Detection of osteoclastic cell-cell fusion through retroviral vector packaging.
Cell-cell fusion generates multinucleated cells such as osteoclasts in bone, myotubes in muscle, and trophoblasts in placenta. Molecular details governing these fusion processes are still largely unknown. As a step toward identification of fusogenic genes, we tested the concept that retroviral vectors can be packaged as a result of cell-cell fusion. First, we introduced replication-deficient retroviral vectors expressing mCAT-1, which mediates fusogenic interaction with the retroviral envelope protein Env, into Chinese hamster ovary (CHO) cells to generate vector cells. Plasmids expressing virion proteins Gag, Pol, and Env were introduced into a separate culture of CHO cells to generate packaging cells. Co-culturing vector and packaging cells resulted in production of infectious retroviruses carrying the mCAT-1 gene as a consequence of cell-cell fusion. Second, we introduced a retroviral vector into primary osteoclast precursors and co-cultured them with established osteoclast precursor RAW264.7 cells, which turned out to harbor packaging activity. Packaged retroviral vector was detected in culture supernatants only where the osteoclast differentiation factor receptor activator for NF-kappaB ligand (RANKL) induced fusion between these two cell types. These data suggest that retrovirus production can occur as a result of cell-cell fusion. This provides a novel approach for isolating and characterizing fusogenic genes using retroviral expression vectors.
Kondo
Takako
T
Department of Geriatric Research, National Institute for Longevity Sciences (NILS), Aichi 474-8522, Japan.
Ikeda
Kyoji
K
Matsuo
Koichi
K
D000818:Animals; D016466:CHO Cells; D002352:Carrier Proteins / Q000494:pharmacology; D027201:Cationic Amino Acid Transporter 1 / Q000235:genetics; D002459:Cell Fusion; D045744:Cell Line, Tumor; D006224:Cricetinae; D003412:Cricetulus; D015870:Gene Expression / Q000235:genetics; D015686:Gene Products, env / Q000235:genetics; D015683:Gene Products, gag / Q000235:genetics; D015684:Gene Products, pol / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics*; D015726:Giant Cells / Q000166:cytology / Q000821:virology; D008264:Macrophages / Q000187:drug effects / Q000821:virology; D008562:Membrane Glycoproteins / Q000494:pharmacology; D051379:Mice; D010010:Osteoclasts / Q000166:cytology* / Q000821:virology; D053245:RANK Ligand; D053246:Receptor Activator of Nuclear Factor-kappa B; D012190:Retroviridae / Q000235:genetics*; D015534:Trans-Activators / Q000235:genetics; D014162:Transfection; D019065:Virus Assembly / Q000235:genetics*
D002352:Carrier Proteins; D027201:Cationic Amino Acid Transporter 1; C070633:Gal-VP16; D015686:Gene Products, env; D015683:Gene Products, gag; D015684:Gene Products, pol; D008562:Membrane Glycoproteins; D053245:RANK Ligand; D053246:Receptor Activator of Nuclear Factor-kappa B; C420826:Slc7a1 protein, mouse; C505687:Tnfrsf11a protein, mouse; C505689:Tnfsf11 protein, mouse; D015534:Trans-Activators