Electroporation-Mediated In Vitro Gene Delivery: An Electric Pulse Technique to Introduce Fluorescent Reporter Protein-Encoding Plasmids Into Cultured Cells

Overview

This video demonstrates the electroporation technique that introduces fluorescent protein-encoding plasmid DNA into mouse primary cerebellar granule cell precursors.

Protocol

1. Electroporation for Hh receptor transgene overexpression:  pEGFP-Smo

1. For electroporation using a 2 mm gap cuvette, prepare the following plasmid-cell electroporation mixture for each reaction of electroporation: 1.2 × 10⁶ mouse primary cerebellar granule cell precursors cells and 10 µg of pEGFP-mSmo (adjust the DNA stock concentration to ~2-5 µg/µL in Tris-EDTA buffer or sterile dH₂O) in 100 µL of Opti-MEM. 
   NOTE: Reduce the total cell number if the cells are insufficient (though this may reduce the electroporation efficiency). However, do not adjust the amount of plasmid nor the total volume of Opti-MEM used per cuvette. If the total cell number required for the experiment exceeds 1.5 × 10⁶ cells, scale up the electroporation mixture accordingly and perform multiple electroporation reactions separately. The cuvette may be reused up to 5 times.
2. Prepare the post-electroporation cell seeding plate by adding 0.5 mL of culture medium into each well of the 24-well culture plate containing coated coverslips and keep it warm at 37 °C in a CO₂ incubator. To prepare the coverslips for cell attachment, incubate autoclaved 12 mm glass coverslips with 100 µg/mL of poly-D-lysine (PDL, 1 mg/mL in sterile dH₂O) for at least 1 h at 37 °C. Keep the coverslips in the same PDL solution at 4 °C until use.
3. Pipette the required number of cells into a sterile 1.5 mL microcentrifuge tube and spin at 200 × g for 5 min at RT. Discard the supernatant and resuspend the cell pellet in 200 µL of Opti-MEM. Repeat this step twice with Opti-MEM to ensure no residual culture medium is present in the tube.  
   NOTE: For every well/coverslip of a 24-well plate, electroporate and seed 1.2-1.3 × 10⁶ cells/well to obtain ~70-75% cell confluency on the next day of culture.
4. Set the parameters of electroporation as shown in Table 1.
5. Pipette the electroporation reaction gently to mix well and use a long P200 tip to transfer an exact volume of 100 µL of the mixture into the 2 mm gap cuvette. Avoid forming bubbles.
6. Place the cuvette in the cuvette chamber.
7. Press the Ω button of the electroporator (see the Table of Materials) and make a note of the impedance value, which should be ~30-35. To ensure the electric impedance value Ω falls within the range of 30-35, adhere to a precise volume of 100 µL.
8. Press the start button to initiate the pulse.
9. Record the values of the measured current and joules shown on the reading frame.
10. Remove the cuvette from the cuvette chamber.
11. Immediately add 100 µL of prewarmed culture medium into the cuvette and resuspend it by gentle pipetting up and down 2-3 times. Immediately transfer the cell suspension to the 24-well plate containing coated coverslips prepared in step 1.2.
    NOTE: To minimize cell death, seed the cells immediately after electroporation.
12. Incubate the cells at 37 °C in a CO₂ incubator. Leave the cells undisturbed for 3 h to avoid cell detachment.
13. At 3 h post seeding, gently aspirate and discard half of the supernatant medium to remove floating dead cells and debris and replace with the same amount of prewarmed culture medium.
14. Incubate the cells at 37 °C in the CO₂ incubator.

	Poring Pulse Setting		Transfer Pulse Setting
	Mouse Primary GCPs	Primary neurons	Mouse Primary GCPs	Primary neurons
Voltage	275 V	275 V	20 V	20 V
Length	1 ms	0.5 ms	50 ms	50 ms
Interval	50 ms	50 ms	50 ms	50 ms
No.	2	2	5	5
D rate	10%	10%	40%	40%
Polarity	+	+	±	±

Table 1: The electroporation parameters of mouse primary GCPs and primary neurons using Super Electroporator NEPA21 TYPE

Materials

Name	Company	Catalog Number	Comments
B27 supplement	Life Technologies LTD	17504044
GlutamMAXTM-I ,100x	Gibco, Life Technologies	35050061	L-glutamine substitute
Matrigel	BD Biosciences	354277	Basement membrane matrix
Neurobasal	Gibco, Life Technologies	21103049
Poly-D-lysine Hydrobromide	Sigma Aldrich	P6407
CU 500 cuvette chamber	Nepagene	CU500
EPA Electroporation cuvette (2 mm gap)	Nepagene	EC-002
Opti-MEM	Life Technologies LTD	31985070	Reduced-serum medium for transfection
pEGFP-mSmo	Addgene	25395
Super Electroporator NEPA21 TYPE II In Vitro and In Vivo Electroporation	Nepagene	NEPA21	Electroporator