A high-throughput cloning system for reverse genetics in Trypanosoma cruzi

Abstract

BackgroundThe three trypanosomatids pathogenic to men, Trypanosoma cruzi, Trypanosoma brucei and Leishmania major, are etiological agents of Chagas disease, African sleeping sickness and cutaneous leishmaniasis, respectively. The complete sequencing of these trypanosomatid genomes represented a breakthrough in the understanding of these organisms. Genome sequencing is a step towards solving the parasite biology puzzle, as there are a high percentage of genes encoding proteins without functional annotation. Also, technical limitations in protein expression in heterologous systems reinforce the evident need for the development of a high-throughput reverse genetics platform. Ideally, such platform would lead to efficient cloning and compatibility with various approaches. Thus, we aimed to construct a highly efficient cloning platform compatible with plasmid vectors that are suitable for various approaches.ResultsWe constructed a platform with a flexible structure allowing the exchange of various elements, such as promoters, fusion tags, intergenic regions or resistance markers. This platform is based on Gateway® technology, to ensure a fast and efficient cloning system. We obtained plasmid vectors carrying genes for fluorescent proteins (green, cyan or yellow), and sequences for the c-myc epitope, and tandem affinity purification or polyhistidine tags. The vectors were verified by successful subcellular localization of two previously characterized proteins (TcRab7 and PAR 2) and a putative centrin. For the tandem affinity purification tag, the purification of two protein complexes (ribosome and proteasome) was performed.ConclusionsWe constructed plasmids with an efficient cloning system and suitable for use across various applications, such as protein localization and co-localization, protein partner identification and protein expression. This platform also allows vector customization, as the vectors were constructed to enable easy exchange of its elements. The development of this high-throughput platform is a step closer towards large-scale trypanosome applications and initiatives.

Trypanosoma cruzi Bromodomain Factor 3 Binds Acetylated α-Tubulin and Concentrates in the Flagellum during Metacyclogenesis

“…TcBDF3 and TcBDF3⌬C coding regions were inserted into a pENTR3C vector (Gateway System Invitrogen) and then transferred by recombination to pDEST17 (Gateway System Invitrogen) and pTcCFPN (29), using LR clonase II enzyme mix (Invitrogen), to generate histidine tag and cyan fluorescent protein (CFP) fusions. pDEST17-TcBDF3 was transformed into Escherichia coli BL21 pLysS, and the recombinant protein (fused to a His tag) was obtained by induction with 0.5 mM isopropyl-␤-D-thiogalactopyranoside (IPTG) for 3 h at 37°C.…”

Stage-Regulated GFP Expression in Trypanosoma cruzi: Applications from Host-Parasite Interactions to Drug Screening

“…RNA integrity was assessed with an Agilent 2100 Bioanalyzer and the RNA 6000 Nano LabChip kit, according to the manufacturer’s instructions. First-strand cDNA was synthesized and qPCR was carried out as previously described [12]. The first-strand cDNA was obtained by mixing 1 µg of total RNA and 1 µM oligo dT and incubating at 70°C for 10 min.…”

“…Thus, RNA pol I, which transcribes the pre-rRNA gene cluster, can mediate the expression of exogenously introduced protein-coding genes under the control of the strong ribosomal promoter, provided that the coding region of interest is flanked by intergenic regions (IRs) containing sequence elements mediating trans -splicing and polyadenylation. Several studies have made use of constructs including the rRNA promoter, to induce high levels of exogenous gene transcription in T. cruzi , in both transient [9], [10], [11], [12] and stable transfection conditions [11], [13], [14], [15].…”

“…GFP has already been expressed in several Leishmania [18], [19], [20], Trypanosoma [13], [21], [22], [23], Toxoplasma [24] and Plasmodium species [25], [26]. The construction of parasite cell lines producing fluorescent proteins has wider applications, in studies of subjects ranging from pathogenesis to protein function [12], [16], [27].…”

Plasmid Vectors and Molecular Building Blocks for the Development of Genetic Manipulation Tools for Trypanosoma cruzi

“…In order to take advantage of the available sequence data, genetic systems based on more efficient and less time consuming construction technologies were developed for the generation of targeted gene replacement vectors (TGRVs) [10] and the expression of fusion proteins [11]. In some cases, attention was paid on producing flexible tools capable of exchanging different constituting elements [11], which is a property absent from most of the existing designs. With TGRVs it has been possible to generate single and double null mutant T. cruzi strains [10].…”