An enhanced toolkit for the generation of knockout and marker-free fluorescent Plasmodium chabaudi

Wellcome Open Research

article

Edward J Marr¹  Rachel M Milne¹  Burcu Anar²  Gareth Girling²  Frank Schwach²  Jason P Mooney³  Wiebke Nahrendorf¹  Philip J Spence¹  Deirdre Cunningham⁴  David A Baker⁵  Jean Langhorne⁴  Julian C Rayner²  Oliver Billker²  Ellen S Bushell²  Joanne Thompson¹ 

[1] Institute of Immunology and Infection Research, School of Biological Sciences, University of Edinburgh, Ashworth Laboratories;Parasites and Microbes, Wellcome Sanger Institute, Wellcome Genome Campus;The Roslin Institute and Royal ,(Dick) School of Veterinary Studies, University of Edinburgh;The Francis Crick Institute;Faculty of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine;Cambridge Institute for Medical Research, University of Cambridge;Laboratory for Molecular Infection Medicine Sweden, Department of Molecular Biology, Umeå University

关键词: Plasmodium chabaudi;    malaria;    Transfection;    PlasmoGem;   

DOI  :  10.12688/wellcomeopenres.15587.2

学科分类：内科医学

来源: Wellcome

【 摘 要 】

The rodent parasitePlasmodium chabaudi is an importantin vivo model of malaria. The ability to produce chronic infections makes it particularly useful for investigating the development of anti-Plasmodium immunity, as well as features associated with parasite virulence during both the acute and chronic phases of infection.P. chabaudi also undergoes asexual maturation (schizogony) and erythrocyte invasion in culture, so offers an experimentally-amenablein vivo to in vitro model for studying gene function and drug activity during parasite replication. To extend the usefulness of this model, we have further optimised transfection protocols and plasmids forP. chabaudi and generated stable, fluorescent lines that are free from drug-selectable marker genes. These mother-lines show the same infection dynamics as wild-type parasites throughout the lifecycle in mice and mosquitoes; furthermore, their virulence can be increased by serial blood passage and reset by mosquito transmission. We have also adapted the large-insert, linearPlasmoGEM vectors that have revolutionised the scale of experimental genetics in another rodent malaria parasite and used these to generate barcodedP. chabaudi gene-deletion and –tagging vectors for transfection in our fluorescentP. chabaudi mother-lines. This produces a tool-kit ofP. chabaudi lines, vectors and transfection approaches that will be of broad utility to the research community.

【 授权许可】

CC BY