A modular and optimized single marker system for generatingTrypanosoma bruceicell lines expressing T7 RNA polymerase and the tetracycline repressor

Abstract

Here, we present a simple modular extendable vector system for introducing the T7 RNA polymerase and tetracycline repressor genes into Trypanosoma brucei. This novel system exploits developments in our understanding of gene expression and genome organization to produce a streamlined plasmid optimized for high levels of expression of the introduced transgenes. We demonstrate the utility of this novel system in bloodstream and procyclic forms of Trypanosoma brucei, including the genome strain TREU927/4. We validate these cell lines using a variety of inducible experiments that recapture previously published lethal and non-lethal phenotypes. We further demonstrate the utility of the single marker (SmOx) TREU927/4 cell line for in vivo experiments in the tsetse fly and provide a set of plasmids that enable both whole-fly and salivary gland-specific inducible expression of transgenes.

Two related trypanosomatid eIF4G homologues have functional differences compatible with distinct roles during translation initiation

“…For the p2T7-177 (RNAi) and p2216 (eYFP fusions) derived plasmids, the procyclic cell line Lister 427 29-13 was used 71 and transfected cells were selected after growth in the presence of phleomycin (2.5 mg/ml). For transfections of plasmids derived from p3927, 68 a 427 KG cell line expressing a modified version of pSMOx 72 as background was used and transfected cells were selected after growth in the presence of blasticidin (10 mg/ml). Expression of TY-tagged proteins was induced after tetracycline addition (1 mg/ml) and cellular growth was monitored by counting the number of viable cells at regular intervals.…”

Tubulin-binding cofactor C domain-containing protein TBCCD1 orchestrates cytoskeletal filament formation

“…Trypanosomes (SmOxP927; Poon et al, 2012) were cultured in SDM-79 medium supplemented with 10% (v/v) heat-inactivated fetal bovine serum (Brun and Schönenberger, 1979). Stable transformation used standard methods.…”

An efficient cumate-inducible system for procyclic and bloodstream form Trypanosoma brucei

“…Next, we modified the inducible expression vector pDEX777 [18] by replacing the 3× tetracycline operator (TetO) with 3× cumate operator (CuO) to create the cumateinducible expression vector pDEX-CuO (Fig. 1B).…”

“…S2). The recombinant gene was then inserted into the pSmOx vector [18] between the T7 RNA polymerase (RNAP) and tetracycline repressor (TetR), to create a pSmOxNus (Single Marker Oxford, NUS modified) vector (Fig. 1A).…”