Tertiary screening in the bloodstream stage. Analysis of RNAi cell lines generated in BS T. brucei for dynein heavy chain (Tb927.3.930, Panel A), hypothetical ORF (Tb927.5.3260, Panel B) and protein phosphatase 1 (Tb11.01.0450, Panel C). Panels A and B, upper left and Panel C, left: cumulative growth curves for each clone cultured in HMI9 with appropriate selective drugs [13, 14] in the absence (-tet) or presence (+tet) of tetracycline; Panels A and B, lower left and Panel C, middle: analysis of nuclei and kinetoplast numbers as determined over time by DAPI staining; Panels A and B, upper right and Panel C, right: flow cytometry profiles at the time points indicated; Panels A and B, lower right: examples of abnormal cells generated (left, DIC image; right, DAPI image. Black bar: 5 μm. The number of nuclei (N) and kinetoplasts (K) in each cell is given). Panel D: real time PCR analysis of mRNA levels for each gene (standardised against a GPI8 endogenous control, using oligonucleotides detailed in Additional file 5) at 6.5 hrs (dynein heavy chain) and 16 hrs (hypothetical ORF and PP1) post-induction.