The overall goal of this procedure is to perform an in vivo electroporation experiment on neonatal mice in order to stably introduce genetic material into the cells of the retina. This is accomplished by first anesthetizing, a neonatal mouse pop on ice for approximately five to 10 minutes. The second step of the procedure is to identify the junction of the fused eyelids and to open the eye by cutting along this junction.
An incision is then made in the eye to facilitate insertion of the microinjection syringe. The micro injection syringe is then inserted through the incision and the DNA solution injected into the subretinal space. Finally, a tweezer electrode is placed onto the pop's head and a series of electrical pulses are delivered.
The main advantage of this technique over existing methods such as retroviral transduction, is that in vivo electroporation does not require the generation and handling of biological agents as tools for gene delivery. As a result, the technique requires less labor, fewer reagents, and can be conducted in any workstation approved for animal procedures. Generally, individuals new to the technique will struggle because it takes time and repetition to develop a feel for the placement of the microinjection syringe into the subretinal space.
Demonstrating the procedure will be Jimmy DeMilo, a postdoctoral fellow in my lab. Because the DNA concentration required for electroporation is relatively high, the desired plasmid DNA is first amplified incompetent cells extracted using a maxi prep, then purified and concentrated to about five micrograms per microliter. Fast green FCF dye is added as an injection tracer.
Once the plasmid DNA has been prepared, anesthetized newborn mouse pups on ice for approximately five minutes, monitoring them until unconsciousness is confirmed by footpad compression. Swap the area of the eye to be injected with 70%isop profile alcohol, and use a dissecting microscope to identify the FUS junctional epithelium where the eyelids come together. Using a sharp 30 gauge needle, carefully open the eye by cutting along the fused junctional epithelium without applying excessive downward pressure or cutting beyond the range of the eyelid.
Once the eyelid is opened and the eye is exposed, use the tip of the needle to make a small incision in the sclera near the junction with the cornea being careful not to penetrate too deeply and puncture the lens. Insert the blunt ended injection needle into the incision. Just until the resistance of the opposing scleral wall is felt slowly inject 0.3 microliters of the viscous DNA solution into the subretinal space.
Being careful not to press too tightly against the scleral wall, rotate the animal to check for an even spread of the DNA solution within the retina. First, soak the tweezer electrode in PBS to maximize electrical conductivity. Then place the head of the injected pup between the electrodes with the injected eye adjacent to the positive pole electrode and the non injected eye adjacent to the negative pole electrode.
Apply five square pulses using a pulse generator with each pulse being 80 volts and 50 milliseconds in duration with a 950 millisecond interval between pulses. Finally warm the pups under a warming lamp until they recover from the ice anesthesia. Upon recovery, return the electroporated pups to their mother at postnatal day three.
The majority of EGFP electroporated cells sit in the retinal neuroplastic layer and do not demonstrate distinct morphological characteristics of any of the differentiated neural glial cells of the retina. By postnatal day 14, electroporated cells can be found in the outer nuclear layer and the inner nuclear layer of the retina and the various labeled cells. Now display characteristic morphological hallmarks of differentiated neurons.
While attempting this procedure, it's important to perform all the steps smoothly and evenly. It's very easy to damage the retinas during the microinjection, and practices required to develop the manual dexterity needed to avoid tissue damage. Following this procedure.
Other methods such as immunohistochemistry or in situ hybridization may be performed in order to determine if the expression of genes of interest are up or down regulated in the electro retinas.
