Development of an Editable Approach to the Study Parasite-Erythroid Interactions
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Author
Jiang, JingyuDate
2020Affiliation
Medical BiologyMetadata
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Masters Research thesisAccess Status
This item is embargoed and will be available on 2022-11-24.Description
© 2020 Jingyu Jiang
Abstract
Malaria remains responsible for an enormous health burden worldwide; considerable
research effort is being devoted to finding ways to combat the disease and its
transmission. Malaria is caused by Plasmodium species, and P. falciparum causes the
most serious disease. The blood stage of the P. falciparum remains critically important
to understand for development of treatments and vaccines. To initiate invasion, the P.
falciparum merozoite recognises specific proteins on the host red cell membrane,
known as invasion receptors. In order to study parasite–host interactions, laboratory adapted P. falciparum strains that invade mature human red cells have been used. Gene
modification methods are well established for P. falciparum; however, genetic
manipulation of the red cell has not been extensively applied because erythrocytes are
not nucleated. The in vitro cultivation of erythroid cell lines facilitates both the scalable
production of host cells to support P. falciparum invasion and editing of nucleated
precursors that can be genetically modified in a precise manner.
In this project, two erythroid cell lines – the Human Umbilical cord blood Derived
Erythroid Progenitors (HUDEP-2) and the Bristol Erythroid Line- Adult (BEL-A), both
of which can differentiate to more mature forms in vitro – were studied as possible host
models. A FACS antibody panel, based on the stage-specific profile of HUDEP-2 and
BEL-A cells, provided the means to analyse host invasion receptors as well as erythroid
maturation markers.
Band 3 is a red cell membrane protein with an uncertain role in merozoite invasion. A
gene knockout was constructed in expansion stage BEL-A cells using the lentiviral
CRISPR/Cas9 system, targeting band 3 which may be involved in merozoite invasion of
human erythrocytes. Single-cell-derived clones were isolated and preliminary validation
using PCR and flow cytometry was performed to verify disruption of band 3.
Completion of work to validate and functionally characterise the band 3 knockout, and
experiments to assess effects on invasion, were curtailed by COVID-19 stay-at-home
orders issued to Melbourne between March and July 2020.
In summary, a genetically editable in vitro erythroid model was defined to study the
function of host invasion receptors for P. falciparum merozoite invasion. Clonal band 3-
deficient BEL-A cells were generated, thus paving the way for studying their role as
invasion receptors.
Keywords
P. falciparum; erythroid cell lines; invasion; CRISPR knock outExport Reference in RIS Format
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