Retroviral pgfp 5 rs vectors

C2C12 cells were seeded in 6-well culture plates in DMEM containing 10% FBS. At 50–70% confluence, medium was replaced by serum-free OptiMEM and cells were transfected with Septin7-specific shRNA constructs in retroviral pGFP-V-RS vectors (Origene, Cambridge, UK) using Lipofectamine 2000 transfection reagent. For controls, a noneffective scrambled shRNA cassette vector was employed (Scr). 3 hr after transfection, the medium was replaced by complete DMEM, cells were allowed to recover and synthesize the coded shRNA for 48 hr. Puromycin (2 µg/ml)-containing medium was applied to select cells containing the specific shRNA sequences until well-defined cell clones were visible within the culture plates. Individual clones were then separated and cultured further to analyze the effective gene silencing by Western blot. Appropriate clones presenting significant Septin7 expression change were tested throughout increasing passage numbers, and continuously detectable lower Septin7 expression was required to use the cell clone for further investigation (S7-KD cells).

The modification of Septin7 expression in C2C12 cells was evaluated using the plasmid-based shRNA technique as described earlier [41 (link)]. Briefly, C2C12 myoblasts were transfected with Septin7-specific shRNA constructs in retroviral pGFP-V-RS vectors (Origene, Cambridge, UK) using Lipofectamine 2000 transfection reagent in serum-free OptiMEM (Life Technologies). For controls, an ineffective scrambled shRNA cassette vector was employed (Scr). Briefly, 48 h after transfection, a Puromycin (2 µg/mL)-containing medium was applied to select cells containing the specific shRNA sequences. Individual clones were then separated and cultured further to analyze the effective gene silencing via Western blotting. Appropriate clones presenting a significant Septin7 expression change were tested throughout increasing passage numbers, and continuously detectable lower Septin7 expression was required to use the cell clone for further investigation (S7-KD cells).