All authors read and approved the final manuscript.”
“Background 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 , learn more protein interaction identification , protein overexpression , gene knockout  and gene silencing . 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 H 89 cell line precludes the use of forward genetics to study gene function in these parasites. In addition, their protein-coding genes are transcribed in polycistronic mRNAs, not related to bacterial operons, which are further processed to mature monocistronic mRNAs by a trans-splicing mechanism . This process MAPK inhibitor results in a short nucleotide sequence (miniexon) being added to the 5′ end of trypanosomatid mRNAs . The same machinery probably scans the intergenic region (IR) to process the upstream transcript and add the poly-A tail . However, no consensus sequence for poly-A tail addition
has been found in trypanosomes. Furthermore, gene expression in these microorganisms is mostly controlled by post-transcriptional events involving RNA processing and stability . Hence, to be expressed in trypanosomatids, transgenes need to be flanked by intergenic regions that contain sequence elements promoting miniexon and poly-A tail addition. however Generally, IRs in trypanosomatid plasmid vectors are derived from constitutively expressed genes, such as those encoding glyceraldehyde 3-phosphate dehydrogenase [11, 12], actin, aldolase [5, 13, 14], α-tubulin  or ubiquitin . Gene expression in trypanosomatids appears to be ubiquitous and is not dependent on the presence of a typical
RNA polymerase II (pol II) promoter . Although typical pol II promoters have not been found in trypanosomatids, it has been shown that pol II transcription of an entire polycistronic unit initiates upstream of the first gene of the polycistron (in strand-switch regions) . To enhance gene expression, vectors for use in trypanosomatids were constructed to ensure that transcription is directed by strong promoters like RNA polymerase I (pol I) promoters [3, 14, 19–21]. Some vectors were also designed to control gene expression, by combining T7 or pol I promoters with tetracycline-inducible systems [5, 12, 14, 16, 22–26]. These features require the development of reverse genetics strategies to deal with trypanosomatid biology. There are a few examples of vectors designed for use in T.