This article describes a method for transfecting wild-type Dictyostelium discoideum amoeba cells using electroporation. The process involves preparing the cells in a cold buffer, introducing a plasmid, and allowing for recovery in a nutrient-rich medium.
Begin with wild-type Dictyostelium discoideum amoeba cells suspended in a cold buffer.
The buffer provides an ionic environment that maintains membrane stability.
Next, add the amoeba cells to a tube containing a plasmid with the target gene.
Gently mix the solution to bring the plasmids into close contact with the amoeba surface.
Transfer this mixture into a pre-chilled electroporation cuvette and perform electroporation.
During electroporation, the electric pulses create transient pores in the cell membrane.
The plasmid enters through these pores into the cytoplasm and subsequently translocates to the nucleus.
Next, transfer the amoeba cells to a Petri dish containing a liquid medium supplemented with feeder bacteria.
The amoeba consumes bacteria via phagocytosis, providing essential nutrients for cell recovery and enabling plasmid expression.
Feeder bacteria support transfection in wild-type amoeba cells, without the need for special strains that grow without bacteria.
Resuspend the cells in H40 buffer.
Next, add 100 microliters of the cells to a tube containing one to two micrograms of DNA. Pipette up and down to mix and transfer the cell DNA mixture to a pre-chilled electroporation cuvette.
Do not add more than two microgram of DNA. Higher DNA concentration are toxic for the cells and reduce efficiency.
After electroporation, immediately transfer the cells to a prepared 10 centimeter petri dish and allow the cells to recover for five hours.
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