Generation of transgenic rodent malaria parasites by transfection of cell culture-derived merozoites

Gesine Kaiser,Volker Heussler,Livia Niklaus,Paul-Christian Burda,Mariana De Niz,Rebecca Limenitakis Stanway

doi:10.1186/s12936-017-1949-y

Abstract

BackgroundMalaria research is greatly dependent on and has drastically advanced with the possibility of genetically modifying Plasmodium parasites. The commonly used transfection protocol by Janse and colleagues utilizes blood stage-derived Plasmodium berghei schizonts that have been purified from a blood culture by density gradient centrifugation. Naturally, this transfection protocol depends on the availability of suitably infected mice, constituting a time-based variable. In this study, the potential of transfecting liver stage-derived merozoites was explored. In cell culture, upon merozoite development, infected cells detach from the neighbouring cells and can be easily harvested from the cell culture supernatant. This protocol offers robust experimental timing and temporal flexibility.MethodsHeLa cells are infected with P. berghei sporozoites to obtain liver stage-derived merozoites, which are harvested from the cell culture supernatant and are transfected using the Amaxa Nucleofector® electroporation technology.ResultsUsing this protocol, wild type P. berghei ANKA strain and marker-free PbmCherryHsp70-expressing P. berghei parasites were successfully transfected with DNA constructs designed for integration via single- or double-crossover homologous recombination.ConclusionAn alternative protocol for Plasmodium transfection is hereby provided, which uses liver stage-derived P. berghei merozoites for transfection. This protocol has the potential to substantially reduce the number of mice used per transfection, as well as to increase the temporal flexibility and robustness of performing transfections, if mosquitoes are routinely present in the laboratory. Transfection of liver stage-derived P. berghei parasites should enable generation of transgenic parasites within 8–18 days.

Highlights

Malaria research is greatly dependent on and has drastically advanced with the possibility of geneti‐ cally modifying Plasmodium parasites
Transfection of deoxyribonucleic acid (DNA) constructs into P. berghei parasites is performed into blood stage-derived schizonts and merozoites, and benefits from the fact that schizonts do not rupture in in vitro blood cultures and can be enriched and purified
This work presents an established protocol that allows efficient transfection of liver stage-derived merozoites in so-called detached cells and merosomes (Fig. 1). This protocol is based on the fact that in vitro, P. berghei-infected cells detach from their neighbouring cells and float into the cell culture supernatant

Summary

Introduction

Malaria research is greatly dependent on and has drastically advanced with the possibility of geneti‐ cally modifying Plasmodium parasites. The commonly used transfection protocol by Janse and colleagues utilizes blood stage-derived Plasmodium berghei schizonts that have been purified from a blood culture by density gradient centrifugation. This transfection protocol depends on the availability of suitably infected mice, constituting a time-based variable. At the end of exo-erythrocytic parasite development, merozoites are released from the parasitophorous vacuole (PV) into the hepatocyte cytoplasm This leads to the detachment of the infected host cell from its neighbouring cells and in in vitro cultures, to detachment of the infected cells, which float freely in the culture supernatant. This work presents an established and optimized protocol for transfection of liver stage-derived schizonts and merozoites, which aims to reduce the number of animals used for the generation of transgenic parasite lines

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