Endogenous Gene Tagging of pfr2 and pfr5 in Trypanosoma cruzi Using CRISPR/Cas9

The flagellum of <em>Trypanosoma cruzi </em>contains the paraflagellar rod (PFR) an extra-axonemal scaffolding. The PFR consists of a lattice of cytoskeletal filaments that lies alongside the (9 + 2) microtubular axoneme, beginning at the flagellar pocket and extending to the flagellar tip. The PFR has only been observed within the phylums Euglenozoa and Dinoflagellata, although many eukaryotic organisms with long flagella have extra-axonemal structures that accommodate enzymes and regulatory proteins along with serving as scaffolding. The exact function and basic molecular composition of the PFR has yet to be determined although the major structural components, PFR1 and PFR2 and several minor proteins have been identified. The PFR is not only a complex structure that has been shown to be critical for motility, it also constitutes a unique set of proteins that are known to be immunogenic and provide protective immunity to <em>T. cruzi</em>. PFR5, a hypothetical minor component of the PFR, contains a PFR internal domain and an SH3 binding domain. Currently, it is unknown if the protein product of <em>pfr5</em> localizes to the flagellum. We have adapted a CRISPR/Cas9 endogenous gene tagging protocol to tag <em>pfr5</em> and investigate the subcellular localization of the protein. PFR2 localization serves as a proof of principle for this system as localization is well established. This technique allows for the precise insertion of a small 3x hemagglutinin tag at the C-terminus of the gene of interest, with subsequent protein product also containing the tag. Localization of the tagged proteins is can then be visualized using immunofluorescence. Successful utilization of this technique, as well as localization of PFR5, will contribute to further the research and understanding of this unique structure.

Name thesis:degree_name

Master of Science (MS)

Discipline thesis:degree_discipline

Biology

Year dc:date.available

2019

Author dc:creator

• Bryant, Naomi

Contributors dc:contributor

• Gabrielle Stryker
• Lucinda Carnell
• Blaise Dondji

• Acadmemic Theses
• Genetics
• Immunology and Infectious Disease
• Life Sciences
• Parasitology

Language dc:language

English

Repository record dc:identifier

https://digitalcommons.cwu.edu/etd/1248

OAI identifier oai:identifier

oai:digitalcommons.cwu.edu:etd-2268