FM
Franziska Mohring
  • Post-Doc, London School of Hygiene and Tropical Medicine, UK
Multiplication and Growth Inhibition Activity Assays for the Zoonotic Malaria Parasite, Plasmodium knowlesi
Authors:  Franziska Mohring, Thomas A. Rawlinson, Simon J. Draper and Robert W. Moon, date: 09/05/2020, view: 4283, Q&A: 0
Malaria remains a major cause of morbidity and mortality globally. Clinical symptoms of the disease arise from the growth and multiplication of Plasmodium parasites within the blood of the host. Thus in vitro assays to determine how drug, antibody and genetic perturbations affect the growth rate of Plasmodium parasites are essential for the development of new therapeutics and improving our understanding of parasite biology. As both P. falciparum and P. knowlesi can be maintained in culture with human red blood cells, the effect of antimalarial drugs and inhibitory antibodies that target the invasion or growth capacity of Plasmodium parasites are routinely investigated by using multiplication assays or growth inhibition activity (GIA) assays against these two species. This protocol gives detailed step-by-step procedures to carry out flow cytometry-based multiplication assays and growth inhibition activity assays to test neutralizing antibodies based on the activity of the parasite enzyme lactate dehydrogenase of Plasmodium knowlesi adapted to human red blood cell culture. Whilst similar assays are well established for P. falciparum, P. knowlesi is more closely related to all other human infective species (Pacheco et al., 2018) and so can be used as a surrogate for testing drugs and vaccines for other malaria species such as P. vivax, which is the most widespread cause of malaria outside of Africa, but cannot yet be cultured under laboratory conditions.
CRISPR-Cas9 Genome Editing of Plasmodium knowlesi
Authors:  Franziska Mohring, Melissa N. Hart, Avnish Patel, David A. Baker and Robert W. Moon, date: 02/20/2020, view: 5808, Q&A: 1
Plasmodium knowlesi is a zoonotic malaria parasite in Southeast Asia that can cause severe and fatal malaria in humans. The main hosts are Macaques, but modern diagnostic tools reveal increasing numbers of human infections. After P. falciparum, P. knowlesi is the only other malaria parasite capable of being maintained in long term in vitro culture with human red blood cells (RBCs). Its closer ancestry to other non-falciparum human malaria parasites, more balanced AT-content, larger merozoites and higher transfection efficiencies, gives P. knowlesi some key advantages over P. falciparum for the study of malaria parasite cell/molecular biology. Here, we describe the generation of marker-free CRISPR gene-edited P. knowlesi parasites, the fast and scalable production of transfection constructs and analysis of transfection efficiencies. Our protocol allows rapid, reliable and unlimited rounds of genome editing in P. knowlesi requiring only a single recyclable selection marker.
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