title: A toolkit enabling efficient, scalable and reproducible gene tagging in trypanosomatids.
abstract: One of the first steps in understanding a protein's function is to determine its localization; however, the methods for localizing proteins in some systems have not kept pace with the developments in other fields, creating a bottleneck in the analysis of the large datasets that are generated in the post-genomic era. To address this, we developed tools for tagging proteins in trypanosomatids. We made a plasmid that, when coupled with long primer PCR, can be used to produce transgenes at their endogenous loci encoding proteins tagged at either terminus or within the protein coding sequence. This system can also be used to generate deletion mutants to investigate the function of different protein domains. We show that the length of homology required for successful integration precluded long primer PCR tagging in Leishmania mexicana. Hence, we developed plasmids and a fusion PCR approach to create gene tagging amplicons with sufficiently long homologous regions for targeted integration, suitable for use in trypanosomatids with less efficient homologous recombination than Trypanosoma brucei. Importantly, we have automated the primer design, developed universal PCR conditions and optimized the workflow to make this system reliable, efficient and scalable such that whole genome tagging is now an achievable goal.
authors.affiliation: Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, UK samuel.dean@path.ox.ac.uk.
authors.forename: Samuel
authors.identifier: http://orcid.org/0000-0002-4792-2198
authors.initials: S
authors.lastname: Dean
authors.affiliation: Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, UK.
authors.forename: Jack
authors.initials: J
authors.lastname: Sunter
authors.affiliation: Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, UK.
authors.forename: Richard J
authors.identifier: http://orcid.org/0000-0002-4270-8360
authors.initials: RJ
authors.lastname: Wheeler
authors.affiliation: Department of Computing, Imperial College London, South Kensington Campus, London SW7 2AZ, UK.
authors.forename: Ian
authors.identifier: http://orcid.org/0000-0003-3650-1942
authors.initials: I
authors.lastname: Hodkinson
authors.affiliation: Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, UK.
authors.forename: Eva
authors.initials: E
authors.lastname: Gluenz
authors.affiliation: Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, UK.
authors.forename: Keith
authors.initials: K
authors.lastname: Gull
mesh_terms: D018390:Gene Targeting / Q000379:methods*; D005818:Genetic Engineering / Q000379:methods*; D007891:Leishmania / Q000235:genetics*; D015800:Protozoan Proteins / Q000235:genetics
chemical_list: D015800:Protozoan Proteins
keywords: fusion PCR; homologous recombination; molecular tools; optimization; tagging; trypanosomatid