Laboratory of Innate Immunity and Signal Transduction, Department of Pathology, Division of Microbiology and Immunology, University of Utah, Salt Lake City, UT, USA.
Methods Mol Biol. 2023;2618:93-107. doi: 10.1007/978-1-0716-2938-3_7.
Mouse dendritic cells (DCs) are routinely generated based on cells isolated form the bone marrow (BM) and cultured in the presence of growth factors that support DC development, such as FMS-like tyrosine kinase 3 ligand (FLT3L) and granulocyte-macrophage colony-stimulating factor (GM-CSF) (Guo et al., J Immunol Methods 432:24-29, 2016). In response to these growth factors, DC progenitors expand and differentiate, while other cell types die during the in vitro culture period, ultimately leading to relatively homogenous DC populations. An alternative method, which is discussed in detail in this chapter, relies on conditional immortalization of progenitor cells with DC potential in vitro using an estrogen-regulated form of Hoxb8 (ERHBD-Hoxb8). Such progenitors are established by retroviral transduction of largely unseparated BM cells with a retroviral vector expressing ERHBD-Hoxb8. Treatment of ERHBD-Hoxb8-expressing progenitors with estrogen results in Hoxb8 activation, which blocks cell differentiation and allows for expansion of homogenous progenitor cell populations in the presence of FLT3L. These cells, referred to as Hoxb8-FL cells, retain lineage potential for lymphocyte and myeloid lineages, including the DC lineage. Upon removal of estrogen (inactivation of Hoxb8), Hoxb8-FL cells differentiate into highly homogenous DC populations in the presence of GM-CSF or FLT3L akin to their endogenous counterparts. Given their unlimited proliferative capacity and amenability for genetic manipulation, for example, by CRISPR/Cas9, these cells provide a large number of options to investigate DC biology. Here, I am describing the method to establish Hoxb8-FL cells from mouse BM, as well as procedures for DC generation and gene deletion using lentivirally delivered CRISPR/Cas9.
小鼠树突状细胞 (DC) 通常是基于从骨髓 (BM) 中分离出来的细胞，并在支持 DC 发育的生长因子存在下培养而产生的，例如 FMS 样酪氨酸激酶 3 配体 (FLT3L) 和粒细胞-巨噬细胞集落刺激因子 (GM-CSF)（Guo 等人，J Immunol Methods 432:24-29, 2016）。在这些生长因子的作用下，DC 前体细胞扩增和分化，而其他细胞类型在体外培养期间死亡，最终导致相对同质的 DC 群体。另一种方法，在本章中详细讨论，依赖于使用雌激素调节形式的 Hoxb8 (ERHBD-Hoxb8) 在体外对具有 DC 潜能的祖细胞进行条件永生化。通过逆转录病毒载体表达 ERHBD-Hoxb8 转导基本上未分离的 BM 细胞来建立这种祖细胞。用雌激素处理表达 ERHBD-Hoxb8 的祖细胞会导致 Hoxb8 激活，从而阻止细胞分化，并允许在存在 FLT3L 的情况下扩增同质祖细胞群体。这些细胞被称为 Hoxb8-FL 细胞，保留淋巴细胞和髓系谱系（包括 DC 谱系）的谱系潜能。在去除雌激素（Hoxb8 失活）后，Hoxb8-FL 细胞在 GM-CSF 或 FLT3L 的存在下分化为高度同质的 DC 群体，类似于它们的内源性对应物。鉴于其无限的增殖能力和遗传操作的易感性，例如通过 CRISPR/Cas9，这些细胞为研究 DC 生物学提供了大量选择。在这里，我描述了从小鼠 BM 中建立 Hoxb8-FL 细胞的方法，以及使用慢病毒递送的 CRISPR/Cas9 进行 DC 生成和基因缺失的程序。
