Electroporation of plasmids into MEFs

Warning

This protocol has not been tested!

Note

This protocol is specific for electroporation to test settings for the Amaxa II electroporator and plasmid concentrations for MEFs. This is a good starting place for any electroporation protocol including for iPSCs. Settings will differ by cell type.

Overexpression

In order to determine which conditions allow the best efficiency and highest viability for the Amaxa cell electroporator, cells first need to be tested for compatibility with a specific buffer and voltage setting. This is achieved by setting up a matrix experiment with controls as follows:

  • MEFs + DNA with no electroporation

  • MEFs – DNA with electroporation

  • MEFs + 2 µg pMax-GFP with MEF buffer 1, under setting A-23

  • MEFs + 2 µg pMax-GFP with MEF buffer 1, under setting T-20

  • MEFs + 2 µg pMax-GFP with MEF buffer 2, under setting A-23

  • MEFs + 2 µg pMax-GFP with MEF buffer 2, under setting T-20

The procedure for electroporation is as follows:

  1. Add 100 µl of Buffer supplement to each buffer type. This reagent will expire in 3 months. Also pre-warm some MEF media and have available.

  2. Thaw MEFs from a desired strain, and pass once. After the passage, cells will be ready for electroporation when they reach 50-60% confluency.

  3. Grow enough cells so that you have 2 x 106 cells for each condition

  4. For each condition, have 4 wells of a 6-well culture plate filled with 2 mL of pre-warmed media, and in store in the incubator at 37°C.

  5. Trypsinize and count cells.

  6. Divide cells into 50 mL tubes so that each tube has 2 x 106 cells. Spin cells down and remove media.

  7. Resuspend cells in 100 µl of desired MEF buffer, and place slurry in cuvette, making sure all liquid is in the bottom and there are no air bubbles.

  8. Call up desired program on the Amaxa electroporator and insert cuvette. It may only be loaded in one direction. Press the “x” button and wait for the program to conclude.

  9. Remove cuvette, and in the BSC, add 500 µl of pre-warmed media. Remove entire mixture and add to a 1.5 mL eppendorf tube.

  10. Add 150 µl each of the above mixture to 4 wells of the culture plate. Make sure plate is labeled on the bottom, as the lid may get mixed up with another lid.

  11. Allow cells to equilibrate in the incubator (37°C, 5% CO2 ) for 24 hours, then examine cells under the fluorescent microscope to estimate confluency and percent GFP positive.

  12. The experimental parameter that yields the best uptake efficiency and highest confluency will be the program that we use for our experiment. For C57BL/6J, the correct buffer/program mixture is MEF Buffer 1 and T-20.

Plasmid Concentration Experiment

Next, we need to determine the proper amount of overexpression plasmid to add to our cells for our planned experiment. We will standardize this by adding 3, 6, and 10 µg of each to three different sets of cells. We will also have a DNA– control and a pMax-GFP control. Below are our parameters:

  • Cells + no DNA with MEF buffer 1, under setting T-20

  • Cells + 2 µg pMax-GFP with MEF buffer 1, under setting T-20

  • Cells + 3 µg experimental plasmid with MEF buffer 1, under setting T-20

  • Cells + 6 µg experimental plasmid with MEF buffer 1, under setting T-20

  • Cells + 10 µg experimental plasmid with MEF buffer 1, under setting T-20

The experiment will be run under the same conditions as above, starting at step #1. Since each parameter will have 4 wells, we can perform 4 different tests on them.

  1. At 24 hours post-electroporation, from the first well, remove supernatant, but do not dispose of it. Wash cells with 0.5 mL of PBS, remove, but do not dispose of it. Then, trypsinize cells with an appropriate amount of trypsin, incubate at 37°C for 5 min., then save supernatant. This saved supernatant from the trypsin step may be used to count the viable cells left on the plate on the countess. After cells are counted, combine the original, PBS and trypsin supernatants and run on the flow cytometer.

  2. At 24 hours post-electroporation, from the second well, remove supernatant and wash well once with 1 ml of PBS. Remove PBS and add 0.5 mL of Trizol to the well and pipette up and down several times to lyse cells. Freeze Trizol/cell mixture immediately.

  3. At 48 hours post-electroporation, repeat step 2 with third well.

  4. At 96 hours post-electroporation, repeat step 2 with forth well.