Using siRNAs to genetically manipulate immune cells is important to both basic immunological studies and therapeutic applications. However, siRNA delivery is challenging because primary immune cells are often sensitive to the delivery materials and generate immune responses. We have recently developed an amphiphilic dendrimer that is able to deliver siRNA to a variety of cells, including primary immune cells. We provide here a protocol for the synthesis of this dendrimer, as well as siRNA delivery to immune cells such as primary T and B cells, natural killer cells, macrophages, and primary microglia. The dendrimer synthesis entails straightforward click coupling followed by an amidation reaction, and the siRNA delivery protocol requires simple mixing of the siRNA and dendrimer in buffer, with subsequent application to the primary immune cells to achieve effective and functional siRNA delivery. This dendrimer-mediated siRNA delivery largely outperforms the standard electroporation technique, opening a new avenue for functional and therapeutic studies of the immune system. The whole protocol encompasses the dendrimer synthesis, which takes 10 days; the primary immune cell preparation, which takes 3–10 d, depending on the tissue source and cell type; the dendrimer-mediated siRNA delivery; and subsequent functional assays, which take an additional 3–6 d.

使用 siRNA 对免疫细胞进行基因操作对于基础免疫学研究和治疗应用都很重要。然而，siRNA 递送具有挑战性，因为初级免疫细胞通常对递送材料敏感并产生免疫反应。我们最近开发了一种两亲树状大分子，它能够将 siRNA 传递到各种细胞，包括原代免疫细胞。我们在这里提供了一种用于合成这种树枝状聚合物的方案，以及将 siRNA 递送到免疫细胞，如原代 T 和 B 细胞、自然杀伤细胞、巨噬细胞和原代小胶质细胞。树枝状大分子合成需要直接点击偶联，然后进行酰胺化反应，而 siRNA 递送方案需要将 siRNA 和树枝状大分子在缓冲液中简单混合，随后应用于初级免疫细胞以实现有效和功能性的 siRNA 递送。这种树枝状大分子介导的 siRNA 递送在很大程度上优于标准电穿孔技术，为免疫系统的功能和治疗研究开辟了一条新途径。整个协议包括树枝状大分子的合成，这需要 10 天；初级免疫细胞制备，需要 3-10 天，取决于组织来源和细胞类型；树枝状大分子介导的 siRNA 递送；和随后的功能测定，这需要额外的 3-6 天。这需要10天；初级免疫细胞制备，需要 3-10 天，取决于组织来源和细胞类型；树枝状大分子介导的 siRNA 递送；和随后的功能测定，这需要额外的 3-6 天。这需要10天；初级免疫细胞制备，需要 3-10 天，取决于组织来源和细胞类型；树枝状大分子介导的 siRNA 递送；和随后的功能测定，这需要额外的 3-6 天。