Name: adenoviral transduction of naive cd4 t cells to study treg differentiation
Uploaded: 2013-08-13T13:00:00
Description: Watch this Scientific Journal Video about Adenoviral Transduction of Naive CD4 T Cells to Study Treg Differentiation at JoVE.com

笔者想感谢杜利瑞建设pCAGAdDU载体和奥利弗·戈尔卡提供的固定协议。

1。感兴趣的基因的克隆到入门载体<ol><li>感兴趣的基因的克隆到一个条目载体。 PCR扩增的基因，然后通过平末端连接到一个拓扑异构酶耦合向量（ 例如载体pENTR / D-TOPO）或限制性内切酶介导的克隆可以用于此过程。 </li></ol> 2。感兴趣的基因转移到的pCAGAdDu目标向量<ol><li>从输入矢量的兴趣基因转移到目标向量由LR重组（ 例如网关LR克隆酶II酶混合物）。这将创建?...

<ol>
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病毒生成和滴定为了获得最佳的转染结果，线性化载体的质量和数量的出现最重要的。我们并没有观察到初级裂解生产的负面影响，从最初的文化繁茂，因为感染会迅速进行一次高效的病毒生产发生。然而，HEK293A细胞的病毒产量可受长的插入效率下降。一些的开放阅读框架，实际上发现干扰病毒生产。这些病毒的股票通常可以通过几轮的扩增获救，只有少数的开放阅读框被认?...

调节性T细胞是维持免疫耐受，抑制过度的免疫反应的关键。调节性T细胞抑制T细胞活化的旁观者。因此，调节性T细胞的消融导致致命的自身免疫和自我毁灭的驱动由活化的T细胞1。调节性T细胞在胸腺中发展CD4单阳性前体的阴性选择过程中，但他们也可以从幼稚后，低剂量的抗原刺激的CD4 + T细胞具有次优的协同刺激1,2分化的周围。胸腺调节性T细胞似乎抑制组织对自身抗原的自身免疫性疾病，而周边有牵连的调节性T细胞在肠道或肺部提供宽容。这些诱导Tregs的有效阻止T细胞活化后在粘膜外来抗原的识别，包括环境抗原的食物和空气，共生细菌，过敏原3,4。此外，调节性T细胞是关键，建立孕产妇胎儿肽的耐受性和预先发泄移植物抗宿主病。与此同时，调节性T细胞介导不良影响肿瘤细胞的免疫监视7,8衰减。调节性T细胞的共同特征，是指定的子集转录因子Foxp3，叉头域的转录因子，是必要的，足以赋予Treg细胞功能9,10的表达。一些信号转导通路，可诱导Foxp3的表达是众所周知的。然而，分子的过程控制，调节，或调节调节性T细胞分化的T细胞受体触发的了解较少。 调节性T细胞可以非常有效地在体外诱导幼稚的CD4 + T细胞与抗-CD3和抗CD28抗体的存在下，TGF-β和IL-2 11通过刺激。由于新兴的调节性T细胞在体内的功能，促进调节性T细胞分化的分子操纵承担巨大的潜力，未来的therapi上课，例如，治疗哮喘，克罗恩病，移植11,12。相反，治疗调制阻止Treg细胞分化的分子可能在未来的肿瘤患者的综合治疗方法中受益。 一直在体外诱导分化实验的描述都与T细胞亚群分化的分子变化。在此刻，实验尝试搜索或基因产物的屏幕控制T细胞分化异位基因表达的最常用的方法失败，在幼稚T细胞的事实而受到阻碍。例如，电穿孔，逆转录病毒转导是唯一有效的活化的T细胞中。与此相反的最初预期的慢病毒转导，这是通常在静息细胞中有效，需要预活化幼稚T细胞的细胞因子是13。此外，在电穿孔过程中的cDNA或mRNA的转移涉及质膜的去极化，其本身赋予T细胞活化的功能，甚至可能动员的Ca 2 +信号 并且激活NFAT蛋白（未发表的观察和参考文献14）。类似地，对于逆转录病毒转导，幼稚T细胞被激活为18 - 40小时。在这段时间内，在细胞分裂过程中，核膜破裂的发生，并允许为随后的基因组整合的逆转录病毒载体15。因此，这些方法是不能够解决早期的分子调控初始T细胞与抗原相遇，这是辅助性T细胞分化的决定性阶段。 腺病毒转导是已知的，赋予短暂异位基因表达的人类细胞类型中表达的人柯萨奇腺病毒受体（CAR）的一些。进行没有要求细胞活化或细胞周期进程。表面表达的CAR是必不可少的有效的病毒附件和内在，发现转基因表达的截断版本CARΔ1的T细胞特异性启动子下使小鼠胸腺细胞和T细胞受到腺病毒感染16。重要的是，转基因不改变胸腺细胞发育或在体外分化幼稚的CD4 T细胞分成不同的子集（数据未显示;文献17）。以前使用的腺病毒介导的转导T细胞过表达17,18和击倒方法19,20。的转基因T细胞可被纯化从市售的DO11.10的TG;：CARΔ1的TG（Taconic公司，Inc。和参考文献17）。重要的是，腺病毒转导允许在幼稚T细胞中感兴趣的基因的高表达而诱导激活的明显迹象。 T细胞保持天真（CD44 低 ，CD62L 高 ），休息（CD25 - ，CD69 - ）后infecti和可以被激活和调节性T细胞分化成类似的非感染的细胞。 腺病毒介导的生产可以实现HEK293A细胞转染后，腺病毒载体（图1）。这些质粒通常含有E1和E3基因删除，以使重组病毒无法复制的21人5型腺病毒基因组。 HEK293A细胞补充不足，因为他们已经通过稳定整合剪切腺病毒22永生复制。由于腺病毒载体是大的（〜40 kb的），因此不适合于传统的限制性内切酶介导的克隆，我们采用的网关系统。感兴趣的基因的最初克隆到一个较小的输入矢量，从它可以很容易地转移到腺病毒的目标向量通过拉姆达重组反应（LR）23。我们构建了pCAGAdDu载体相结合的的CAG启动（鸡肌动蛋白启动子和CMV增强）的表达盒含有LR网站侧翼原核CCDB选择标记24。此表达盒的一个内部核糖体进入位点（IRES）元件，允许共表达真核感染标记增强型绿色荧光蛋白（EGFP），这是含有牛生长激素多聚（A）信号序列融合到融合。我们选择的CAG顺式调控序列，由于原型的CMV启动子被认为是高度活化相关的，因此不利于在幼稚T细胞的基因表达。 在这里，我们提供了一个协议，用于高效在体外调节性T细胞的分化和幼稚的CD4 T细胞转导方法，无需激活（图2）。该方法使异位基因表达或击倒前面的CD4 T细胞分化的幼稚状态。它允许测试一个overexpresse的效果d基因的兴趣在早期信号事件在初始TCR刺激，直到T细胞亚群的承诺。我们的验证实验也提供了基础，建立类似的腺病毒应用程序在其他T细胞亚群，如Th1细胞和Th2，TH9，Th17细胞，TH22，或TFH细胞的分化。

