T-DNA Mediated Gene Tagging in Arabidopsis

Faculty Opinions recommendation of A recombineering-based gene tagging system for Arabidopsis.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.9786956.10485054 ◽

2011 ◽

Author(s):

Brian Forde

Keyword(s):

Gene Tagging ◽

Tagging System

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Seamless Gene Tagging by Endonuclease-Driven Homologous Recombination

PLoS ONE ◽

10.1371/journal.pone.0023794 ◽

2011 ◽

Vol 6 (8) ◽

pp. e23794 ◽

Cited By ~ 43

Author(s):

Anton Khmelinskii ◽

Matthias Meurer ◽

Nurlanbek Duishoev ◽

Nicolas Delhomme ◽

Michael Knop

Keyword(s):

Homologous Recombination ◽

Gene Tagging

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The Use of Mutator for Gene-Tagging: Cross-Referencing between Transposable Element Systems

Plant Transposable Elements ◽

10.1007/978-1-4684-5550-2_10 ◽

1988 ◽

pp. 137-147 ◽

Cited By ~ 4

Author(s):

N. S. Shepherd ◽

W. F. Sheridan ◽

M. G. Mattes ◽

G. Deno

Keyword(s):

Transposable Element ◽

Gene Tagging

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

Open Biology ◽

10.1098/rsob.140197 ◽

2015 ◽

Vol 5 (1) ◽

pp. 140197 ◽

Cited By ~ 97

Author(s):

Samuel Dean ◽

Jack Sunter ◽

Richard J. Wheeler ◽

Ian Hodkinson ◽

Eva Gluenz ◽

...

Keyword(s):

Trypanosoma Brucei ◽

Large Datasets ◽

Gene Tagging ◽

Deletion Mutants ◽

Whole Genome ◽

Leishmania Mexicana ◽

Protein Coding ◽

Achievable Goal ◽

Endogenous Loci ◽

Pcr Conditions

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.

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