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Open Biol. 2015 Jan;5(1):140197. doi: 10.1098/rsob.140197.

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

Author information

  • 1Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, UK samuel.dean@path.ox.ac.uk.
  • 2Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, UK.
  • 3Department of Computing, Imperial College London, South Kensington Campus, London SW7 2AZ, UK.

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.

KEYWORDS:

fusion PCR; homologous recombination; molecular tools; optimization; tagging; trypanosomatid

PMID:
25567099
PMCID:
PMC4313374
DOI:
10.1098/rsob.140197
[PubMed - indexed for MEDLINE]
Free PMC Article
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