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
		</tr>
		<tr>
			<td>pENTR/D-TOPO Cloning Kit</td>
			<td>Invitrogen</td>
			<td>K240020</td>
		</tr>
		<tr>
			<td>Gateway LR Clonase II Enzyme mix</td>
			<td>Invitrogen</td>
			<td>11791020</td>
		</tr>
		<tr>
			<td>PacI</td>
			<td>New England Biolabs</td>
			<td>R057S</td>
		</tr>
		<tr>
			<td>jetPEI</td>
			<td>Polyplus-transfection</td>
			<td>101-10N</td>
		</tr>
		<tr>
			<td>HEK293A Cell Line</td>
			<td>Invitrogen</td>
			<td>R705-07</td>
		</tr>
		<tr>
			<td>A549</td>
			<td>ATCC</td>
			<td>CCL-185</td>
		</tr>
		<tr>
			<td>6-well plates</td>
			<td>BD Falcon</td>
			<td>353046</td>
		</tr>
		<tr>
			<td>14 cm tissue culture dish</td>
			<td>Nunc</td>
			<td>168381</td>
		</tr>
		<tr>
			<td>BALB/cJ-Tg(DO11.10)10Dlo Tg(CAR&Delta;1)1Jdgr</td>
			<td>Taconic Farms</td>
			<td>Model Nr. 4285</td>
		</tr>
		<tr>
			<td>Naive CD4+ T Cell Isolation Kit II</td>
			<td>Miltenyi Biotech</td>
			<td>130-094-131</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Invitrogen</td>
			<td>41966-052</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>PAN Biotech</td>
			<td>1502-P110704</td>
		</tr>
		<tr>
			<td>PenStrep</td>
			<td>Invitrogen</td>
			<td>15140-122</td>
		</tr>
		<tr>
			<td>RPMI 1640</td>
			<td>Lonza</td>
			<td>BE12-167F</td>
		</tr>
		<tr>
			<td>NaPyruvate</td>
			<td>Lonza</td>
			<td>BE13-115E</td>
		</tr>
		<tr>
			<td>NEAA 100x</td>
			<td>Lonza</td>
			<td>BE13-114E</td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Invitrogen</td>
			<td>25030</td>
		</tr>
		<tr>
			<td>HEPES Buffer Solution (1 M)</td>
			<td>Invitrogen</td>
			<td>15630-056</td>
		</tr>
		<tr>
			<td>&beta;-Mercaptoethanol</td>
			<td>Sigma-Aldrich</td>
			<td>M-7522</td>
		</tr>
		<tr>
			<td>MEM Essential vitamin mixture (100x)</td>
			<td>Lonza</td>
			<td>13-607C</td>
		</tr>
		<tr>
			<td>Dynabeads Mouse T-Activator CD3/CD28 for Cell Expansion and Activation</td>
			<td>Invitrogen</td>
			<td>114-56D</td>
		</tr>
		<tr>
			<td>Recombinant Human TGF-beta 1</td>
			<td>R&amp;D Systems</td>
			<td>240B</td>
		</tr>
		<tr>
			<td>Proleukin S (18 x 106IE)</td>
			<td>Novartis</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>LIVE/DEAD Fixable Blue Dead Cell Stain Kit</td>
			<td>Invitrogen</td>
			<td>L-23105</td>
		</tr>
		<tr>
			<td>Mouse BD Fc BlockT</td>
			<td>BD Pharmingen</td>
			<td>553141</td>
		</tr>
		<tr>
			<td>Anti-Mouse/Rat Foxp3 PE</td>
			<td>eBioscience</td>
			<td>12-5773-82</td>
		</tr>
		<tr>
			<td>Mmu-miR-155 TaqMan MicroRNA Assay</td>
			<td>Roche Applied Biosystems</td>
			<td>4427975</td>
		</tr>
		<tr>
			<td>LightCycler 480 Probes Master</td>
			<td>Roche Applied Biosystems</td>
			<td>04902343001</td>
		</tr>
		<tr>
			<td>TaqMan MicroRNA Reverse Transcription Kit</td>
			<td>Roche Applied Biosystems</td>
			<td>4366596</td>
		</tr>
	</tbody>
</table>

