A toolkit enabling efficient, scalable and reproducible gene tagging in trypanosomatids

Dean, S., Sunter, J., Wheeler, R. J., Hodkinson, I., Gluenz, E. and Gull, K. (2015) A toolkit enabling efficient, scalable and reproducible gene tagging in trypanosomatids. Open Biology, 5(1), 140197. (doi: 10.1098/rsob.140197) (PMID:25567099) (PMCID:PMC4313374)

[img]
Preview
Text
211910.pdf - Published Version
Available under License Creative Commons Attribution.

1MB

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.

Item Type:Articles
Status:Published
Refereed:Yes
Glasgow Author(s) Enlighten ID:Gluenz, Dr Eva
Authors: Dean, S., Sunter, J., Wheeler, R. J., Hodkinson, I., Gluenz, E., and Gull, K.
College/School:College of Medical Veterinary and Life Sciences > Institute of Infection Immunity and Inflammation
Journal Name:Open Biology
Publisher:Royal Society
ISSN:2046-2441
ISSN (Online):2046-2441
Published Online:01 January 2015
Copyright Holders:Copyright © 2015 The Authors
First Published:First published in Open Biology
Publisher Policy:Reproduced under a Creative Commons license

University Staff: Request a correction | Enlighten Editors: Update this record