Expression of exogenous genes in Trypanosoma cruzi : improving vectors and electroporation protocols

Abstract

To improve transfection efficiency in Trypanosoma cruzi, we developed a new electroporation protocol and expression vectors which use luciferase and green and red fluorescent proteins as reporter genes. In transient transfections, the electroporation conditions reported here resulted in luciferase expression 100 times higher than the levels obtained with previously described protocols. To verify whether sequences containing different trans-splicing signals influence reporter gene expression, we compared DNA fragments corresponding to 5' untranslated plus intergenic (5' UTR plus Ig) regions from GAPDH, TcP2beta, alpha- and beta- tubulin and amastin genes. Vectors containing sequences derived from the first four genes presented similar efficiencies and resulted in luciferase expression in transiently transfected epimastigotes that was up to 10 times higher than that for a control vector. In contrast, the amastin 5' UTR plus Ig resulted in lower levels of reporter gene expression. We also constructed a vector containing an expression cassette designed to be targeted to the tubulin locus of the parasite.

Trypanosoma cruzias an effective cancer antigen delivery vector

“…We engineered the CL-14 to express NY-ESO-1, a well-defined and highly immunogenic CTA, which is currently being tested as a vaccine candidate against cancer in different clinical trials (16)(17)(18)(19). For homologous recombination, we used the pROCKNeo plasmid (20) that promotes insertion of the transgene in one of the many copies of the β-tubulin gene in the T. cruzi genome (Fig. S1A).…”

“…Transgenic parasites were obtained by cloning the NY-ESO-1 Open reading frame (ORF), fused to His tag or to the gp63 SP sequences into the pROCKNeo plasmid. T. cruzi epimastigotes were transfected and maintained as previously described (20). The integration of the exogenous gene into the parasite genome was confirmed by Southern blot analysis of genomic DNA digested with BamHI and SacI and hybridized with NY-ESO-1 and amastin probes labeled with …”

Developmental Expression of a Trypanosoma cruzi Phosphoinositide-Specific Phospholipase C in Amastigotes and Stimulation of Host Phosphoinositide Hydrolysis

“…However, our previous attempts to knockout TcPI-PLC gene were unsuccessful, and we concluded that the enzyme is essential for T. cruzi (18). In addition, T. cruzi lacks the machinery for RNA interference, which is present in T. brucei (4). To further complicate things, no comparable experiments could be done using T. brucei conditional knockout parasites because, although this parasite possesses an orthologue of TcPI-PLC, it lacks an intracellular stage.…”

Cloning and characterization of DNA polymerase η from Trypanosoma cruzi: Roles for translesion bypass of oxidative damage

“…Cells were grown at 288C in liver infusion tryptose (LIT) medium supplemented with 10% fetal calf serum (FCS), as described by Camargo [1964]. Empty pROCKNeo [DaRocha et al, 2004] transfected population were used as controls.…”

“…Fragments obtained after digestion with Spe I and EcoR I were purified and inserted into the Xba I and EcoR I sites of pTREXGFP vector [Vazquez and Levin, 1999] to produce the plasmid pTREXGFP_Polh. Parasites were transfected with pTREXGFP_Polh as described by DaRocha [DaRocha et al, 2004]. Twenty-four hours after electroporation, 5 3 10 6 transfected parasite cells were harvested, washed twice in PBS, and centrifuged at 3,000g for 10 min.…”