title: High-throughput Gene Tagging in Trypanosoma brucei.
abstract: Improvements in mass spectrometry, sequencing and bioinformatics have generated large datasets of potentially interesting genes. Tagging these proteins can give insights into their function by determining their localization within the cell and enabling interaction partner identification. We recently published a fast and scalable method to generate Trypanosoma brucei cell lines that express a tagged protein from the endogenous locus. The method was based on a plasmid we generated that, when coupled with long primer PCR, can be used to modify a gene to encode a protein tagged at either terminus. This allows the tagging of dozens of trypanosome proteins in parallel, facilitating the large-scale validation of candidate genes of interest. This system can be used to tag proteins for localization (using a fluorescent protein, epitope tag or electron microscopy tag) or biochemistry (using tags for purification, such as the TAP (tandem affinity purification) tag). Here, we describe a protocol to perform the long primer PCR and the electroporation in 96-well plates, with the recovery and selection of transgenic trypanosomes occurring in 24-well plates. With this workflow, hundreds of proteins can be tagged in parallel; this is an order of magnitude improvement to our previous protocol and genome scale tagging is now possible.
authors: Sir William Dunn School of Pathology, University of Oxford.
authors: Philip
authors: P
authors: Dyer
authors: Sir William Dunn School of Pathology, University of Oxford; samuel.dean@path.ox.ac.uk.
authors: Samuel
authors: S
authors: Dean
authors: Sir William Dunn School of Pathology, University of Oxford; jack.sunter@path.ox.ac.uk.
authors: Jack
authors: J
authors: Sunter
mesh_terms: D002846:Chromatography, Affinity; D017931:DNA Primers; D018390:Gene Targeting / Q000379:methods*; D010957:Plasmids; D011506:Proteins; D015800:Protozoan Proteins / Q000235:genetics*; D014346:Trypanosoma brucei brucei / Q000235:genetics*
chemical_list: D017931:DNA Primers; D011506:Proteins; D015800:Protozoan Proteins