Rac1 and Rac2 in Osteoclastogenesis: A Cell Immortalization Model

Background: Cell lines drawn from primary cells with a particular gene deletion are useful models for examining gene functions. This may be an especially useful technique for osteoclast studies as these are terminally differentiated cells that are often difficult and time consuming to generate. Objectives: establish osteoclast precursor cell lines from Rac1 null, Rac2 null and wildtype (WT) mice and evaluate the contribution of these genes in osteoclastogenesis by using these newly established cell lines. Methods: Bone marrow monocytes from mice were primed with macrophage colony stimulation factor and soluble receptor activator of NF-ęB ligand followed by transformation using a retrovirus containing SV40 large T-antigen and a neomycin-resistant cassette. Results: 7 to 19 immortal cell lines from each genotype were established. Among them, WT2, Rac1 null-D9 and Rac2 null-A2 were characterized and used to study the contribution of Rac1 and Rac2 to osteoclastogenesis. In vitro osteoclastogenesis showed that immortalized WT cells possess a high potential to differentiate into mature osteoclasts (fusion index 59.3 ± 6.9%). However, Rac null cells demonstrated defects in osteoclastogenesis (fusion index for Rac2 null and Rac1 null cells were 35.2 ± 5.4% and 15.0 ± 3.3%, respectively). Conclusions: We describe here a simple and effective protocol for generating immortalized mouse osteoclast precursor cell lines. Using this approach we show that both Rac1 and Rac2 small GTPases are required for osteoclastogenesis but that Rac1 is more critical. Grants: CIHR Grant to MG.