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Construction of three new Gateway® expression plasmids for Trypanosoma cruzi.

Alonso VL, Ritagliati C, Cribb P, Serra EC - Mem. Inst. Oswaldo Cruz (2014)

Bottom Line: Both plasmids were assayed by introducing green fluorescent protein (GFP) by recombination and the integrity of the double-tagged protein was determined by western blotting and immunofluorescence microscopy.The third Gateway adapted vector assayed was the inducible pTcINDEX.When tested with GFP, pTcINDEX-GW showed a good response to tetracycline, being less leaky than its precursor (pTcINDEX).

View Article: PubMed Central - PubMed

Affiliation: Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Rosario, Argentina.

ABSTRACT
We present here three expression plasmids for Trypanosoma cruzi adapted to the Gateway® recombination cloning system. Two of these plasmids were designed to express trypanosomal proteins fused to a double tag for tandem affinity purification (TAPtag). The TAPtag and Gateway® cassette were introduced into an episomal (pTEX) and an integrative (pTREX) plasmid. Both plasmids were assayed by introducing green fluorescent protein (GFP) by recombination and the integrity of the double-tagged protein was determined by western blotting and immunofluorescence microscopy. The third Gateway adapted vector assayed was the inducible pTcINDEX. When tested with GFP, pTcINDEX-GW showed a good response to tetracycline, being less leaky than its precursor (pTcINDEX).

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: pTcINDEX-GW experimental validation. A: fluorescencemicroscopy of Dm28c epimastigotes transfected withpTcINDEX-GW-green fluorescent protein (GFP) andpTcINDEX-Red before and after induction with tetracycline (0.5µg mL-1 for 96 h). Parasites were fixed with 4% paraformaldehyde inphosphate-buffered saline (PBS), permeabilised with 0.1% Triton X-100 in PBS andincubated with 2 µg mL-1 4’-6-diamidino-2-phenylindole (DAPI) in PBS beforemounting the slides with VectaShield (Vector Laboratories). Images were acquiredwith a Nikon Eclipse TE-2000-E2 confocal microscope; B: western blot analysis ofDm28c pTcINDEX-GW-GFP total lysates induced with 0.5 µg mL-1tetracycline for 0, 24, 48 and 72 h. Anti-GFP (a-GFP) and anti-αtubulin antibodies were used. Bound antibodies were detected withperoxidase-labelled anti-mouse or anti-rabbit immunoglobulin G (GE Healthcare) andECL Prime (GE Healthcare) using standard protocols; C: growth curve of Dm28cpTcINDEX-GW-GFP induced with different concentrations oftetracycline (0-5 µg mL-1); D: expression of GFP after induction (0.5 µg mL-1 oftetracycline for 96 h) in Dm28c and CL Brener strains transfectedwith pTcINDEX-GW-GFP; E: fluorescence microscopy ofDm28c pTcINDEX-GW-GFP at different stages ofthe life cycle. Parasites were fixed with 4% paraformaldehyde in PBS,permeabilised with 0.1% Triton X-100 in PBS and incubated with 2 µg mL-1 DAPI inPBS before mounting the slides with VectaShield (Vector Laboratories). Images wereacquired with a Nikon Eclipse E300 microscope.
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f03: : pTcINDEX-GW experimental validation. A: fluorescencemicroscopy of Dm28c epimastigotes transfected withpTcINDEX-GW-green fluorescent protein (GFP) andpTcINDEX-Red before and after induction with tetracycline (0.5µg mL-1 for 96 h). Parasites were fixed with 4% paraformaldehyde inphosphate-buffered saline (PBS), permeabilised with 0.1% Triton X-100 in PBS andincubated with 2 µg mL-1 4’-6-diamidino-2-phenylindole (DAPI) in PBS beforemounting the slides with VectaShield (Vector Laboratories). Images were acquiredwith a Nikon Eclipse TE-2000-E2 confocal microscope; B: western blot analysis ofDm28c pTcINDEX-GW-GFP total lysates induced with 0.5 µg mL-1tetracycline for 0, 24, 48 and 72 h. Anti-GFP (a-GFP) and anti-αtubulin antibodies were used. Bound antibodies were detected withperoxidase-labelled anti-mouse or anti-rabbit immunoglobulin G (GE Healthcare) andECL Prime (GE Healthcare) using standard protocols; C: growth curve of Dm28cpTcINDEX-GW-GFP induced with different concentrations oftetracycline (0-5 µg mL-1); D: expression of GFP after induction (0.5 µg mL-1 oftetracycline for 96 h) in Dm28c and CL Brener strains transfectedwith pTcINDEX-GW-GFP; E: fluorescence microscopy ofDm28c pTcINDEX-GW-GFP at different stages ofthe life cycle. Parasites were fixed with 4% paraformaldehyde in PBS,permeabilised with 0.1% Triton X-100 in PBS and incubated with 2 µg mL-1 DAPI inPBS before mounting the slides with VectaShield (Vector Laboratories). Images wereacquired with a Nikon Eclipse E300 microscope.

