骨髓祖细胞的逆转录病毒转导到生成T细胞受体Retrogenic小鼠
Summary
We present a rapid and flexible protocol for a single T cell receptor (TCR) retroviral-based in vivo expression system. Retroviral vectors are used to transduce bone marrow progenitor cells to study T cell development and function of a single TCR in vivo as an alternative to TCR transgenic mice.
Abstract
T cell receptor (TCR) signaling is essential in the development and differentiation of T cells in the thymus and periphery, respectively. The vast array of TCRs proves studying a specific antigenic response difficult. Therefore, TCR transgenic mice were made to study positive and negative selection in the thymus as well as peripheral T cell activation, proliferation and tolerance. However, relatively few TCR transgenic mice have been generated specific to any given antigen. Thus, studies involving TCRs of varying affinities for the same antigenic peptide have been lacking. The generation of a new TCR transgenic line can take six or more months. Additionally, any specific backcrosses can take an additional six months. In order to allow faster generation and screening of multiple TCRs, a protocol for retroviral transduction of bone marrow was established with stoichiometric expression of the TCRα and TCRβ chains and the generation of retrogenic mice. Each retrogenic mouse is essentially a founder, virtually negating a founder effect, while the length of time to generate a TCR retrogenic is cut from six months to approximately six weeks. Here we present a rapid and flexible alternative to TCR transgenic mice that can be expressed on any chosen background with any particular TCR.
Introduction
人类和小鼠的T细胞受体（TCR）剧目估计为1×10 8 2×10 6个独特的TCR分别1,2。这个大多样性使T细胞能够识别来自自身肽，以及从由抗原呈递细胞（APC）的主要组织相容性复合体（MHC）颁发的病原体衍生的抗原表位的繁多。在这个独特的肽MHC复合物的TCR的相互作用的细微差别决定T细胞是否发生凋亡，无能，活化，分化，细胞因子的产生和细胞毒性。但是，由于大的T细胞受体库的一个特异性TCR将如何对特定抗原作出反应的分析需要使用单一的TCR系统。
各种TCR转基因小鼠已为了研究单一的TCR的功能在体内模型3-9生成的。然而，有警告到TCR转基因小鼠，包括成本，时间，以产生一个单一的转基因小鼠和随机转基因插入的所谓的创始人作用到种系的DNA 10的长度。因此，已经产生相对少的TCR转基因小鼠为任何给定抗原和高和低TCR的亲和力为相同的表位的功能影响很少讨论。为了满足迅速的方法来屏的需要，单独地或组合研究多种的TCR，（来自逆转录病毒的“逆向”和从转基因“基因”）retrogenic小鼠已被利用作为替代TCR转基因小鼠的11-13。
内几种病毒中发现的2A肽共有基序包含一个2A-ASP-缬氨酸/异亮氨酸 – 戊二醛-X-ASN-Pro的基-Gly-2B-Pro的，其中的2A的甘氨酸和2B的脯氨酸之间发生裂解从顺式短效水解酶活性，导致核糖体跳绳翻译10,14-16中。有关详细的图描绘的C各种2A肽（F2A，E2A，T2A和P2A）的leavage请参阅参考文献10 – 12以这种方式，2顺反子（TCR的α和TCR测试版）可以链接导致单个载体化学计量的翻译。利用这种方法，我们都能够表达和体内直接比较多个抗原特异性的TCR。
Protocol
Representative Results
Discussion
在协议中，我们详细介绍几个关键步骤，以确保最佳的骨髓健康，传递效率和重建。第一个关键步骤是产生和GP + E86病毒生产细胞的适当的维修。使用早期传代生产细胞系，并在80％汇合或使用较低前保持。当制作新鲜的GP + E86病毒生产细胞，保证了293T细胞是早期的通道，并在培养24成长 – 48小时。在转步电镀太多的GP + E86细胞会降低病毒滴度。如在霍尔斯等 11中所述，以确保GP + E86?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作是由赠款美国国立卫生研究院（5K22A1119151-01和1R56DK104903-01），以美国职棒大联盟，糖尿病研究中心（P30-DK079638）在BCM的试点/可行性方案，JDRF 1-FAC-2014-243-AN APF，ADA支持1-15-JF-07，免疫学奖学金到MB，罗伯特和Janice捷基金会AAI招聘。
Materials
| DMEM, high glucose + glutamine | Corning Cellgro | 10-013-CV | Dulbecco's Modification of Eagle's Medium with 4.5 g/L glucose, L-glutamine & sodium pyruvate |
| FBS | Atlanta Biological | S11550 | |
| Trypsin-Versene | Lonza | 17-161F | |
| 0.45 um syringe filter | Thermo Scientific | 194-2545 | |
| polybrene | Sigma | H9268-10G | Sterile Filtered in dH2O |
| Ciprofloxacin | VWR | AAJ61970-06 | |
| 5-fluorouracil (5-FU) | VWR | AAA13456-06 | |
| Sodium Pyruvate | Corning Cellgro | 25-000-CI | |
| MEM nonessential Amino Acids | Corning Cellgro | 25-025-CI | |
| HEPES 1M solution | Corning Cellgro | 25-060-CI | |
| 2-Mercaptoethanol | Gibco by Life Technologies | 21985-023 | |
| Pen/Strept | Corning Cellgro | 30-002-CI | |
| L-glutamine | Corning Cellgro | 25-005-CI | |
| 150 mm tissue culture dishes | Greiner Bio-one | 639160 | |
| Tisue culture-treated 6-well flat plate | Greiner Bio-one | 657160 | |
| 70 um nylon cell strainers | Falcon | 352350 | |
| Mouse IL-3 | Invitrogen | PMC0033 | |
| Human IL-6 | Invitrogen | PHC0063 | |
| Mouse Stem Cell Factor | Invitrogen | PMC2113L | |
| 10x PBS | Corning Cellgro | 46-D13-CM | |
| HANKS Buffer | Corning Cellgro | 21020147 | |
| BD 10 mL Syringe | BD | 300912 | |
| BD 1 mL Syringe | BD | 309659 | |
| 27G x 1/2 BD Precision Glide Needle | BD | 305109 | |
| 30G x 1/2 BD Precision Glide Needle | BD | 305106 |
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