adenoviral transduction of naive cd4 t cells to study treg differentiation

调节性T细胞（Treg细胞）是必不可少的，提供自我以及某些外来抗原的免疫耐受。调节性T细胞可产生从幼稚的CD4 T细胞在体外与T细胞受体和TGF-β和IL-2的存在下的共刺激。承担巨大的潜力，未来的治疗，但是，分子和信号通路，控制分化在很大程度上是未知。 原发性T细胞可以通过操纵异位基因表达，但常用的方法没有针对前最重要的幼稚状态的T细胞抗原识别初级。在这里，我们提供了一个协议异位基因表达在幼稚的CD4 T细胞在体外诱导调节性T细胞的分化之前。它适用于复制缺陷型腺病毒转导和解释它的产生和生产。腺病毒可以占用大量插入（最多7 KB），并可以配备促销员实现高瞬态曝光过度在T细胞ression。它有效传导天真的小鼠T细胞，如果他们表达的转基因柯萨奇腺病毒受体（CAR）。重要的是，感染后T细胞仍然幼稚（CD44，CD62L 的高点 ）和休息（CD25 - CD69 - ），并且可以被激活和调节性T细胞分化成类似的非感染的细胞。因此，该方法从一开始使CD4 + T细胞分化的操纵。确保异位基因表达已经到位，当早期信号事件的初始TCR刺激诱导细胞的变化，最终导致Treg细胞分化。

病毒生产新一代的病毒滴度高，在初级生产病毒或病毒扩增HEK293A细胞收获的时机是至关重要的。代表荧光和相位与病毒生产的视觉迹象的对比度的图像如图3所示。 CPE观察转染的HEK293A细胞与控制pCAGAdDu向量没有插入后10天。 CPE的特点是外观方面扩大和圆形细胞开始分离，并表现出高表达的感染标记GFP。 CPE的程度是表示高效的病毒的产生。细胞培养板，显示类似CPE可...

