Figure 2.

The pattern of expression levels of FEZ1 transcripts correlates with the levels of retroviral resistance in the various Rat2 lines. (A–C) Overexpression of FEZ1 blocks retrovirus infection in Rat2 cells. Cells transduced with the empty vector pcDNA4 or the pcDNA4 vector expressing FEZ1 originated from the mutant R3-2 line, m-FEZ1, were incubated with various amounts of MLV-N-neo (A), MLV-B-neo (B), and HIV-1-puro (C). Cells were selected in medium containing G418 (A,B; Neoviruses) or puromycin (C; HIV-1) and the number of the transduced colonies were then counted after 8–12 d. The wild-type Rat2 cells and the parental R3-2 line were included as negative and positive controls, respectively. Similar results were obtained in at least three independent experiments. (D,E) Quantitative RT–PCR showing the level of FEZ1 expression in Rat2 cells. Cytoplasmic RNA was prepared from the same cell lines used in the transduction assay. The ds cDNA was generated and used as template in RT–PCR assays. Primers unique to each cDNA sequences were used to distinguished the transgene FEZ1 (D) from the total (endogenous and transgene) (E) FEZ1 transcripts. The FC ratio between the Rat2 mutant lines and the control empty vector line (pcDNA4:3) are given as median copy numbers normalized to GAPDH copy numbers obtained in duplicate from two independent RNA preparations.