Mentions: Once the lines were selected, we monitored the expression of the fluorescent proteins ininduced and un-induced cultures by fluorescence microscopy (Fig. 3A). GFP was observed in the cultures induced with tetracycline, but not inthose under un-induced conditions. In contrast, the pTcINDEX-Red strain(used as a transfection control) showed leaky expression because we detected redfluorescence in un-induced parasites. The expression of GFP was also tested by western blotassays (Fig. 3B). We observed that between 48-72 hpost-induction, there were no significant changes in the expression level of GFP. Then, weperformed growth curves in the presence of different concentrations of tetracycline (Fig. 3C) and observed that the parasites grew normallyusing up to 5 µg mL-1 of the antibiotic. We demonstrated that the attB1 andattB2 sequences generated by recombination did not affect the expression of exogenousproteins in T. cruzi. If anything, these sequences appear to have made thevector more efficient because there was no leaky expression in the Dm28cpTcINDEX-GW-GFP strain.


Construction of three new Gateway® expression plasmids for Trypanosoma cruzi.

Alonso VL, Ritagliati C, Cribb P, Serra EC - Mem. Inst. Oswaldo Cruz (2014)

: pTcINDEX-GW experimental validation. A: fluorescencemicroscopy of Dm28c epimastigotes transfected withpTcINDEX-GW-green fluorescent protein (GFP) andpTcINDEX-Red before and after induction with tetracycline (0.5µg mL-1 for 96 h). Parasites were fixed with 4% paraformaldehyde inphosphate-buffered saline (PBS), permeabilised with 0.1% Triton X-100 in PBS andincubated with 2 µg mL-1 4’-6-diamidino-2-phenylindole (DAPI) in PBS beforemounting the slides with VectaShield (Vector Laboratories). Images were acquiredwith a Nikon Eclipse TE-2000-E2 confocal microscope; B: western blot analysis ofDm28c pTcINDEX-GW-GFP total lysates induced with 0.5 µg mL-1tetracycline for 0, 24, 48 and 72 h. Anti-GFP (a-GFP) and anti-αtubulin antibodies were used. Bound antibodies were detected withperoxidase-labelled anti-mouse or anti-rabbit immunoglobulin G (GE Healthcare) andECL Prime (GE Healthcare) using standard protocols; C: growth curve of Dm28cpTcINDEX-GW-GFP induced with different concentrations oftetracycline (0-5 µg mL-1); D: expression of GFP after induction (0.5 µg mL-1 oftetracycline for 96 h) in Dm28c and CL Brener strains transfectedwith pTcINDEX-GW-GFP; E: fluorescence microscopy ofDm28c pTcINDEX-GW-GFP at different stages ofthe life cycle. Parasites were fixed with 4% paraformaldehyde in PBS,permeabilised with 0.1% Triton X-100 in PBS and incubated with 2 µg mL-1 DAPI inPBS before mounting the slides with VectaShield (Vector Laboratories). Images wereacquired with a Nikon Eclipse E300 microscope.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4325611&req=5

