two different cell populations to more distinctly characterize their interactions in vivo. This method of derivation, expansion, and dual in vivo imaging provides a reliable approach for producing NK cells and their evaluation which is necessary to improve current NK cell adoptive therapies.

Development, Expansion, and In vivo Monitoring of Human NK Cells from Human Embryonic Stem Cells hESCs and Induced Pluripotent Stem Cells iPSCs

This protocol describes the development, expansion, and in vivo imaging of NK cells derived from hESCs and iPSCs.

开发，扩展，<em&gt;在体内</em&gt;从人类胚胎干细胞（hESCs）监测人类NK细胞和诱导多能干细胞（iPS细胞）

We present a method for deriving natural killer (NK) cells from undifferentiated hESCs and iPSCs using a feeder-free approach. This method gives rise to ...

科研

生物工程

Lentiviral Vector Platform for the Efficient Delivery of Epigenome-editing Tools into Human Induced Pluripotent Stem Cell-derived Disease Models

The use of hiPSC-derived cells represents a valuable approach to study human neurodegenerative diseases. Here, we describe an optimized protocol for the differentiation of hiPSCs derived from a patient with the triplication of the alpha-synuclein gene (SNCA) locus into Parkinson&#8217;s disease (PD)-relevant dopaminergic neuronal populations. Accumulating evidence has shown that high levels of SNCA are causative for the development of PD. Recognizing the unmet need to establish novel therapeutic approaches for PD, especially those targeting the regulation of SNCA expression, we recently developed a CRISPR/dCas9-DNA-methylation-based system to epigenetically modulate SNCA transcription by enriching methylation levels at the SNCA intron 1 regulatory region. To deliver the system, consisting of a dead (deactivated) version of Cas9 (dCas9) fused with the catalytic domain of the DNA methyltransferase enzyme 3A (DNMT3A), a lentiviral vector is used. This system is applied to cells with the triplication of the SNCA locus and reduces the SNCA-mRNA and protein levels by about 30% through the targeted DNA methylation of SNCA intron 1. The fine-tuned downregulation of the SNCA levels rescues disease-related cellular phenotypes. In the current protocol, we aim to describe a step-by-step procedure for differentiating hiPSCs into neural progenitor cells (NPCs) and the establishment and validation of pyrosequencing assays for the evaluation of the methylation profile in the SNCA intron 1. To outline in more detail the lentivirus-CRISPR/dCas9 system used in these experiments, this protocol describes how to produce, purify, and concentrate lentiviral vectors and to highlight their suitability for epigenome- and genome-editing applications using hiPSCs and NPCs. The protocol is easily adaptable and can be used to produce high titer lentiviruses for in vitro and in vivo applications.

Targeted DNA epigenome editing represents a powerful therapeutic approach. This protocol describes the production, purification, and concentration of all-in-one lentiviral vectors harboring the CRISPR-dCas9-DNMT3A transgene for epigenome-editing applications in human induced pluripotent stem cell (hiPSC)-derived neurons.

慢病毒载体平台, 用于将表观基因编辑工具高效地传递到人类诱导的多能干细胞衍生疾病模型中

The use of hiPSC-derived cells represents a valuable approach to study human neurodegenerative diseases. Here, we describe an optimized protocol for the ...

遗传学

A Tetracycline-regulated Cell Line Produces High-titer Lentiviral Vectors that Specifically Target Dendritic Cells

Lentiviral vectors (LVs) are a powerful means of delivering genetic material to many types of cells. Because of safety concerns associated with these HIV-1 derived vectors, producing large quantities of LVs is challenging. In this paper, we report a method for producing high titers of self-inactivating LVs. We retrovirally transduce the tet-off stable producer cell line GPR to generate a cell line, GPRS, which can express all the viral components, including a dendritic cell-specific glycoprotein, SVGmu. Then, we use concatemeric DNA transfection to transfect the LV transfer plasmid encoding a reporter gene GFP in combination with a selectable marker. Several of the resulting clones can produce LV at a titer 10-fold greater than what we achieve with transient transfection. Plus, these viruses efficiently transduce dendritic cells in vitro and generate a strong T cell immune response to our reporter antigen. This method may be a good option for producing strong LV-based vaccines for clinical studies of cancer or infectious diseases.

Here, we use retroviral transduction and concatemeric transfection to create a cell line that can express the components of a lentiviral vector (LV) in the absence of tetracycline. This LV encodes GFP and is pseudotyped with a glycoprotein, SVGmu, which is specific for a receptor on dendritic cells.

四环素调节细胞株产生高滴度的慢病毒载体，专门针对树突状细胞

Lentiviral  vectors (LVs) are a powerful means of delivering genetic material to many types  of cells. Because of safety concerns associated with these ...

