Keizo Nishikawa1,2,3, Masaru Ishii4,5. 1. Laboratory of Cell Biology and Metabolic Biochemistry, Department of Medical Life Systems, Graduate School of Life and Medical Sciences, Doshisha University, Tatara Miyakodani 1-3, Kyotanabe, Kyoto, 610-0394, Japan. kenishik@mail.doshisha.ac.jp. 2. Department of Immunology and Cell Biology, Graduate School of Medicine/Frontier Biosciences, Osaka University, Yamada-oka 2-2, Suita, Osaka, 565-0871, Japan. kenishik@mail.doshisha.ac.jp. 3. WPI-Immunology Frontier Research Center, Osaka University, Yamada-oka 2-2, Suita, Osaka, 565-0871, Japan. kenishik@mail.doshisha.ac.jp. 4. Department of Immunology and Cell Biology, Graduate School of Medicine/Frontier Biosciences, Osaka University, Yamada-oka 2-2, Suita, Osaka, 565-0871, Japan. 5. WPI-Immunology Frontier Research Center, Osaka University, Yamada-oka 2-2, Suita, Osaka, 565-0871, Japan.
Abstract
INTRODUCTION: Overexpression 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 METHODS: BMMs 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. RESULTS: We 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. CONCLUSION: RNA-mediated gene delivery is quick and easy method for performing gain-of-function analyses in primary osteoclast precursors and mature osteoclasts.
INTRODUCTION: Overexpression 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 METHODS: BMMs 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. RESULTS: We 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. CONCLUSION: RNA-mediated gene delivery is quick and easy method for performing gain-of-function analyses in primary osteoclast precursors and mature osteoclasts.