Novel method for gain-of-function analyses in primary osteoclasts using a non-viral gene delivery system.
Novel method for gain-of-function analyses in primary osteoclasts using a non-viral gene delivery system.
复制标题
使用非病毒基因传递系统对原代破骨细胞进行功能获得分析的新方法。
DOI：
10.1007/s00774-020-01161-7
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发表时间：
2021
期刊：
影响因子：
--
通讯作者：
Ishii M
中科院分区：
文献类型：
--
作者：
Nishikawa K;Ishii M
过表达研究通常用于细胞生物学的重大进展。体外破骨细胞培养涉及骨髓源性单核巨噬细胞前体（BMMs）在添加巨噬细胞集落刺激因子和核因子- kb配体受体激活剂（RANKL）的培养基中分化为成熟的破骨细胞。逆转录病毒载体是有效将基因传递到BMMs的黄金标准。虽然该策略对处于分化早期阶段的骨髓瘤有效，但对rankl处理的骨髓瘤（如单核和多核破骨细胞）无效。本研究试图通过脂质体介导的RNA转染来增强基因传递到分化的bmm中。材料和方法用EYFP过表达质粒或EYFP RNA脂质转染bmms，或用表达EYFP的逆转录病毒载体转导bmms。流式细胞术检测EYFP的表达。结果使用增强黄色荧光蛋白（enhanced yellow fluorescent protein， EYFP）进行过表达分析。虽然在含EYFP的重组逆转录病毒感染BMMs后24小时观察到EYFP的表达，但在转染EYFP RNA后3小时内观察到EYFP的表达。此外，EYFP RNA将基因传递到BMMs的效率与EYFP的逆转录病毒转导相当。相反，在逆转录病毒感染后，RANKL刺激2天的bmm很少表达EYFP，而在转染EYFP RNA后，超过一半的细胞表达EYFP。结论rna介导的基因传递是一种快速简便的破骨细胞前体细胞和成熟破骨细胞功能获得分析方法。
IntroductionOverexpression studies have been commonly used to yield significant advances in cell biology. In vitro osteoclast culturing involves the differentiation of bone marrow-derived monocyte macrophage precursors (BMMs) in medium supplemented with macrophage colony-stimulating factor and receptor activator of nuclear factor-kB ligand (RANKL) into mature osteoclasts. Retroviral vectors are the gold standards for efficient gene delivery into BMMs. While this strategy is effective in BMMs that are in the early stages of differentiation, it is ineffective in RANKL-treated BMMs such as mono- and multinucleated osteoclasts. This study attempted to enhance gene delivery into differentiated BMMs using liposome-mediated RNA transfection.Material and methodsBMMs were transfected with an EYFP overexpression plasmid or EYFP RNA by lipofection, or transduced with a retroviral vector expressing EYFP. EYFP expression was assessed by flow cytometry.ResultsWe performed overexpression analyses using enhanced yellow fluorescent protein (EYFP). Although EYFP expression was observed 24 h after infection of BMMs with a recombinant retrovirus containing EYFP, expression of EYFP was observed within 3 h of transfection with EYFP RNA. Moreover, the efficiency of EYFP RNA for gene delivery into BMMs was comparable to that of retroviral transduction of EYFP. In contrast, while very few BMMs stimulated by RANKL for two days expressed EYFP after retroviral infection, more than half of the cells expressed EYFP after transfection with EYFP RNA.ConclusionRNA-mediated gene delivery is quick and easy method for performing gain-of-function analyses in primary osteoclast precursors and mature osteoclasts.