Figure 3

Cre-mediated hIgG expression in chicken oviduct primary cultured cells. (A) Expression of ovalbumin protein in oviduct primary cultured cells. Cell culture supernatants were collected 2 days after culture (culture sup.) or at the beginning of cell culture (control sup.) and analyzed by anti-ovalbumin immunoblotting. (B) Expression of hIgG in oviduct primary cultured cells. Cells were isolated from laying hen oviducts as described in Materials and methods. Oviduct primary cultured cells were singly or doubly transfected with the expression vectors, as shown in the panel. The cell culture supernatants were collected and analyzed by anti-human IgG Fc immunoblotting in the non-reduced condition. Obvious expression of hIgG was observed when pBS-DS-hIgG was co-transfected with pBS-Ova2.8-Cre or pCMV-Cre. (C) Expression of hIgG in chicken embryonic fibroblast cells. Fibroblast cells from 9-10 day chicken embryos were transfected with the expression vectors, as shown in the panel. hIgG expression was analyzed as described above. Expression of hIgG was observed when pBS-DS-hIgG was co-transfected with pCMV-Cre but not with pBS-Ova2.8-Cre (D) The induction levels of hIgG were examined by ELISA for human IgG. hIgG is abundant in the media of pBS-DS-hIgG and pBS-Ova2.8-Cre co-transfected oviduct primary culture cells (20 ng/ml) compared to levels in pBS-Ova2.8-hIgG-transfected cells (0.5 ng/ml). Essentially identical results were obtained in three independent experiments.