免疫与感染

Generation of Natural Killer Cells from Human Expanded Potential Stem Cells

The differentiation of natural killer (NK) cells from human pluripotent stem cells allows for research on and the manufacture of clinical-grade cellular products for immunotherapy. Described here is a two-phase protocol that uses a serum-free commercial medium and a cocktail of cytokines (interleukin [IL]-3, IL-7, IL-15, stem cell factor [SCF], and FMS-like tyrosine kinase 3 ligand [Ftl3L]) to differentiate human expanded potential stem cells (hEPSCs) into cells that possess NK cell properties in vitro with both 3-dimensional (3D) and 2-dimensional (2D) culture technology. Following this protocol, CD3&#8722;CD56+ or CD45+CD56+ NK cells are consistently generated. When cocultured with tumor targets for 3 h, the differentiated products display mild cytotoxicity as compared to an IL-2-independent permanent cell line, NK92mi cells. The protocol preserves the complexity of the differentiation microenvironment by the generation of 3D structures, thus facilitating the study of the spatial relationships between immune cells and their niches. Meanwhile, the 2D culture system enables the routine phenotypical validation of cell differentiation without harming the delicate differentiation niche.

The present protocol shows how to differentiate CD3&#8722;/CD45+CD56+ cells with mild cytotoxicity from human expanded potential stem cells (hEPSCs) under both 3D and 2D culture conditions. This allows for routine phenotypical validation without the destruction of the complex microenvironment.

从人类扩增的潜在干细胞中产生自然杀伤细胞

The differentiation of natural killer (NK) cells from human pluripotent stem cells allows for research on and the manufacture of clinical-grade cellular ...

发育生物学

Lentiviral-mediated Knockdown During Ex Vivo Erythropoiesis of Human Hematopoietic Stem Cells

Erythropoiesis is a commonly used model system to study cell differentiation. During erythropoiesis, pluripotent adult human hematopoietic stem cells (HSCs) differentiate into oligopotent progenitors, committed precursors and mature red blood cells 1. This process is regulated for a large part at the level of gene expression, whereby specific transcription factors activate lineage-specific genes while concomitantly repressing genes that are specific to other cell types 2. Studies on transcription factors regulating erythropoiesis are often performed using human and murine cell lines that represent, to some extent, erythroid cells at given stages of differentiation 3-5. However transformed cell lines can only partially mimic erythroid cells and most importantly they do not allow one to comprehensibly study the dynamic changes that occur as cells progress through many stages towards their final erythroid fate. Therefore, a current challenge remains the development of a protocol to obtain relatively homogenous populations of primary HSCs and erythroid cells at various stages of differentiation in quantities that are sufficient to perform genomics and proteomics experiments.
Here we describe an ex vivo cell culture protocol to induce erythroid differentiation from human hematopoietic stem/progenitor cells that have been isolated from either cord blood, bone marrow, or adult peripheral blood mobilized with G-CSF (leukapheresis). This culture system, initially developed by the Douay laboratory 6, uses cytokines and co-culture on mesenchymal cells to mimic the bone marrow microenvironment. Using this ex vivo differentiation protocol, we observe a strong amplification of erythroid progenitors, an induction of differentiation exclusively towards the erythroid lineage and a complete maturation to the stage of enucleated red blood cells. Thus, this system provides an opportunity to study the molecular mechanism of transcriptional regulation as hematopoietic stem cells progress along the erythroid lineage. 
Studying erythropoiesis at the transcriptional level also requires the ability to over-express or knockdown specific factors in primary erythroid cells. For this purpose, we use a lentivirus-mediated gene delivery system that allows for the efficient infection of both dividing and non-dividing cells 7. Here we show that we are able to efficiently knockdown the transcription factor TAL1 in primary human erythroid cells. In addition, GFP expression demonstrates an efficiency of lentiviral infection close to 90%. Thus, our protocol provides a highly useful system for characterization of the regulatory network of transcription factors that control erythropoiesis.

Lentiviral-mediated Knockdown During Ex Vivo Erythropoiesis of Human Hematopoietic Stem Cells

An ex vivo protocol to generate mature human red blood cells from hematopoietic stem/progenitors is described. Additionally we describe an efficient lentiviral-delivery method to knockdown the transcription factor TAL1 in primary erythroid cells. The efficiency of lentivirus mediated gene delivery is demonstrated using GFP expressing viruses.

慢病毒介导的击倒在体外</em人造血干细胞>红细胞

Erythropoiesis is a commonly used model system to study cell differentiation. During erythropoiesis, pluripotent adult human hematopoietic stem cells ...

