Generation, characterization and validation of a T.brucei overexpression library.

Rockefeller University, New York NY

Investigators

Linked publications, trials & patents

Abstract

? DESCRIPTION (provided by applicant): The African trypanosomes (including T. brucei sp) are tsetse-transmitted protozoans that infect both humans (causing sleeping sickness) and livestock (causing nagana). Our understanding of T. brucei biology increased dramatically after the generation of RNAi libraries amenable to loss-of-function studies, as well as the optimization of those strains for high transfection efficiencies. Coupled to next-generation sequencing approaches, these RNAi libraries have given rise to novel screens for genome-scale functional analyses that are both interesting (highlighting the unique biology of this model system) and useful (allowing for the direct genetic validation of anti-trypanocidal therapies and the potential generation of novel ones, which are sorely needed due to the high toxicity of the compounds currently in use). In contrast to genome-wide, loss-of-function experiments, complementary gain-of-function approaches have not been available for T. brucei. This is partly because control of gene expression in trypanosomes is post-transcriptional, and the levels of expression of transcripts are exquisitely dependent on the sequence of their untranslated regions, thus rendering the standard construction of cDNA libraries from reverse-transcribed polyA+ mRNA completely useless. cDNA and random-shotgun libraries of genomic DNA fragments have been attempted in other labs and proven to be problematic, both in regards to the coverage of the library and practicality. Here we propose to remedy this situation by generating, characterizing and validating an overexpression library for T. brucei, by individually amplifying all ORFs >100 bp from genomic DNA (Note that T. brucei genes are intronless, except for two characterized cases). Overexpression of the CDS alone should overcome the problems that arise when UTRs are included in the overexpressed constructs. We expect that such a library will be a significant new resource in the field, adding much-needed gain-of-function capabilities to the tools currently available, and almost certainly allowing for new trypanosome biology to be accessed.