Name: dextran enhances the lentiviral transduction efficiency of murine and human primary nk cells
Uploaded: 2018-01-15T12:30:00
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...

<ol>
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K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lentiviral+vectors+mediate+stable+and+efficient+gene+delivery+into+primary+murine+natural+killer+cells.">Lentiviral vectors mediate stable and efficient gene delivery into primary murine natural killer cells.</a> Mol. Ther. 15, 1331-1339 (2007).</li><li>Maasho, K., Marusina, A., Reynolds, N. M., Coligan, J. E., Borrego, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+gene+transfer+into+the+human+natural+killer+cell+line,+NKL,+using+the+Amaxa+nucleofection+system.">Efficient gene transfer into the human natural killer cell line, NKL, using the Amaxa nucleofection system.</a> J. Immunol. Methods. 284, 133-140 (2004).</li><li>Trompeter, H. 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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-&#947; )</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>51Chromium (Cr)-release assays</td>
			<td>perkin elmer&#39;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 ...

Research

JoVE Journal

Immunology and Infection

Expansion, Purification, and Functional Assessment of Human Peripheral Blood NK Cells

人外周血NK细胞的扩展，纯化和功能评估

Natural killer (NK) cells play an important role in immune surveillance against a variety of infectious microorganisms and tumors. Limited availability of ...

科研

免疫与感染

Generation of Induced Regulatory T Cells from Primary Human Na&iuml;ve and Memory T Cells

Generation of Induced Regulatory T Cells from Primary Human NaÃƒÆ’Ã‚Â¯ve and Memory T Cells

从初级人类的幼稚和记忆T细胞的诱导调节性T细胞的生成

The development and maintenance of immunosuppressive CD4+ regulatory T cells (Tregs) contribute to the peripheral tolerance needed to remain in ...

piggyBac Transposon System Modification of Primary Human T Cells

piggyBac Transposon System Modification of Primary Human T Cells

piggyBac转座子初级人类T细胞系统改造

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most ...

Isolation and Intravenous Injection of Murine Bone Marrow Derived Monocytes

隔离和小鼠骨髓的静脉注射单核细胞衍生

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of ...

Reliable and High Efficiency Extraction of Kidney Immune Cells

肾脏免疫细胞的可靠和高效率的提取

Immune system activation occurs in multiple kidney diseases and pathophysiological processes.  The immune system consists of both adaptive and innate ...

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

辅助性T细胞作为一种遗传方法来研究逆转录病毒转导机制控制其分化和功能

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

A Flow Cytometry-Based Cytotoxicity Assay for the Assessment of Human NK Cell Activity

Within the innate immune system, effector lymphocytes known as natural killer (NK) cells play an essential role in host defense against aberrant cells, ...

Assessment of the Synaptic Interface of Primary Human T Cells from Peripheral Blood and Lymphoid Tissue

人外周血和淋巴组织中原发性 T 细胞突触界面的评价

The current understanding of the dynamics and structural features of T-cell synaptic interfaces has been largely determined through the use of ...

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

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 ...

High-throughput Measurement of Plasma Membrane Resealing Efficiency in Mammalian Cells

哺乳动物细胞血浆膜检测效率的高通量测量

In their physiological environment, mammalian cells are often subjected to mechanical and biochemical stresses that result in plasma membrane damage. In ...