%0 Journal Article
%T A high-throughput cloning system for reverse genetics in Trypanosoma cruzi
%A Michel Batista
%A Fabricio K Marchini
%A Paola AF Celedon
%A Stenio P Fragoso
%A Christian M Probst
%A Henrique Preti
%A Luiz S Ozaki
%A Gregory A Buck
%A Samuel Goldenberg
%A Marco A Krieger
%J BMC Microbiology
%D 2010
%I BioMed Central
%R 10.1186/1471-2180-10-259
%X We 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.We 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.Currently, reverse genetics-based tools have been largely employed to obtain biological information on genes of unknown function. Nowadays genomic sequence data are easily obtained, but gene function is not always obviously extracted from these data. These tools have been used for many purposes, such as protein subcellular localization [1], protein interaction identification [2], protein overexpression [3], gene knockout [4] and gene silencing [5]. These techniques are particularly important in the study of trypanosomatid protozoa. Sexual reproduction, although not frequent, may play a role in the heterogeneity of several trypanosomatid species. However, these parasites mostly have a clonal population structure [6,7]. This characteristic precludes the use of forward genetics to study gene function in these
%U http://www.biomedcentral.com/1471-2180/10/259