Cloning and expression of transgenes using linear vectors in Trypanosoma cruzi

• Published in: International journal for parasitology 2014-06, Vol.44 (7), p.447-456
• Main Authors: Curto, María de los Ángeles, Lorenzi, Hernán A., Moraes Barros, Roberto R., Souza, Renata T., Levin, Mariano J., Da Silveira, José Franco, Schijman, Alejandro G.
• Format: Article
• Language: English
• Subjects: Animals; Artificial chromosomes; Chromosomes, Artificial; Cloning, Molecular; Crithidia fasciculata; Functional genetic complementation; Gene Expression Regulation - physiology; GFP; Linear vectors; Mice; Organisms, Genetically Modified; Ornithine decarboxylase; Stable transfection; Trypanosoma cruzi; Trypanosoma cruzi - genetics; Trypanosoma cruzi - metabolism
• ISSN: 0020-7519; 1879-0135
• DOI: 10.1016/j.ijpara.2014.03.009

[Display omitted] •We have developed a series of Trypanosoma artificial chromosomes (TACs) as tools for stable transfection in T. cruzi.•These linear vectors were stably replicated and segregated even in the absence of selective pressure.•pTAC-odc (ornithine decarboxylase) transfected parasites could overcome the natural auxotrophy of T. cruzi for polyamines.•These transgenic trypanosomes could constitute interesting models to deepen understanding of T. cruzi chromosome behaviour. The identification of new targets for vaccine and drug development for the treatment of Chagas’ disease is dependent on deepening our understanding of the parasite genome. Vectors for genetic manipulation in Trypanosoma cruzi basically include those that remain as circular episomes and those that integrate into the parasite’s genome. Artificial chromosomes are alternative vectors to overcome problematic transgene expression often occurring with conventional vectors in this parasite. We have constructed a series of vectors named pTACs (Trypanosome Artificial Chromosomes), all of them carrying telomeric and subtelomeric sequences and genes conferring resistance to different selection drugs. In addition, one pTAC harbours a modified GFP gene (pTAC-gfp), and another one carries the ornithine decarboxilase gene from Crithidia fasciculata (pTAC-odc). We have encountered artificial chromosomes generated from pTACs in transformed T. cruzi epimastigotes for every version of the designed vectors. These extragenomic elements, in approximately 6–8 copies per cell, remained as linear episomes, contained telomeres and persisted after 150 and 60 generations with or without selection drugs, respectively. The linear molecules remained stable through the different T. cruzi developmental forms. Furthermore, derived artificial chromosomes from pTAC-odc could complement the auxotrophy of T. cruzi for polyamines. Our results show that pTACs constitute useful tools for reverse functional genetics in T. cruzi that will contribute to a better understanding of T. cruzi biology.