The overall goal of the following experiment is to transfect single or small groups of retinal ganglion cells using in vivo electroporation in postnatal mice to study the development of retinal ugal projections. This is achieved by surgically exposing the eyeball and injecting a small volume of plasma DNA solution directly into the retina. As a second step, electrodes are placed on the eyeball directly over the injection site, and electrical pulses are applied, which allows transfer of plasma DNA into retinal neurons.
Next, at the desired age, electroporated retinas and brains are harvested to visualize transfected retinal ganglion cells and their projections to the CNS results are obtained that show fluorescent labeling of both dendrites and axonal ARBs of labeled retinal ganglion cells in their various subcortical target structures. The main advantage of this technique over existing methods, like in-utero retinal electroporation, is that this technique is less invasive and does not interfere with early axon guidance events, such as crossing over at the optic chiasm. It also allows us to label a small population or single retinal ganglion cells in postnatal mice with precise spatial and temporal control.
To make electrodes for electroporation, break one prong off a pair of dumont number five forceps, and then solder a wire at the base of each prong. Wrap the base in wires with insulation, tape and fit a plastic spacer between the prongs to provide spring action. Once the electrodes are put together, connect the wire from one prong to the foot pedal, which is connected in series to the electrical stimulator.
When the foot pedal is pressed, current flows through the electrodes. Next, connect the wire from the other prong to the electrical stimulator. And finally, connect the electrical stimulator to the oscilloscope and audio monitor.
Mount the injector system onto a three axis micro manipulator at the bench using a pipette puller. Make pulled glass pipettes with a long taper and a small tip using a needle provided with the nano Inject two. Backfill a pulled pipette with mineral oil and secure it to the micro injector.
Using sharp microdissection scissors. Cut the tip of the mounted pipette to create a two to three micrometer opening. Fill the pipette with injection solution by drawing it up through the open tip.
Anesthetize mouse pops at H four to five days postnatal by hypothermia. And confirm the anesthetic state using a toe pinch. Place the anesthetized mouse under a dissecting scope and surgically open the eyelid by cutting along the length of the future eyelid Opening with microdissection scissors, protrude the eyeball by gently applying pressure around the eye socket with a pair of forceps.
Position the pipette near the eyeball by adjusting the micro manipulator. Press the foot pedal of the microinjection system to ensure that the pipette tip is not clogged with one hand. Stabilize the protruded eyeball by pinching the skin around the eye socket with forceps with the other.
Move the micro manipulator so that the pipette pierces through the retinal pigment, epithelium and enters the retina. Dispense the desired amount of solution by pressing the foot pedal, retract the pipette and place the electrode tips directly over the injection site. On either side of the eyeball just touching the surface, press the foot pedal attached to the stimulator to complete the circuit and electro parade the eyeball.
Gently push the eyeball back into its socket and put up ophthalmic ointment over the cut. Place the mice on a heating pad maintained at 36 degrees Celsius until they regain consciousness and return them to their mother only RG CS transfected. With both plasmid and coding for Cree Recombinase and flock stop EGFP are able to express.
EGFP single cell labeling is achieved by virtue of the relatively low concentration of Cree plasmid used EGFP labeled single retinal ganglion, cell dendrites and axons can be visualized in a flat mount retina. Other retinal cell types such as starburst omicron cells can also be labeled with this technique. Here we see an example of a cluster of EGFP labeled retinal neurons, including RG CS and omicron cells in a flat mount retina at postnatal day 14.
Using this technique, we can analyze retina topic targeting of RG CS in a whole mount superior colus from all four major retinal coordinates in the same mouse Once mastered. This technique can be done in four to five minutes if it is performed properly. After watching this video, you should have a good understanding of how to make focal injections of solutions such as plasma DNA into the postnatal retina and in vivo transfection of retinal neurons using electroporation.
