This protocol demonstrates a straightforward in vitro genetic modification method for the molecular study of the primary cilium-dependent signaling pathway in the primary granule cell precursor cultures. Due to the cost, low viability, and poor efficiency of the current transfection method, we introduce a simple, cost effective, and efficient electroporation technique for investigating cilium-dependent signaling pathway in primary GCP cultures. To begin, add 0.5 milliliters of culture medium into each well of the 24-well culture plate containing coated cover slips, and keep it warm at 37 degrees Celsius in a carbon dioxide incubator.
Pipette the required number of cells into a sterile 1.5-milliliter microcentrifuge tube and spin at 200x G for five minutes at room temperature. Discard the supernatant and resuspend the cell pellet in 200 microliters of Opti-MEM. Repeat this procedure twice to ensure no residual culture medium is present in the tube.
Set the parameters of electroporation as listed. Pipette the electroporation reaction gently to mix well, and use a long P200 pipette tip to transfer an exact volume of 100 microliters of the mixture into the two-millimeter gap cuvette. Once the cuvette is inside the cuvette chamber, press the omega button of the electroporator and note the impedance value by adhering to a precise volume of 100 microliters.
The range of impedance value should be approximately 30 and 35. Press the start button to initiate the pulse. Record the measured current values in Joules shown on the reading frame.
Remove the cuvette from the chamber. Immediately add 100 microliters of pre-warmed culture medium into the cuvette and resuspended it by pipetting up and down two to three times. Immediately transfer the cell suspension to the 24-well plate previously prepared.
Incubate the cells at 37 degrees Celsius in a carbon dioxide incubator. Observe the cells under the fluorescence microscope on the following day. In the present study, the electroporation efficiency of DMSO and SAG-treated groups appeared comparable.
Immunostaining of the primary cilium marker Arl13b demonstrates that the ciliation rate of GCP at div-2 of culture in both the vehicle and SAG-treated groups. The ciliation rate illustrates the percentage of Pax6 expressing GCPs bearing a primary cilium on the cell surface at div-2 post-electroporation. The figure shows a significant increase in smoothened EGFP localization on the primary cilium axoneme of Pax6 expression GCP cells at 24 hours post-SAG treatment, indicating a profound activation of the primary cilium-dependent hedgehog signaling pathway.
In order to achieve a higher electroporation efficiency, one should ensure high purity of the plasmid used, and no residue culture media is present in the plasmid electroporation mixture. This protocol should be directly applicable and beneficial for in vitro genetic modification experiments on primary cultures and cell types that are difficult to transfect, including neurons and human-induced pluripotent stem cells.
