Analysis of mutant cell lines.

<p>Panels A and C–E show growth curve of the parental 427 90-13 BSF cells (WT) and mutant cell lines incubated in the IMDM medium. The mutant cell lines are <i>^RNAi</i>PDH (A and D), Δ<i>tdh</i> (C) and Δ<i>tdh</i>/<i>^RNAi</i>PDH (E) incubated in the presence (.i, +tet) or in the absence (.ni) of tetracycline. In panel D, the <i>^RNAi</i>PDH.i cell line was incubated in threonine-depleted medium supplemented with 0 to 150 µM threonine. The cells were maintained in the exponential growth phase (between 2×10⁵ and 2×10⁶ cells/mL) and cumulative cell numbers reflect normalization for dilution during cultivation. The insets in panels A and C–E show western blot analyses of the parental (WT) and mutant cell lines with the immune sera indicated in the right margin. The lower inset in panel C shows the TDH activity (milliunits/mg of protein) measured in the WT and Δ<i>tdh</i> cell lines (ND stands for not detectable). Panel B shows a PCR analysis of genomic DNA isolated from the parental WT and Δ<i>tdh</i> cell lines. Amplifications were performed with primers based on sequences that flank the 5′UTR and 3′UTR fragments used to target the <i>TDH</i> gene depletion (black boxes) and internal sequences of the puromycin (<i>PAC</i>, PCR products 3 and 5) or blasticidin (<i>BSD</i>, PCR products 4 and 6) resistance genes. As controls, we also used primers corresponding to the 5′UTR (PCR product 1), the <i>TDH</i>, <i>PAC</i> and <i>BLE</i> genes (PCR products 2, 7 and 8, respectively). As expected, PCR amplification of the 5′UTR was observed for both cell lines (lane 1), while the <i>TDH</i> gene was PCR-amplified only in the WT cells (lane 2) and PCR products with marker-derived primers were observed only in the Δ<i>tdh</i> cell line (lanes 3–8).