(See previous page).Analysis of immortalized bovine fibroblasts. (A) Bright-field pictures of in vitro cultured bovine fibroblasts. BFs at passage 7 were used as a control (a), and BBFs (b) and BTBFs (c) represented non-morphological changes at passage 42 compared with the early passage of BFs. (B) The population doubling time of immortalized bovine fibroblasts. Senescent cells did not show any cellular growth after 80 days of primary culture, but immortalized cells were populated continuously after the BFs' senescence. (C) The doubling time was only increased in BBFs as their passage increased, and they arrived at cellular senescence after passage 43 (b). (D) Clonogenic assay of each cell line; single colonies of BFs, BBFs, and BTBFs were stained using crystal blue, and the number of colonies in each group were counted (E). (F) Karyogram of BTBFs at passage 52. BTBFs have normal karyotype (b) like BFs (a). (BFs: Primary cultured bovine fibroblasts, BBFs: Bmi-1-introduced bovine fibroblasts, BTBFs, Bmi-1+hTert-introduced bovine fibroblasts).