In the past 15 years,
Plasmodium parasites have become greatly accessible for genetic manipulation [
1‐
3], facilitated by the genome sequencing of human and rodent malaria parasites [
4‐
6]. Advances continue and
Plasmodium knowlesi parasites were recently successfully adapted for in vitro culture in human red blood cells including successful transfection, which resulted in efficiencies of up to 30% [
7]. Transfection efficiencies of rodent
Plasmodium berghei parasites have increased up to 1:1000 as a result of implementing the highly efficient non-viral Nucleofector
® technology [
1,
8]. A major advantage of using the model organism
P. berghei for
Plasmodium research is the accessibility of the entire life cycle in vitro as well as in vivo, including the liver stage development. A further advantage is the availability of an almost complete genomic DNA library that originated from phage-based vectors, applicable for generation of knock-outs, and tagging of genes [
9‐
11]. Methods for typical genetic manipulation, such as the generation of knock-outs and complemented parasites, fluorescent tagging of proteins and even conditional knock-outs, are available for both rodent and human
Plasmodium parasites [
9,
12‐
14]. Classically, transfection of 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 thus be enriched and purified. Transfection of schizonts and free merozoites, compared to other asexual blood stages, is facilitated by the fact that DNA used for transfection has to cross only two or three membranes, namely the erythrocyte membrane (depending on whether or not merozoites have been released), the parasite plasma membrane (PM) and the nuclear membrane, instead of four, including the parasitophorous vacuole membrane [
1]. The standard protocol for
P. berghei transfection, requires the infection of two mice, which ideally should have a parasitaemia of about 3% usually achieved between day 5 and 7 after pre-infection. Once the parasitaemia has reached about 3%, blood stage parasites are taken into culture for 16–18 h and following this, schizonts are purified using a density gradient. Purified schizonts and merozoites are subsequently transfected using the Amaxa Nucleofector
® electroporation technology [
1,
8]. This study took advantage of the fact that the merozoite stage of
Plasmodium parasites is not restricted to the blood stage, but is also produced at the end of liver stage development. The
Plasmodium liver stage is characterized by an immense expansion of the parasite population. Intriguingly, a single sporozoite that has infected a host hepatocyte can mature into thousands of progeny merozoites [
15]. 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 then float freely in the culture supernatant. Merosomes, sacs containing infectious merozoites, are subsequently extruded from the detached cell and are also found in the cell culture supernatant [
16,
17]. Single detached cells of in vitro-cultured
P. berghei parasites were recently described to harbour an average of about 4500 individual merozoites [
16,
18]. In a previous study by Stanway et al. individual merosomes or detached cells were collected and used for sub-cloning of transgenic parasites, thereby greatly contributing to the reduction of animals used to achieve clonal transgenic parasite lines [
17]. This work presents an established and optimized protocol for transfection of liver stage-derived schizonts and merozoites, which equally aims to reduce the number of animals used for the generation of transgenic parasite lines.