Figure 3

Enhancement of recombinant protein production at 32°C by MCRE. (A, B) CHO-K1 Flp-In cells were stably transfected with the pcDNA5/FRT vector expressing secreted alkaline phosphatase (SEAP) under the control of the CMV promoter with (+) or without (−) an enhancer containing 48 copies of MCRE and its active mutant (MCRExx48). Cells were cultured at 32°C or 37°C for 3 days, and the SEAP activity in culture media (A) and mRNA levels in cells (B) were analyzed. SEAP mRNAs were quantified by quantitative RT-PCR, and expressed after normalization to those of β-actin. a.u., arbitrary unit. Values are mean ± SE. nd, not different. *, significantly different (p <0.05). (C) CHO-K1 Flp-In cells were stably transfected with the pcDNA5/FRT vector expressing erythropoietin (EPO) under the control of the CMV promoter with (+) or without (−) the MCRExx48 enhancer. Cells were cultured at 32°C or 37°C for 3 days, and the EPO concentration in culture media was determined. Values are expressed as relative to the value obtained in cells transfected with plasmids having CMV promoter alone (−) and cultured at 37°C. (D) U-2 OS Flp-In cells were stably transfected with the indicated pcDNA5/FRT vector expressing firefly luciferase (Luc) under the control of the CMV promoter with (+) or without (−) MCRExx48. Cells were transferred to 32°C or remained at 37°C for indicated times. Cell extracts were assayed for Luc activity and normalized to the protein level. Values are expressed as relative to the value obtained in cells transfected with plasmids having CMV promoter alone (−) at time 0, and mean ± SE. Experiments were repeated twice (A, B) and three times (C, D), with similar results.