Figure 1
From: Searching for novel cell cycle regulators in Trypanosoma brucei with an RNA interference screen
Tertiary screening in the procyclic form. Independent RNAi cell lines generated in PF T. brucei for dynein heavy chain (Tb927.3.930), panel A; TOR1 (Tb10.6k15.2060), panel B; TOR-like 2 kinase (Tb927.1.1930), panel C and a hypothetical ORF (Tb927.5.3260), panel D, were analysed for cell cycle progression defects following RNAi induction. Upper left of each panel: cumulative growth curves for each clone cultured in SDM79 with appropriate selective drugs [13, 14] and the absence (-tet) or presence (+tet) of 1 μgml-1 tetracycline. Lower left of each panel: real time PCR analysis of mRNA levels for each gene (using oligonucleotides detailed in Additional file 5 and standardised against a GPI8 control) at 96 hrs (dynein heavy chain and hypothetical ORF), 161 hrs (TOR-like 2 kinase) or 212 hrs (TOR1) post-induction. Middle of each panel: analysis of nuclei and kinetoplast numbers as determined over time by DAPI staining. Upper right of panels A and D and right of panels B and C: flow cytometry profiles of uninduced (-tet) and induced (+tet) cells at the time points indicated. The DNA content of each peak is given. Lower right panels A and D: 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.