This article details a method for delivering plasmids into sporozoite nuclei using nucleofection. The process involves isolating sporozoites and utilizing a specialized buffer to ensure efficient transfection while maintaining cell viability.
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 fields also disturb the double-layered nuclear membrane surrounding the nucleus that develops transient gaps, facilitating direct entry of plasmids into the nucleus, without need for cell division.
Once nucleofection is complete, cellular and nuclear membrane pores reseal, restoring membrane integrity and homeostasis while trapping plasmids inside the nucleus.
Remove the cuvette from the device. Add suitable media for sporozoite recovery and transfer into a fresh tube. Successfully nucleofected sporozoites with plasmid DNA express fluorescent proteins.
First, centrifuge the sporozoites or merozoites suspension at 600 x g for 10 minutes, then, discard the supernatant. In the following order, add 85 microliters of nuclear transfection buffer, 10 micrograms of plasmid, and 25 units of restriction enzyme into the 1.5-milliliter tube containing sporozoites or merozoites. Transfer the suspension to a nuclear transfection cup.
Put the cup into a nuclear transfer groove, turn on the nucleofection device by using the power button, and select the transfection procedure U-033. When the program finishes, press the X button of the nucleofection device, and the screen displays OK, indicating that the nucleofection is successful. Add 0.5 to 1 milliliter of Dulbecco's Modified Eagle's Medium to the nucleofection cup to stop the reaction.
After mixing gently, transfer the suspension to a 1.5-milliliter tube.
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