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

Dean, Samuel; Sunter, Jack D.; Wheeler, Richard J.; Hodkinson, Ian; Gluenz, Eva; Gull, Keith

doi:10.1098/rsob.140197

Cited by 250 publications

(317 citation statements)

References 32 publications

(45 reference statements)

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“…Bloodstream forms have a lower transfection efficiency 14 , moreover they are likely to die during selection due to density-dependent toxicity that is unrelated to the selective drug. Therefore, our previous protocol represents the current best technology for tagging of bloodstream form trypanosomes 9 . It is also likely that the transfection efficiency will vary dependent upon the specific trypanosome isolate.…”

Section: Discussionmentioning

confidence: 99%

High-throughput Gene Tagging in <em>Trypanosoma brucei</em>

Dyer

Dean

Sunter

2016

JoVE

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Improvements in mass spectrometry, sequencing and bioinformatics have generated large datasets of potentially interesting genes. Tagging these proteins can give insights into their function by determining their localization within the cell and enabling interaction partner identification. We recently published a fast and scalable method to generate Trypanosoma brucei cell lines that express a tagged protein from the endogenous locus. The method was based on a plasmid we generated that, when coupled with long primer PCR, can be used to modify a gene to encode a protein tagged at either terminus. This allows the tagging of dozens of trypanosome proteins in parallel, facilitating the large-scale validation of candidate genes of interest. This system can be used to tag proteins for localization (using a fluorescent protein, epitope tag or electron microscopy tag) or biochemistry (using tags for purification, such as the TAP (tandem affinity purification) tag). Here, we describe a protocol to perform the long primer PCR and the electroporation in 96-well plates, with the recovery and selection of transgenic trypanosomes occurring in 24-well plates. With this workflow, hundreds of proteins can be tagged in parallel; this is an order of magnitude improvement to our previous protocol and genome scale tagging is now possible.

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Section: Discussionmentioning

confidence: 99%

High-throughput Gene Tagging in <em>Trypanosoma brucei</em>

Dyer

Dean

Sunter

2016

JoVE

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“…RNAi plasmids were made using primers designed by RNAi-it (54) and cloned into the pQuadra stem loop vector (55). All tagged cell lines were generated using the pPOT system (30).…”

Section: Methodsmentioning

confidence: 99%

“…SmOxP927 P9 trypanosomes (52) were cultured in SDM79 (53) supplemented with 10% (vol/vol) FCS at 28°C. Transgenic cell lines were generated as described (30). RNAi plasmids were made using primers designed by RNAi-it (54) and cloned into the pQuadra stem loop vector (55).…”

Section: Methodsmentioning

confidence: 99%

“…Live cells and cytoskeletons were prepared for microscopy as described (30). Concentrations of primary antibodies: BBA4 (58): 1/50; α-PFR2 (L8C4) (59): 1/50; α-TbSAXO (Mab25) (60): 1:20; TFP1: 1/50; α-TbMORN1 (61): 1/3,000; α-FTZC (29): 1/2000; and α-TbBILBO1 (36): 1/10.…”

Section: Methodsmentioning

confidence: 99%

“…We selected 80 of the 192 priority TZ candidates based on their bioinformatics profiles (excluding obvious contaminants such as histones and ribosomal proteins), tagged them using a high-throughput approach, and verified the fusion protein's localization by microscopy (30). Of these, 34 proteins (43%) localized at the TZ, 4 proteins (5%) localized to cytoskeletal structures close to the TZ, 7 proteins (8%) localized to the flagellum, and 7 proteins (8%) localized to the cell body ( Fig.…”

Section: Tz and Ciliary Subdomains Defined By Protein Localization Usingmentioning

confidence: 99%

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Cilium transition zone proteome reveals compartmentalization and differential dynamics of ciliopathy complexes

