Generation of T Cell Receptor Retrogenic Mice.
Generation of T Cell Receptor Retrogenic Mice.
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DOI：
10.1002/cpim.76
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发表时间：
2019-06-01
影响因子：
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通讯作者：
Bettini, Maria
中科院分区：
文献类型：
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作者：
Kong, Yuelin;Jing, Yi;Bettini, Maria
体内表达和研究单个 T 细胞受体 (TCR) 的能力是基础和转化免疫学研究的一个重要方面。传统上，这是通过使用 TCR 转基因小鼠来实现的。在过去的十年中，开发了一种更有效的单一 TCR 表达方法。这种相对快速且易于使用的方法利用逆转录病毒介导的基于干细胞的基因转移，通常称为 TCR 逆转录方法。在这种方法中，造血骨髓前体细胞被携带 T 细胞受体 α 和 β 链的逆转录病毒载体转导。成功转导后，将骨髓注射到受体小鼠中，其中 T 细胞的发育由载体编码的 TCR 的表达驱动。本文详细介绍了生成 TCR 逆转录小鼠所需的材料和方法。它分为三个部分，提供了稳定逆转录病毒生产细胞系的生成、造血骨髓细胞的分离和最佳转导以及 TCR 逆转录 T 细胞的后续分析的详细方法。提供了此类分析的详细示例。当前的协议是多年优化的结晶，是迄今为止最有效的方法。现在可以在短短四天内实现骨髓转导并转移到受体小鼠体内。大多数拥有标准生物医学设备的实验室都可以遵循该协议，并有涵盖潜在陷阱和意外结果的故障排除指南提供支持。 © 2019 John Wiley & Sons, Inc. 版权所有
The ability to express and study a single T cell receptor (TCR) in vivo is an important aspect of both basic and translational immunological research. Traditionally, this was achieved by using TCR transgenic mice. In the past decade, a more efficient approach for single TCR expression was developed. This relatively rapid and accessible method utilizes retrovirus-mediated stem cell-based gene transfer and is commonly referred to as the TCR retrogenic approach. In this approach, hematopoietic bone marrow precursors are transduced with retroviral vector carrying both alpha and beta chains of a T cell receptor. After successful transduction, bone marrow is injected into recipient mice, in which T cell development is driven by expression of the vector-encoded TCR. This article details the materials and methods required to generate TCR retrogenic mice. It is divided into three sections and provides detailed methods for generation of stable retroviral producer cell lines, isolation and optimal transduction of hematopoietic bone marrow cells, and subsequent analysis of TCR retrogenic T cells. A detailed example of such analysis is provided. The current protocol is a culmination of many years of optimization and is the most efficient approach to date. Bone marrow transduction and transfer into recipient mice can now be achieved in a short period of four days. The protocol can be followed in most laboratories with standard biomedical equipment, and is supported by a troubleshooting guide that covers potential pitfalls and unexpected results. © 2019 by John Wiley & Sons, Inc.