The DOI matches its Crossref record; 25 of the 25 references it cites were found in a registry or an index.
A toolkit enabling efficient, scalable and reproducible gene tagging in trypanosomatids
Open accessAbstract
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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Cites
TriTrypDB: a functional genomic resource for the Trypanosomatidae
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Flagellar motility is required for the viability of the bloodstream trypanosome
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A novel epitope tag system to study protein targeting and organelle biogenesis in Trypanosoma brucei
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Self-citationHigh-throughput decoding of antitrypanosomal drug efficacy and resistance
Alsford, S., Eckert, S. E., Baker, N., Glover, L., Sánchez‐Flores, A., Leung, K. F., Turner, D. J., Field, M. C., Berriman, M., & Horn, D. (2012) · Nature · doi:10.1038/nature10771 · Cited by 303
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Inducible Gene Expression in Trypanosomes Mediated by a Prokaryotic Repressor
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Highly efficient stable transformation of bloodstream forms of Trypanosoma brucei
Burkard, G., Fragoso, C. M., & Roditi, I. (2007) · Molecular and Biochemical Parasitology · doi:10.1016/j.molbiopara.2007.02.008 · Cited by 246
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Mutations in CFAP43 and CFAP44 cause male infertility and flagellum defects in Trypanosoma and human
Coutton, C., Vargas, A. S., Amiri-Yekta, A., Kherraf, Z.-E., Ben Mustapha, S. F., Le Tanno, P., Wambergue-Legrand, C., Karaouzène, T., Martinez, G., Crouzy, S., et al. (2018) · Nature Communications · doi:10.1038/s41467-017-02792-7 · Cited by 245
A multi-adenylate cyclase regulator at the flagellar tip controls African trypanosome transmission
Bachmaier, S., Giacomelli, G., Calvo-Alvarez, E., Vieira, L. R., Van Den Abbeele, J., Aristodemou, A., Lorentzen, E., Gould, M. K., Brennand, A., Dupuy, J.-W., et al. (2022) · Nature Communications · doi:10.1038/s41467-022-33108-z · Cited by 38
FAP106 is an interaction hub for assembling microtubule inner proteins at the cilium inner junction
Shimogawa, M. M., Wijono, A. S., Wang, H., Zhang, J., Sha, J., Szombathy, N., Vadakkan, S., Pelayo, P., Jonnalagadda, K., Wohlschlegel, J., et al. (2023) · Nature Communications · doi:10.1038/s41467-023-40230-z · Cited by 18
The SPARC complex defines RNAPII promoters in Trypanosoma brucei
Staneva, D. P., Bresson, S., Auchynnikava, T., Spanos, C., Rappsilber, J., Jeyaprakash, A. A., Tollervey, D., Matthews, K. R., & Allshire, R. C. (2022) · eLife · doi:10.7554/elife.83135 · Cited by 13
Kinetoplastid kinetochore proteins KKT14–KKT15 are divergent Bub1/BubR1–Bub3 proteins
Ballmer, D., Carter, W., van Hooff, J. J. E., Tromer, E. C., Ishii, M., Ludzia, P., & Akiyoshi, B. (2024) · Open Biology · doi:10.1098/rsob.240025 · Cited by 13
Metadata
- Authors
- Dean, S., Sunter, J., Wheeler, R. J., Hodkinson, I., Gluenz, E., & Gull, K.
- Venue
- Open Biology
- Publisher
- The Royal Society
- Published
- 2015-01
- Volume
- 5
- Issue
- 1
- Pages
- 140197
- ISSN
- 2046-2441 (ISSN-L)
- OpenAlex
- W2125112712
- Cited by
- 264
- References
- 25
- FWCI
- 9.86
- Type
- journal article
- Language
- en
- Open access
- gold
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Contains information from the Semantic Scholar Academic Graph Datasets, made available under the ODC Attribution License (ODC-BY 1.0).
Semantic Scholar’s data is described in: Kinney, R., et al. (2023). The Semantic Scholar Open Data Platform. arXiv:2301.10140.