f03: : pTcINDEX-GW experimental validation. A: fluorescencemicroscopy of Dm28c epimastigotes transfected withpTcINDEX-GW-green fluorescent protein (GFP) andpTcINDEX-Red before and after induction with tetracycline (0.5µg mL-1 for 96 h). Parasites were fixed with 4% paraformaldehyde inphosphate-buffered saline (PBS), permeabilised with 0.1% Triton X-100 in PBS andincubated with 2 µg mL-1 4’-6-diamidino-2-phenylindole (DAPI) in PBS beforemounting the slides with VectaShield (Vector Laboratories). Images were acquiredwith a Nikon Eclipse TE-2000-E2 confocal microscope; B: western blot analysis ofDm28c pTcINDEX-GW-GFP total lysates induced with 0.5 µg mL-1tetracycline for 0, 24, 48 and 72 h. Anti-GFP (a-GFP) and anti-αtubulin antibodies were used. Bound antibodies were detected withperoxidase-labelled anti-mouse or anti-rabbit immunoglobulin G (GE Healthcare) andECL Prime (GE Healthcare) using standard protocols; C: growth curve of Dm28cpTcINDEX-GW-GFP induced with different concentrations oftetracycline (0-5 µg mL-1); D: expression of GFP after induction (0.5 µg mL-1 oftetracycline for 96 h) in Dm28c and CL Brener strains transfectedwith pTcINDEX-GW-GFP; E: fluorescence microscopy ofDm28c pTcINDEX-GW-GFP at different stages ofthe life cycle. Parasites were fixed with 4% paraformaldehyde in PBS,permeabilised with 0.1% Triton X-100 in PBS and incubated with 2 µg mL-1 DAPI inPBS before mounting the slides with VectaShield (Vector Laboratories). Images wereacquired with a Nikon Eclipse E300 microscope.
Mentions: Once the lines were selected, we monitored the expression of the fluorescent proteins ininduced and un-induced cultures by fluorescence microscopy (Fig. 3A). GFP was observed in the cultures induced with tetracycline, but not inthose under un-induced conditions. In contrast, the pTcINDEX-Red strain(used as a transfection control) showed leaky expression because we detected redfluorescence in un-induced parasites. The expression of GFP was also tested by western blotassays (Fig. 3B). We observed that between 48-72 hpost-induction, there were no significant changes in the expression level of GFP. Then, weperformed growth curves in the presence of different concentrations of tetracycline (Fig. 3C) and observed that the parasites grew normallyusing up to 5 µg mL-1 of the antibiotic. We demonstrated that the attB1 andattB2 sequences generated by recombination did not affect the expression of exogenousproteins in T. cruzi. If anything, these sequences appear to have made thevector more efficient because there was no leaky expression in the Dm28cpTcINDEX-GW-GFP strain.

Bottom Line: Both plasmids were assayed by introducing green fluorescent protein (GFP) by recombination and the integrity of the double-tagged protein was determined by western blotting and immunofluorescence microscopy.The third Gateway adapted vector assayed was the inducible pTcINDEX.When tested with GFP, pTcINDEX-GW showed a good response to tetracycline, being less leaky than its precursor (pTcINDEX).

View Article: PubMed Central - PubMed

Affiliation: Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Rosario, Argentina.

ABSTRACT
We present here three expression plasmids for Trypanosoma cruzi adapted to the Gateway® recombination cloning system. Two of these plasmids were designed to express trypanosomal proteins fused to a double tag for tandem affinity purification (TAPtag). The TAPtag and Gateway® cassette were introduced into an episomal (pTEX) and an integrative (pTREX) plasmid. Both plasmids were assayed by introducing green fluorescent protein (GFP) by recombination and the integrity of the double-tagged protein was determined by western blotting and immunofluorescence microscopy. The third Gateway adapted vector assayed was the inducible pTcINDEX. When tested with GFP, pTcINDEX-GW showed a good response to tetracycline, being less leaky than its precursor (pTcINDEX).

Show MeSH