Figure 2.

CAT expressions in cells infected with ecotropic retroviral vectors carrying an I-CAT gene or an intron-free CAT gene (CAT). (A) Transient expression of the CAT gene. NIH 3T3 cells were plated into 60-mm culture dishes at 5 × 10⁵ cells per dish 24 hr before infection. The cells in one dish were incubated with 1 × 10⁵ infectious retroviral vectors carrying the I-CAT or the intron-free CAT gene at 37°C for 24 hr in the presence of 4 μg/ml polybrene. Then the medium was replaced with fresh medium and the cells were further incubated at 37°C for 28 hr. The CAT expression was measured as described in Materials and Methods. Noninfected NIH 3T3 cells were used as a control. (B) CAT gene expression in G418 selected cells. NIH 3T3 cells were infected with the I-CAT or CAT retroviral vectors and then incubated for 6 days in the presence of 600 μg/ml G418. The cells were trypsinized, replated (1 × 10⁶ cells per 60-mM dish), cultured at 37°C for 24 hr, and tested for CAT activity. (C) CAT gene expression in cell clones. The infected cells were incubated for 11 days in the presence of 600 μg/ml G418. Nine resistant colonies were picked from each infection (CAT 1–9 and I-CAT 1–9) and expanded by incubation for 11–18 days. The CAT activity of the cells was measured as described in B. Bars = SD.