CONICET | Buscador de Institutos y Recursos Humanos

Vectors for genetic manipulation are scarce in Trypanosoma cruzi. We have been engaged in the construction of artificial chromosomes as attractive tools for cloning and expression as well as for functional complementation or studies of required elements for proper chromosomal behaviour. These non-integrating vectors could overcome some of the drawbacks of integrating ones (i.e. local perturbations at the integration sites). The presence of telomeric ends could allow the propagation in absence of selective pressure. Herein, we report the characterization of pTACgfp, a 12kb vector that carries telomeric and subtelomeric sequences, two genes conferring resistance to different selection drugs and a modified reporter gene (gfp(EcoRI)). The added EcoRI site was made up to act as a cloning site interrupting the gfp gene, hence allowing differentiation between parasites carrying recombinant vectors (colorless) and those with empty constructs (fluorescents). Stability tests show that linear molecules generated after transfection of epimastigotes with this vector are still present in the transgenic lines after 120 and 60 generations with and without selection, respectively. This construct was conceived to accept large DNA inserts from other species of kinetoplastids (for example, a complete locus) and to express them in T. cruzi. As a previous step, to demonstrate if such expression is possible, experiments were carried out using as DNA insert, the ornitine decarboxilase gene (odc) and its RNA processing regions from Crithidia fasciculata. These sequences were cloned into a pBluescript vector yielding the construct pBSodc. The odc gene is absent in the Trypanosoma cruzi genome making essential an exogenous source of polyamines in the culture medium. When 50ug of circular pBSodc were transfected into T.cruzi (CL Brener) epimastigotes the resulting transgenic population, in contrast with wild type parasites, was able to survive in the absence of external addition of polyamines. The functionality of RNA processing signals from C. fasciculata in T. cruzi was assessed by mapping the trans-splicing acceptor site by RT-PCR and subsequent fragment sequencing. Altogether, these results encourage us to attempt the cloning of large fragments from C. fasciculata and other trypanosomatids into artificial chromosomes to study protein expression in T.cruzi . Stability tests show that linear molecules generated after transfection of epimastigotes with this vector are still present in the transgenic lines after 120 and 60 generations with and without selection, respectively. This construct was conceived to accept large DNA inserts from other species of kinetoplastids (for example, a complete locus) and to express them in T. cruzi. As a previous step, to demonstrate if such expression is possible, experiments were carried out using as DNA insert, the ornitine decarboxilase gene (odc) and its RNA processing regions from Crithidia fasciculata. These sequences were cloned into a pBluescript vector yielding the construct pBSodc. The odc gene is absent in the Trypanosoma cruzi genome making essential an exogenous source of polyamines in the culture medium. When 50ug of circular pBSodc were transfected into T.cruzi (CL Brener) epimastigotes the resulting transgenic population, in contrast with wild type parasites, was able to survive in the absence of external addition of polyamines. The functionality of RNA processing signals from C. fasciculata in T. cruzi was assessed by mapping the trans-splicing acceptor site by RT-PCR and subsequent fragment sequencing. Altogether, these results encourage us to attempt the cloning of large fragments from C. fasciculata and other trypanosomatids into artificial chromosomes to study protein expression in T.cruzi . Stability tests show that linear molecules generated after transfection of epimastigotes with this vector are still present in the transgenic lines after 120 and 60 generations with and without selection, respectively. This construct was conceived to accept large DNA inserts from other species of kinetoplastids (for example, a complete locus) and to express them in T. cruzi. As a previous step, to demonstrate if such expression is possible, experiments were carried out using as DNA insert, the ornitine decarboxilase gene (odc) and its RNA processing regions from Crithidia fasciculata. These sequences were cloned into a pBluescript vector yielding the construct pBSodc. The odc gene is absent in the Trypanosoma cruzi genome making essential an exogenous source of polyamines in the culture medium. When 50ug of circular pBSodc were transfected into T.cruzi (CL Brener) epimastigotes the resulting transgenic population, in contrast with wild type parasites, was able to survive in the absence of external addition of polyamines. The functionality of RNA processing signals from C. fasciculata in T. cruzi was assessed by mapping the trans-splicing acceptor site by RT-PCR and subsequent fragment sequencing. Altogether, these results encourage us to attempt the cloning of large fragments from C. fasciculata and other trypanosomatids into artificial chromosomes to study protein expression in T.cruzi .