生物学

Transduction of Human Cells with Polymer-complexed Ecotropic Lentivirus for Enhanced Biosafety

Stem and tumor cell biology studies often require viral transduction of human cells with known or suspected oncogenes, raising major safety issues for laboratory personnel. Pantropic lentiviruses, such as the commonly used VSV-G pseudotype, are a valuable tool for studying gene function because they can transduce many cell types, including non-dividing cells. However, researchers may wish to avoid production and centrifugation of pantropic viruses encoding oncogenes due to higher biosafety level handling requirements and safety issues. Several potent oncogenes, including c-Myc and SV40 large T antigen, are known to enhance production of induced pluripotent stem cells (iPSC). All other known iPSC-inducing genetic changes (OCT4, SOX2, KLF4, NANOG, LIN28, and p53 loss of function) also have links to cancer, making them of relatively high safety concern as well. 
While these cancer-related viruses are useful in studying cellular reprogramming and pluripotency, they must be used safely. To address these biosafety issues, we demonstrate a method for transduction of human cells with ecotropic lentivirus, with additional emphasis on reduced cost and convenient handling. We have produced ecotropic lentivirus with sufficiently high titer to transduce greater than 90% of receptor-expressing human cells exposed to the virus, validating the efficacy of this approach. 
Lentivirus is often concentrated by ultracentrifugation; however, this process takes several hours and can produce aerosols infectious to human biomedical researchers. As an alternative, viral particles can be more safely sedimented onto cells by complexation with chondroitin sulfate and polybrene (CS/PB). This technique increases the functional viral titer up to 3-fold in cells stably expressing murine retrovirus receptor, with negligible added time and cost. Transduction of human dermal fibroblasts (HDFs) is maximally enhanced using CS/PB concentrations approximately 4-fold lower than the optimal value previously reported for cancer cell lines, suggesting that polymer concentration should be titrated for the target cell type of interest. We therefore describe the use of methylthiazolyldiphenyl-tetrazolium bromide (MTT) to assay for polymer toxicity in a new cell type. We observe equivalent viability of HDFs after viral transduction using either polymer complexation or the standard dose of polybrene (PB, 6 &mu;g/ml), indicating minimal acute toxicity.
In this protocol, we describe the use of ecotropic lentivirus for overexpression of oncogenes in human cells, reducing biosafety risks and increasing the transduction rate. We also demonstrate the use of polymer complexation to enhance transduction while avoiding aerosol-forming centrifugation of viral particles.

Lentiviruses are a valuable research tool for exploring gene function; however, researchers may wish to avoid production of pantropic lentivirus encoding known or suspected oncogenes. As an alternative, we present a safer protocol for use of ecotropic lentivirus on human cells modified to express the ecotropic receptor mSlc7a1.

增强生物安全与聚合物复合物Ecotropic慢病毒的人体细胞的转导

Stem and tumor cell biology studies often require viral transduction of human cells with known or suspected oncogenes, raising major safety issues for ...

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor microenvironment and the underlying mechanism is still largely unknown. The lack of a consistent and reliable source of NK cells limits the research progress of NK cell immunity. Here, we report an in vitro system that can provide high quality and quantity of bone marrow-derived murine NK cells under a feeder-free condition. More importantly, we also demonstrate that siRNA-mediated gene silencing successfully inhibits the E4bp4-dependent NK cell maturation by using this system. Thus, this novel in vitro NK cell differentiating system is a biomaterial solution for immunity research.

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Here, we present a protocol to mass-produce gene-silencing murine NK cells by using a feeder-free differentiation system for mechanistic study in vitro and in vivo.

一种新型大鼠自然杀伤细胞无饲养系统的研究体外

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor ...

Generation of Knock-out Primary and Expanded Human NK Cells Using Cas9 Ribonucleoproteins

CRISPR/Cas9 technology is accelerating genome engineering in many cell types, but so far, gene delivery and stable gene modification have been challenging in primary NK cells. For example, transgene delivery using lentiviral or retroviral transduction resulted in a limited yield of genetically-engineered NK cells due to substantial procedure-associated NK cell apoptosis. We describe here a DNA-free method for genome editing of human primary and expanded NK cells using Cas9 ribonucleoprotein complexes (Cas9/RNPs). This method allowed efficient knockout of the TGFBR2 and HPRT1 genes in NK cells. RT-PCR data showed a significant decrease in gene expression level, and a cytotoxicity assay of a representative cell product suggested that the RNP-modified NK cells became less sensitive to TGF&#946;. Genetically modified cells could be expanded post-electroporation by stimulation with irradiated mbIL21-expressing feeder cells.

Here, we present a protocol to genetically modify primary or expanded human natural killer (NK) cells using Cas9 Ribonucleoproteins (Cas9/RNPs). By using this protocol, we generated human NK cells deficient for transforming growth factor&#8211;b receptor 2 (TGFBR2) and hypoxanthine phosphoribosyltransferase 1 (HPRT1).

