The Gateway(r) platform has also had a significant impact on

gene characterization in large-scale projects; for example: when a collection of ORFs has been available in GS-7977 cost compatible plasmids [37, 43]. Another interesting selleck chemical feature was achieved during the design of vectors; we selected several one-cut restriction endonuclease sites to insert the elements, with the exception of XhoI whose sites flank the antibiotic resistance marker. This provides the flexibility to exchange all the elements in these vectors, such as promoter, intergenic regions (IRs), tags and antibiotic resistance genes. A good example of this flexibility was the set of experiments performed with the co-localization vector. This flexibility is important for further developments of this platform. Some of these developments have already been defined: First, there is evidence of intra-species ribosomal promoter specificity Selleck GDC 0032 in T. cruzi [44]. Hence, we designed constructs allowing the exchange of the T. cruzi I ribosomal promoter with other promoters, such as the T. cruzi II ribosomal promoter, seeking to expand the use of pTcGW vectors in other T. cruzi strains.

Second, IRs are the other exchangeable elements in pTcGW vectors. Several studies have shown that untranslated regions affect the level of expression of reporter genes in trypanosomatids [45–48]. The vectors described here allow IR exchange, thus modifying mRNA stability in attempts to modify the gene expression profiles in specific situations, for example during specific stages of the T. cruzi life cycle. Finally, we followed a protocol for transfection that minimizes the amount of DNA and medium used. Thus, we obtained transfectants using DNA from a unique plasmid minipreparation. Moreover, our protocol also minimizes the amount of media and antibiotics used for cell cultivation, thus decreasing the cost and time-scale of large projects. Our procedure can be improved further, increasing its efficiency for use in high-throughput projects. Taken together, these observations

demonstrate that our vector platform represents a powerful system for gene characterization in T. cruzi. Conclusions Due to an absence of vectors combining a high-throughput cloning system and flexibility for exchanging its elements in T. cruzi, we developed and constructed destination vectors incorporating these features. Our pTcGW Bumetanide vectors can be used for protein subcellular localization, co-localization and complex purification. These constructs can also be customized. In addition, we standardized some of our protocols, simplifying the use of our platform in large-scale projects. This is a very important step towards improving available methodologies for the characterization of thousands of genes whose functions remain unknown in T. cruzi. Methods Plasmid construction Three cassettes were inserted into the pBluescript(r) II plasmid (Stratagene, San Diego, USA) following the strategy shown in Figure 6.