Tagging a T. brucei RRNA locus improves stable transfection efficiency and circumvents inducible expression position effects

Abstract

In Trypanosoma brucei, RNA interference (RNAi) and recombinant protein expression are established as powerful approaches for functional genomics, particularly when combined with inducible expression. The favoured methods involve exploiting homologous recombination to target expression cassettes to a chromosome sub-set to establish stable cell lines. Unfortunately, bloodstream-form cells, those that cause disease in mammals, exhibit low efficiency stable transfection. Current expression systems can also exhibit other undesirable features, including variable position effects and leaky, inducible expression. We have developed systems in bloodstream-form cells that alleviate these problems. Using constructs for RNAi and expression of (GFP) tagged proteins, we target a (hyg) tagged ribosomal RNA (RRNA) locus which circumvents position effects and allows increased targeting efficiency. We also report a compatible double-inducible system for tight regulation of highly toxic products. This system exploits a new inducible RRNA promoter to drive T7 RNA polymerase (T7RNAP) transcription which then drives expression from inducible T7 promoters. The developments described should facilitate functional analysis and increased throughput.

Fig 1

Expression of T7RNAP from a Tet-inducible RRNA promoter. (A) NotI-digestion targets pRP^T7pol to an RRNA locus. BLA, blasticidin resistance. Arrows represent promoters. RNA processing regions and promoters: ALD, aldolase; PRO, procyclin; TUB, tubulin. (B and C) Western blot analysis of T7RNAP expression. The blots were probed with primary mouse α-T7RNAP (1:5000). The lower panels show coomassie-stained gels as loading controls. (B) Six independent cell-lines expressing TetR and transfected with pRP^T7pol express different levels of T7 polymerase upon induction with 1μg ml⁻¹ Tet. Lane 1: un-induced cell line 1. (C) Induction of cell line 1 with 1ng to 1μg ml⁻¹ Tet showed that T7 polymerase expression was dose dependent, peaking in the presence of 50ng ml⁻¹ Tet. (D) Growth analysis was carried out in triplicate and populations were diluted as shown. Error bars: standard deviation.

Fig. 2

Tagging an RRNA locus. (A) NotI-digested pRP^GFP and SalI/SacI-digested ph3E were used to identify an RRNA locus with no evidence of position effect repression and to add the hyg-tag to that locus respectively. The resultant tag can then be replaced using AscI-digested pRPa^GFP. HYG, hygromycin resistance; PAC, puromycin resistance; ACT, actin; ES, variant surface glycoprotein Expression-Site. (B) Western blot analysis of inducible expression in three independent clones generated using non-tagged cells and pRP^cMyc with a reporter gene (GeneDB: Tb927.4.2520). The blot was probed with primary mouse α-cMyc (1:2000). (C) Western blot analysis of induced expression in three independent and representative clones generated using tagged RRNA locus cells and pRPa^GFP with a reporter gene (GeneDB: Tb11.01.3380). Lane 1: un-induced cell line 1. The blot was probed with primary rabbit α-GFP (1:4000). Other details as in Fig. 1.

Fig. 3

Increased transfection efficiency in cells with a hyg-tagged RRNA locus. HYG selection was administered <6h following electroporation to minimise cell division prior to distribution of cells in 96-well plates. Data for multiple targets was derived over several time points. Error bars: standard deviation.

Fig. 4

Double-inducible expression in cells with a hyg-tagged RRNA locus. (A and B) A reporter gene (GeneDB: Tb10.6k15.3240) was cloned in pT7a^GFP (similar to pRPa^GFP but with a T7 promoter in place of an RRNA promoter) and the nuclear GFP-tagged protein was expressed in T7ⁱ:Tag cells. (A) Induction was with 5ng ml⁻¹ Tet for 24h. The western blot was probed with primary rabbit α-GFP (1:4000). The left hand panel shows a coomassie-stained gel as a loading control. (B) Direct detection of the GFP-tagged protein. DNA counterstained with (DAPI); k, kinetoplast; n, nucleus. (C) The p2T7a^TAblue RNAi construct. Details as in Fig. 1A. (D and E) dsRNA against α/β-tubulin was expressed in T7ⁱ:Tag cells using p2T7a^TUB, a derivative of p2T7a^TAblue. Details as above. (D) Microscopy. Induction was with 5ng ml⁻¹ Tet for 20h. (E) Growth analysis. Details as in Fig. 1D.