利用 Cas9 Ribonucleoproteins 生成人 NK 细胞的研究

CRISPR/Cas9 technology is accelerating genome engineering in many cell types, but so far, gene delivery and stable gene modification have been challenging ...

Expression of Fluorescent Fusion Proteins in Murine Bone Marrow-derived Dendritic Cells and Macrophages

Dendritic cells and macrophages are crucial cells that form the first line of defense against pathogens. They also play important roles in the initiation of an adaptive immune response. Experimental work with these cells is rather challenging. Their abundance in organs and tissues is relatively low. As a result, they cannot be isolated in large numbers. They are also difficult to transfect with cDNA constructs. In the murine model, these problems can be partially overcome by in vitro differentiation from bone marrow progenitors in the presence of M-CSF for macrophages or GM-CSF for dendritic cells. In this way, it is possible to obtain large amounts of these cells from very few animals. Moreover, bone marrow progenitors can be transduced with retroviral vectors carrying cDNA constructs during early stages of cultivation prior to their differentiation into bone marrow derived dendritic cells and macrophages. Thus, retroviral transduction followed by differentiation in vitro can be used to express various cDNA constructs in these cells. The ability to express ectopic proteins substantially extends the range of experiments that can be performed on these cells, including live cell imaging of fluorescent proteins, tandem purifications for interactome analyses, structure-function analyses, monitoring of cellular functions with biosensors and many others. In this article, we describe a detailed protocol for retroviral transduction of murine bone marrow derived dendritic cells and macrophages with vectors coding for fluorescently-tagged proteins. On the example of two adaptor proteins, OPAL1 and PSTPIP2, we demonstrate its practical application in flow cytometry and microscopy. We also discuss the advantages and limitations of this approach.

In this article, we provide a detailed protocol for the expression of fluorescent fusion proteins in murine bone marrow derived dendritic cells and macrophages. The method is based on the transduction of bone marrow progenitors with retroviral constructs followed by differentiation into macrophages and dendritic cells in vitro.

荧光融合蛋白在小鼠骨髓基质树突状细胞和巨噬细胞中的表达

Dendritic cells and macrophages are crucial cells that form the first line of defense against pathogens. They also play important roles in the initiation ...

Delivery of the Cas9/sgRNA Ribonucleoprotein Complex in Immortalized and Primary Cells via Virus-like Particles ("Nanoblades")

The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system has democratized genome-editing in eukaryotic cells and led to the development of numerous innovative applications. However, delivery of the Cas9 protein and single-guide RNA (sgRNA) into target cells can be technically challenge. Classical viral vectors, such as those derived from lentiviruses (LVs) or adeno-associated viruses (AAVs), allow for efficient delivery of transgenes coding for the Cas9 protein and its associated sgRNA in many primary cells and in vivo. Nevertheless, these vectors can suffer from drawbacks such as integration of the transgene in the target cell genome, a limited cargo capacity, and long-term expression of the Cas9 protein and guide RNA in target cells.
To overcome some of these problems, a delivery vector based on the murine Leukemia virus (MLV) was developed to package the Cas9 protein and its associated guide RNA in the absence of any coding transgene. By fusing the Cas9 protein to the C-terminus of the structural protein Gag from MLV, virus-like particles (VLPs) loaded with the Cas9 protein and sgRNA (named &#34;Nanoblades&#34;) were formed. Nanoblades can be collected from the culture medium of producer cells, purified, quantified, and used to transduce target cells and deliver the active Cas9/sgRNA complex. Nanoblades deliver their ribonucleoprotein (RNP) cargo transiently and rapidly in a wide range of primary and immortalized cells and can be programmed for other applications, such as transient transcriptional activation of targeted genes, using modified Cas9 proteins. Nanoblades are capable of in vivo genome-editing in the liver of injected adult mice and in oocytes to generate transgenic animals. Finally, they can be complexed with donor DNA for &#34;transfection-free&#34; homology-directed repair. Nanoblade preparation is simple, relatively low-cost, and can be easily carried out in any cell biology laboratory.

Delivery of the Cas9/sgRNA Ribonucleoprotein Complex in Immortalized and Primary Cells via Virus-like Particles "Nanoblades"

We have developed a simple and inexpensive protocol to load Cas9/single-guide RNA (sgRNA) ribonucleoprotein complexes within virus-like particles. These particles, called "Nanoblades", allow efficient delivery of the Cas9/sgRNA complex in immortalized and primary cells as well as in vivo.

通过病毒样颗粒（"纳米刀片"）在永生化和原代细胞中递送Cas9 / sgRNA核糖核蛋白复合物

The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system has democratized genome-editing in eukaryotic cells and led to the ...

葡聚糖增强小鼠和人原发性 NK 细胞的慢转导效率

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