For large-scale biochemistry, we electroporate neurons before plating and grow them in 75-cm2 flasks.
Hercules, CA, USA)
Rad)
of 1 to 4 mg/mL
Coating of Flasks: Prepare at Least 1 Day Before Preparing the Neurons 1. Add 10 mL of a 10 μg/mL polylysine solution to the flask and incubate for 1 hour at room temperature. 2. Wash 3 times with water and add 10 mL horse-MEM. Incubate overnight in a 37°C, 5% CO2 incubator. 3. Replace the medium with N2 medium and equilibrate in the incubator for at least 3 hours. 4. Dissect hippocampi, triturate, and count. 5. Add 2.5 to 3 × 106 cells to an electroporation cuvette. 6. Add 50 μg DNA and HBSS if necessary, to a final volume of 0.8 mL and mix with a pasteur pipet. Leave at room temperature for 3 to 5 minutes with occasional mixing. 7. Electroporate at 850 V, 25 μF, and 200 Ohm. Time constant should be 0.8. 8. Carefully resuspend cells by pipettingtwice with the pasteur and transfer cells into flask. To check efficiency, a small aliquot can be plated in a 3.5-cm coated dish. Neurons can be fixed and processed for immunofluorescence directly in the dish. For microscopy, a coverslip can be mounted with Mowiol (Merck, West Point, PA, USA), and the neurons can be analyzed for transfection efficiency. We analyze neurons typically 6 to 7 days after EP, but probably later time points are also possible.
Expression of Cytomegalovirus (CMV)-
Promoter Driven Genes Can be Enhanced
by Induction with Sodium Butyrate
Add sodium butyrate to a final concentration
of 2 mM and incubate for 17 to 24
hours. A stock solution of butyrate is prepared
by dissolving butyrate in water to a
final concentration of 0.5 M. Stock solution
is stored at 4°C.
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