Cytokines and Immunity Section, Cancer Innovation Laboratory, National Cancer Institute, National Institutes of Health.
Cytokines and Immunity Section, Cancer Innovation Laboratory, National Cancer Institute, National Institutes of Health; Comparative Biomedical Scientist Training Program, NIH; Department of Pathobiology and Diagnostic Investigation, Veterinary Diagnostic Laboratory, Michigan State University.
J Vis Exp. 2023 Jun 9(196). doi: 10.3791/64271.
Transduced mouse immature thymocytes can be differentiated into T cells in vitro using the delta-like 4-expressing bone marrow stromal cell line co-culture system (OP9-DL4). As retroviral transduction requires dividing cells for transgene integration, OP9-DL4 provides a suitable in vitro environment for cultivating hematopoietic progenitor cells. This is particularly advantageous when studying the effects of the expression of a specific gene during normal T cell development and leukemogenesis, as it allows researchers to circumvent the time-consuming process of generating transgenic mice. To achieve successful outcomes, a series of coordinated steps involving the simultaneous manipulation of different types of cells must be carefully performed. Although these are very well-established procedures, the lack of a common source in the literature often means a series of optimizations are required, which can be time-consuming. This protocol has been shown to be efficient in transducing primary thymocytes followed by differentiation on OP9-DL4 cells. Detailed here is a protocol that can serve as a quick and optimized guide for the co-culture of retrovirally transduced thymocytes on OP9-DL4 stromal cells.
利用表达 Delta 样配体 4 的骨髓基质细胞系共培养系统（OP9-DL4），可将转导的小鼠未成熟胸腺细胞在体外分化为 T 细胞。由于逆转录病毒转导需要分裂细胞进行转基因整合，因此 OP9-DL4 为培养造血祖细胞提供了合适的体外环境。当研究特定基因在正常 T 细胞发育和白血病发生过程中的表达效应时，这尤其有利，因为它可以避免生成转基因小鼠所需的耗时过程。为了获得成功的结果，必须仔细执行一系列涉及同时操作不同类型细胞的协调步骤。尽管这些都是非常成熟的程序，但由于文献中缺乏通用来源，通常意味着需要进行一系列优化，这可能很耗时。该方案已被证明可有效地转导原代胸腺细胞，然后在 OP9-DL4 细胞上进行分化。本文详细介绍了一种方案，可作为在 OP9-DL4 基质细胞上共培养逆转录病毒转导的胸腺细胞的快速优化指南。
