Cloning and expression of transgenes using linear vectors in Trypanosoma cruzi

Curto, Maria de Los Angeles; Lorenzi, Hernán; Barros, Roberto R. Moraes; Souza, Renato T.; Levìn, Mariano J.; Silveira, José Franco da; Schijman, Alejandro G.

doi:10.1016/j.ijpara.2014.03.009

Cited by 4 publications

(6 citation statements)

References 52 publications

(72 reference statements)

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“…As previously demonstrated, pTAC vectors can exist as highly stable linear extrachromosomal elements, showing accurate replication and segregation comparable to that of natural T. cruzi chromosomes (Curto et al, 2014). We were also able to introduce a DSB at a targeted subtelomeric locus carried in an artificial chromosome by means of heterologous expression of yeast meganuclease I-SceI even though T. cruzi is generally considered to be refractory to genetic manipulation.…”

Section: Discussionmentioning

confidence: 56%

“…Transfectants were selected in the presence of G418 at 500 μg/mL in LIT medium with 10% fetal bovine serum. Next, epimastigotes expressing I-SceI meganuclease or not were transfected with pTAC, pTAC-D6C ∗ or pTAC-D6C ∗I-SceI vectors and grown in the presence of G418 (100 μg/mL) and puromycin (10 μg/mL; Curto et al, 2014). Double transfectants were then cloned by serial dilution in a 96-well plate, and clones were evaluated by fluorescence microscopy to confirm expression of GFP in the nucleus of the mutant cell lines.…”

Section: Methodsmentioning

confidence: 99%

“…Recently, a linear vector series termed pTAC, for Trypanosoma Artificial Chromosome, has proven to be a useful set of tools for genetic studies in T. cruzi because of the high stability of these vectors as linear episomes and their faithful segregation in all the parasite developmental forms (Curto et al, 2014). In the study by Curto et al (2014) T. cruzi epimastigotes were transfected with a pTAC carrying a 1.7-kb fragment of a subtelomeric ψTS sequence, and after transgenic parasites were subjected to different conditions, no recombination events involving the displacement of the ψTS sequence from the pTAC to the parasite genome were detected.…”

Section: Introductionmentioning

confidence: 99%

“…In the study by Curto et al (2014) T. cruzi epimastigotes were transfected with a pTAC carrying a 1.7-kb fragment of a subtelomeric ψTS sequence, and after transgenic parasites were subjected to different conditions, no recombination events involving the displacement of the ψTS sequence from the pTAC to the parasite genome were detected. Their results therefore suggest that the presence of a ψTS sequence in the construct was not sufficient to induce recombination between the artificial chromosome pTAC and an endogenous chromosome.…”

Section: Introductionmentioning

confidence: 99%

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Subtelomeric I-SceI-Mediated Double-Strand Breaks Are Repaired by Homologous Recombination in Trypanosoma cruzi

et al. 2016

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Trypanosoma cruzi chromosome ends are enriched in surface protein genes and pseudogenes (e.g., trans-sialidases) surrounded by repetitive sequences. It has been proposed that the extensive sequence variability among members of these protein families could play a role in parasite infectivity and evasion of host immune response. In previous reports we showed evidence suggesting that sequences located in these regions are subjected to recombination. To support this hypothesis we introduced a double-strand break (DSB) at a specific target site in a T. cruzi subtelomeric region cloned into an artificial chromosome (pTAC). This construct was used to transfect T. cruzi epimastigotes expressing the I-SceI meganuclease. Examination of the repaired sequences showed that DNA repair occurred only through homologous recombination (HR) with endogenous subtelomeric sequences. Our findings suggest that DSBs in subtelomeric repetitive sequences followed by HR between them may contribute to increased variability in T. cruzi multigene families.

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Section: Discussionmentioning

confidence: 56%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Subtelomeric I-SceI-Mediated Double-Strand Breaks Are Repaired by Homologous Recombination in Trypanosoma cruzi

et al. 2016

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“…Further, the recent generation of novel genetic manipulation tools, such as improved and easy-to-use T. cruzi expression vectors [163], T. cruzi artificial chromosomes [164] and the nuclease-mediated gene-targeting technology, CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats) [165] will contribute significantly to the development of this area in the next 5 years.…”

Section: Five-year Viewmentioning

confidence: 99%

Gene-deleted live-attenuatedTrypanosoma cruziparasites as vaccines to protect against Chagas disease

Sánchez-Valdéz

Brandán

Ferreira

et al. 2014

Expert Review of Vaccines

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Chagas disease is a neglected tropical disease caused by the protozoan parasite Trypanosoma cruzi. This illness is now becoming global, mainly due to congenital transmission, and so far, there are no prophylactic or therapeutic vaccines available to either prevent or treat Chagas disease. Therefore, different approaches aimed at identifying new protective immunogens are urgently needed. Live vaccines are likely to be more efficient in inducing protection, but safety issues linked with their use have been raised. The development of improved protozoan genetic manipulation tools and genomic and biological information has helped to increase the safety of live vaccines. These advances have generated a renewed interest in the use of genetically attenuated parasites as vaccines against Chagas disease. This review discusses the protective capacity of genetically attenuated parasite vaccines and the challenges and perspectives for the development of an effective whole-parasite Chagas disease vaccine.

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