High-efficiency transfection of Acanthamoeba castellanii using a cationic polymer

The free-living amoeba Acanthamoeba castellanii is an ecologically, clinically, and evolutionarily important microorganism. A. castellanii amoebae are directly pathogenic to humans and serve as reservoirs for bacterial pathogens (e.g., Legionella pneumophila ) but also regulate the proliferation of other microorganisms in the soil. Despite their importance, no reliable genetic system has been developed, hampering the use of A. castellanii and related species as model organisms. Transfecting A. castellanii with plasmids is possible with commercial kits, but it is expensive, inefficient, and vulnerable to product discontinuation. In this contribution, we present a method for efficient transfection of A. castellanii with readily available and inexpensive polyethylenimines. We systematically explore the method’s parameters, obtaining up to 100-fold higher efficiency than currently used protocols. The method presented here provides a robust step towards a complete genetic toolbox for A. castellanii , hence expanding its use as a model organism. ### Competing Interest Statement The authors have declared no competing interest.