Dean

Moreira-Leite

Varga

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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The transition zone (TZ) of eukaryotic cilia and flagella is a structural intermediate between the basal body and the axoneme that regulates ciliary traffic. Mutations in genes encoding TZ proteins (TZPs) cause human inherited diseases (ciliopathies). Here, we use the trypanosome to identify TZ components and localize them to TZ subdomains, showing that the Bardet-Biedl syndrome complex (BBSome) is more distal in the TZ than the Meckel syndrome (MKS) complex. Several of the TZPs identified here have human orthologs. Functional analysis shows essential roles for TZPs in motility, in building the axoneme central pair apparatus and in flagellum biogenesis. Analysis using RNAi and HaloTag fusion protein approaches reveals that most TZPs (including the MKS ciliopathy complex) show long-term stable association with the TZ, whereas the BBSome is dynamic. We propose that some Bardet-Biedl syndrome and MKS pleiotropy may be caused by mutations that impact TZP complex dynamics.transition zone | cilium/flagellum | BBSome | MKS/B9 complex | trypanosome C ilia, or flagella (the two terms are used here interchangeably), are multifunctional organelles that were present in the last common eukaryotic ancestor (1). Defects in cilia are responsible for pleiotropic human diseases (called ciliopathies) and their function is essential for pathogenesis in protozoan parasites (2, 3).During ciliogenesis, a microtubule organizing center called the "basal body" docks at the membrane. Basal bodies are built from nine microtubule triplets and two microtubules from each triplet extend to form a transition zone (TZ) and, ultimately, the axoneme. Thus, the TZ represents a structural junction between the basal body and the axoneme.As expected from its strategic position between the basal body and the axoneme, the TZ acts as a "ciliary gate" that controls ciliary composition and function (4). Many ciliopathies are caused by defects in complexes that associate with the TZ, including Meckel syndrome (MKS), Joubert syndrome and Bardet-Biedl syndrome (BBS). Studies using murine kidney epithelial cells and embryos suggest that the MKS complex demarcates the ciliary membrane by forming a diffusion barrier at the base of the cilium (5-7). On the other hand, the BBS complex (BBSome) is an adapter for intraflagellar transport (IFT) complexes that cross the TZ barrier to transport sensory proteins between the ciliary membrane and the cell body (8, 9).The TZ has distinctive structural features. At the proximal boundary of the TZ, where the basal body C tubule terminates, a "terminal plate" crosses the TZ. Studies in Tetrahymena suggest that the terminal plate contains pores for the passage of IFT "trains" that deliver axonemal components to the distal tip of flagella (10). Striated transitional fibers radiate from the distal end of the basal body triplets to join the plasma membrane (11-14), forming blades thought to create a physical barrier preventing vesicles from entering the ciliary lumen. Electron microscopy (EM) studies in Chlamydomonas suggest that...

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Transduction and transfection of difficult‐to‐transfect cells: Systematic attempts for the transfection of protozoa Leishmania

Keller

Scheiding

Breitling

et al. 2018

J of Cellular Biochemistry

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Cell-penetrating peptides (CPPs) are used to internalize different cargoes, including DNA, into live mammalian and plant cells. Despite many cells being easily transfected with this approach, other cells are rather "difficult" or "hard to transfect," including protist cells of the genus Leishmania. Based on our previous results in successfully internalizing proteins into Leishmania tarentolae cells, we used single CPPs and three different DNA-binding proteins to form protein-like complexes with plasmids covered with CPPs. We attempted magnetofection, electroporation, and transfection using a number of commercially available detergents. While complex formation with negatively charged DNA required substantially higher amounts of CPPs than those necessary for mostly neutral proteins, the cytotoxicity of the required amounts of CPPs and auxiliaries was thoroughly studied. We found that Leishmania cells were indeed susceptible to high concentrations of some CPPs and auxiliaries, although in a different manner compared with that for mammalian cells. The lack of successful transfections implies the necessity to accept certain general limitations regarding DNA internalization into difficult-to-transfect cells. Only electroporation allowed reproducible internalization of large and rigid plasmid DNA molecules through electrically disturbed extended membrane areas, known as permeable membrane macrodomains.

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

Cited by 250 publications

References 32 publications

High-throughput Gene Tagging in <em>Trypanosoma brucei</em>

High-throughput Gene Tagging in <em>Trypanosoma brucei</em>

Cilium transition zone proteome reveals compartmentalization and differential dynamics of ciliopathy complexes

Transduction and transfection of difficult‐to‐transfect cells: Systematic attempts for the transfection of protozoa Leishmania

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