Watch this Scientific Journal Video about Adenoviral Transduction of Naive CD4 T Cells to Study Treg Differentiation at JoVE.com

Adenoviral Transduction of Naive CD4 T Cells to Study Treg Differentiation

腺病毒基因转移到幼稚的CD4 T细胞与柯萨奇腺病毒受体的转基因表达，使调节性T细胞分化的分子分析<em&gt;在体外</em&gt;。

腺病毒转导朴素的CD4 T细胞调节性T细胞分化研究

Regulatory T cells (Tregs) are essential to provide immune tolerance to self as well as to certain foreign antigens. Tregs can be generated from naive CD4 ...

Research

JoVE Journal

Immunology and Infection

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

T细胞受体在小鼠T细胞的逆转录病毒转导

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

科研

免疫与感染

Directed Differentiation of Induced Pluripotent Stem Cells towards T Lymphocytes

定向分化诱导性多潜能干细胞向T淋巴细胞

Adoptive cell transfer (ACT) of antigen-specific CD8+  cytotoxic T lymphocytes (CTLs) is a promising treatment for a variety of  malignancies 1. CTLs can  ...

Accelerated Type 1 Diabetes Induction in Mice by Adoptive Transfer of Diabetogenic CD4+ T Cells

加速1型糖尿病感应在小鼠体内的过继转移致糖尿病的CD4 + T细胞

The nonobese diabetic (NOD) mouse spontaneously develops autoimmune diabetes after 12 weeks of age and is the most extensively studied animal model of ...

Isolation and Th17 Differentiation of Na&iuml;ve CD4 T Lymphocytes

Isolation and Th17 Differentiation of NaÃƒÆ’Ã‚Â¯ve CD4 T Lymphocytes

隔离和幼稚的CD4 T细胞的Th17细胞分化

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets ...

Transplantation of Tail Skin to Study Allogeneic CD4 T Cell Responses in Mice

尾部皮肤来研究异基因CD4 + T细胞应答的小鼠移植

The study of T cell responses and their consequences during allo-antigen recognition requires a model that enables one to distinguish between donor and ...

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

诱导小鼠肠道炎症由效应CD4的过继转移<sup&gt; +</sup&gt; CD45RB<sup&gt;高</sup&gt; T细胞免疫缺陷小鼠

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages ...

Mouse Na&#239;ve CD4+ T Cell Isolation and In vitro Differentiation into T Cell Subsets

Mouse NaÃƒÆ’Ã‚Â¯ve CD4+ T Cell Isolation and In vitro Differentiation into T Cell Subsets

鼠标朴素CD4<sup&gt; +</sup&gt; T细胞分离和<em&gt;在体外</em&gt;分化为T细胞亚群

Antigen inexperienced (na&#239;ve) CD4+ T cells undergo expansion and differentiation to effector subsets at the time of T cell receptor (TCR) recognition ...

Isolation and Ex Vivo Culture of V&#948;1+CD4+&#947;&#948; T Cells, an Extrathymic &#945;&#946;T-cell Progenitor

Isolation and Ex Vivo Culture of VÃƒÅ½Ã‚Â´1+CD4+ÃƒÅ½Ã‚Â³ÃƒÅ½Ã‚Â´ T Cells, an Extrathymic ÃƒÅ½Ã‚Â±ÃƒÅ½Ã‚Â²T-cell Progenitor

分离和<em&gt;离体</emVδ1的&gt;企业文化<sup&gt; +</sup&gt; CD4<sup&gt; +</sup&gt;γδT细胞，胸腺外的αβT细胞祖

The thymus, the primary organ for the generation of &#945;&#946; T cells and backbone of the adaptive immune system in vertebrates, has long been ...

Retroviral Transduction of Bone Marrow Progenitor Cells to Generate T-cell Receptor Retrogenic Mice

骨髓祖细胞的逆转录病毒转导到生成T细胞受体Retrogenic小鼠

T cell receptor (TCR) signaling is essential in the development and differentiation of T cells in the thymus and periphery, respectively.  The vast array ...

Preparation of CD4+ T Cells for Analysis of GD3 and GD2 Ganglioside Membrane Expression by Microscopy

Preparation of CD4+ T Cells for Analysis of GD3 and GD2 Ganglioside Membrane Expression by Microscopy

CD4的制备<sup&gt; +</sup&gt; T细胞通过显微镜分析GD3和GD2神经节苷脂膜表达的

The methods described herein for activation of na&#239;ve CD4+ T cells in suspension and their adherence in coverslips for confocal microscopy analysis ...