Agarose-Embedded Electroporation-Assisted Gene Transfer in Mouse Cortical Interneuron Progenitors

Fill a micropipette with a DNA mixture containing plasmid vectors carrying the gene of interest and a tracking dye. Secure it in a micromanipulator.

Position an agarose block under the microscope and place an agarose-embedded mouse embryonic brain slice with cortical interneurons over it.

Lower the micropipette and inject the DNA mixture into the interneuron progenitor region. The tracking dye marks the injection site.

Position another agarose block on a Petri dish electrode and an agarose column on the cover electrode.

Transfer the slice onto the block and align the cover electrode over the injection site.

Apply brief electrical pulses to permeabilize cell membranes temporarily, allowing the plasmids to enter the nucleus.

Incubate the slice in a medium to maintain cell viability.

Inside the nucleus, the plasmid triggers gene expression, produces the target protein, and modulates interneuron progenitor functions.

For the injection, mix the expression and control vector DNA at a 1 microgram per microliter concentration for each vector, and add fast green stock solution at a 1 to 10th dilution. Fill a pulled 0.5 millimeter inner diameter, 1 millimeter outer diameter glass micropipette with 10 microliters of the DNA mixture, and load the micropipette into a pneumatic pico pump injector. Place the bigger block of agarose under a stereomicroscope, and place the slice to be injected onto the piece of agarose.

Then, inject 25 to 50 nanoliter volumes into the selected medial ganglionic or caudal ganglionic eminence region of the slice. Immediately after the injection, place the small agarose block on the Petri dish electrode, and use a flat ended micro spatula to attach the agarose column to the mobile cover electrode.

Transfer the injected slice with its supporting membrane onto the agarose block, and place the top electrode with the agarose column on top of the selected region of the slice. Then electroplate the region with two 5 millisecond pulses of 125 volts, 500 milliseconds apart. After the electroporation, place the slice with its supporting membrane back into its holding dish and return the dish to the tissue culture incubator.