FLA3 is an essential protein in the bloodstream form o<i>f T. brucei.</i>

<p>Panel A. Northern blot analysis of 15 µg of total RNA from a cloned FLA3 bloodstream RNAi cell line grown under induced or noninduced conditions for 24 h. The probe used for hybridisation was the same fragment employed for RNAi. The lower part of the panel presents the ethidium bromide staining of the RNA as a control for loading. The arrow indicates the presence of the dsRNA (∼ 600 bp) in the induced cells. Panel B. An immunoblot analysis confirmed loss of the FLA3 protein in the induced cells after 30 h induction. Each lane contained 5×10⁶ cells and a loading control was performed with antibodies against trypanosome actin. Panel C. The effect of knock down of FLA3 on the growth of a representative cloned FLA3 RNAi cell line cultured in the presence (○) or absence of tetracycline (•). Panel D. Analysis of the number of nuclei and kinetoplasts per cell in a FLA3 RNAi cell line cultured in the presence of tetracycline. For each time point individual cells were assessed for the presence of kinetoplast and nucleus and scored as 1N1K, 1N2K, 2N2K and cells with clearly more than 2N. The results were expressed as the mean ± SD of four separate surveys of 50 cells.