Cre-mediated hIgG expression in 293T cells. (A) Cre-mediated reduction of the stuffer gene products in 293T cells. 293T cells were transfected with the control vector (pBS, top row), pBS-DS-hIgG (second row), or co-transfected with pBS-DS-hIgG and a low or high dose of the Cre expression vector (pCMV-Cre, third and bottom rows). Forty-eight hours after transfection, cells were visualized under a fluorescent microscope. Simultaneous expression of EGFP (green) and mCherry (red) was induced from two stuffer genes when cells were transfected only with pBS-DS-hIgG (second row). The expression of EGFP and mCherry were dramatically reduced by Cre expression in 293T cells (third row). The reduction of the stuffer gene expression was Cre-dose dependent (third and bottom rows). (B, C) Cre-dependent hIgG induction in 293T cells. pBS-DS-hIgG was transfected into 293T cells with or without pCMV-Cre. Forty-eight hours after transfection, cell culture supernatants were collected and analyzed by anti-human IgG Fc immunoblotting in the non-reduced condition (B) or by anti-human IgG (H+L) immunoblotting in the reduced condition (C). Expression of hIgG was observed when pBS-DS-hIgG was co-transfected with the Cre expression vector (lane 3, A and C) but not when the IgG expression vector was transfected alone (lane 2, B and C). In the non-reduced condition, hIgG is detectable as a single band of approximately 150 kDa, and in the reduced condition, two bands of approximately 50 and 25 kDa are detected. Trastuzumab was used as a recombinant hIgG control (lane 4, B and C).