A tightly regulated inducible expression system for conditional gene knock-outs and dominant-negative genetics in Trypanosoma brucei

Targeted depletion of a mitochondrial nucleotidyltransferase suggests the presence of multiple enzymes that polymerize mRNA 3′ tails in Trypanosoma brucei mitochondria

“…Procyclic T. brucei brucei strain 29-13, which contains integrated genes for T7 RNA polymerase (T7RNAP) and tetracycline repressor (TetR), were grown in SDM-79 medium supplemented with 15% fetal bovine serum (FBS) at 27°C as described previously (36,39) in the presence of G418 (15 μg/ ml) and hygromycin (50 μg/ml). The bloodstream single-marker cell line, which also expresses T7RNAP and TetR, were maintained in HMI-9 medium supplemented with 10% FBS and 10% Serum Plus (JRH Biosciences) at 37°C/5% CO 2 as described previously (37) in the presence of G418 (2.5 μg/ml).…”

“…A 476-bp fragment of the kPAP2 coding region corresponding to nts 200-675 from the start codon was cloned into p2T7-177 vector between opposing T7 promoters (40). The resulting construct was transfected into either procyclic T. brucei 29-13 cells or bloodstream single marker cells, both of which express TetR protein and T7RNAP (39). Stable clonal cells that had integrated the kPAP2 fragment into the 177 bp repeat locus on the minichromosomes were selected by phleomycin treatment and used for subsequent analyses.…”

The transcriptomes of Trypanosoma brucei Lister 427 and TREU927 bloodstream and procyclic trypomastigotes

“…brucei TREU 927 was adapted relatively recently to in vitro culture and can be transmitted readily by Tsetse [8]; it also shows limited resistance to human serum [9]. We were therefore interested in comparing its gene regulation with that of strain Lister 427, which has been subjected to in vitro culture or animal passage for over 30 years and is a standard strain for genetic manipulation [10]. Since comparisons of mRNAs do not detect translational regulation, we were also interested to find out whether additional information could be obtained from a polysomal RNA fraction.…”

Telomere shortening and cell cycle arrest in Trypanosoma brucei expressing human telomeric repeat factor TRF1

“…brucei strain 427, antigenic type 1.2 (MITat 1.2), clone 221a [13], had been previously manipulated to create the PF cell line 29-13 and the BF cell line 13-90 that constitutively express T7 RNA polymerase from the tubulin locus and TetR from the RNP1 locus [35]. PF were cultured at 27°C in SDM-79 supplemented with fetal bovine serum and containing 15 mg ml − 1 G418 (Sigma) and 25 mg ml − 1 hygromycin (Sigma), to a maximum cell density of 10 7 trypanosomes ml − 1 .…”

“…BF were cultured in HMI-9 containing 5 mg ml − 1 G418 and 5 mg ml − 1 hygromycin to a maximum cell density of 2× 10 6 trypanosomes ml − 1 . The FLAG-TRF1 fusion was cloned in the HindIII-digested plasmid pLew111 [36], a derivative of pLew82 [35]. All transfections were done as previously described [37].…”