Nucleofection of Parasite Forms: An Electroporation-Based Transfection Method to Deliver Fluorescent Protein Encoding-Plasmids Into Sporozoite Nucleus

To deliver plasmids into sporozoite nuclei by nucleofection, begin with a suspension of isolated, viable sporozoites - motile, infectious stage of parasites - in buffer. Sporozoites are slender-shaped cells enclosed by an outer plasma membrane and an underlying inner membrane complex consisting of a series of flattened vesicles and proteins critical for structural stability and motility.

Centrifuge the suspension to pelletize the sporozoites. Resuspend the sporozoite pellet in a suitable volume of nuclear transfection buffer, supplemented with fluorescent protein-encoding plasmids. Nuclear transfection buffer with high buffering capacity and ionic strength ensures efficient transfection while preserving sporozoite functionality.

Transfer the suspension to a nuclear transfection cuvette. Place the cuvette into the holder of a nucleofection device. Run the appropriate nucleofection program.

During nucleofection, specifically applied, short, high-voltage pulses, along with buffer constituents, temporarily disrupt the plasma membrane and inner membrane complex, forming transmembrane pores, which provide plasmids access into the cytoplasm.

Simultaneously, these high-strength electric fi