# High-efficiency transfection of Acanthamoeba castellanii using a cationic polymer

## TL;DR

A new, efficient, and low-cost method for transfecting Acanthamoeba castellanii using a common polymer could help study this important amoeba.

## Contribution

A 100-fold more efficient and affordable transfection method for Acanthamoeba castellanii using polyethylenimine.

## Key findings

- Transfection efficiency increased up to 100-fold compared to commercial kits.
- The method uses inexpensive and readily available polyethylenimine polymer.
- Multiple plasmids can be co-transfected into individual amoebal cells.

## Abstract

The free-living amoeba Acanthamoeba castellanii is an ecologically, clinically, and evolutionarily important microorganism. A. castellanii amoebae can be directly pathogenic to humans and serve as reservoirs for bacterial pathogens (e.g., Legionella pneumophila), but they 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, achieving up to 100-fold higher efficiency compared to currently used protocols. This method provides a robust step toward a complete genetic toolbox for A. castellanii, thereby expanding its utility as a model organism.

•A method for transfection of A. castellanii up to 100-fold more efficient than commercial kits•Method is based on polyethylenimine, a readily available and inexpensive polymer•We demonstrate feasibility of co-transfection of multiple plasmids per amoebal cell

A method for transfection of A. castellanii up to 100-fold more efficient than commercial kits

Method is based on polyethylenimine, a readily available and inexpensive polymer

We demonstrate feasibility of co-transfection of multiple plasmids per amoebal cell

Studies of the free-living amoeba Acanthamoeba castellanii, a model for pathogenic amoebae and a reservoir of bacterial pathogens, are limited by the lack of a genetic system. Transfection with plasmids is therefore a crucial alternative. Here, we describe a robust method to transfect A. castellanii using widely used and inexpensive polyethylenimines, resulting in a 100-fold increased efficiency compared with current, commercially available kits.

Moreno et al. present a robust, cost-effective transfection method for Acanthamoeba castellanii employing the common polymer polyethylenimine, yielding up to 100-fold improvement in genetic delivery efficiency. By overcoming a major technical limitation, this advance facilitates molecular genetic manipulation of an evolutionarily, ecologically, and clinically significant amoeba.