The overall goal of this procedure is to genetically label and manipulate peel surface neural progenitors. This is accomplished by first inserting a glass pipette filled with plasma DNA solution into the peel surface region. The second step is to inject the DNA into the meningeal space, overlying the peel surface.
Next, the electrodes are oriented to appropriately deliver current to the peel surface. The final step is that DNA is electroporated into peel surface progenitors by application of current. Ultimately, this method can be combined with other techniques to show the natural history and molecular determinants of peel surface, neural progenitor proliferation, migration, and or differentiation.
The main advantages of this technique over existing methods such as viral transduction, is that electroporation is safer, more flexible, and does not require creation of virus particles or packaging. This method can answer key questions in the field of neural stem and progenitor cells, such as what is the contribution of these peel surface progenitors to tissue, histo genesis and neural circuit function. To begin, use a pipette puller to form pipettes from glass capillary tubes once pulled, cut the tip with sharp scissors.
Next, prepare a 1%fast green stock solution using filtered nuclease free water. After diluting endotoxin free plasma DNA interest EDTA buffer, combine it in the appropriate proportions to the 1%fast green solution. Using a standard pipette place the desired amount of plasmid mix to be injected onto a piece of paraffin wax film for reference.
Next, using a micro injector, carefully insert the assembled glass pipette into the tube containing the plasmid mixture. Avoid touching the tip to the edges of the tube to prevent breaking. Aspirate the solution into the pipette by slowly dialing back the transfer dial.
Once a sufficient volume has been loaded, dial forward until the pressure returns to the neutral position of zero. Hector Pascals. Next, place the tip next to the reference injection and use the foot pedal from the micro injector to eject one volume.
Adjust the pressure until the amount equals the previously pipetted reference volume. After calibrating the injection volume, retrieve the animals for plasmid injection. Once the depth of anesthesia is confirmed, hold the pup using the thumb and index finger of the less dominant hand.
Use the dominant hand to target the desired region, the cortical hemispheres and other superficial structures such as the superior colliculi should be readily visible. Once the correct location has been identified, insert the pipette past the skin and skull. Taking care to avoid cerebral arteries.
To target the peel surface immediately cease advancing the pipette As soon as the skull has been penetrated with the pipette tip in the correct position, inject the plasmid solution using the foot pedal of the micro injector After the injection, carefully remove the pipette. Prevent the tip from drying and clogging in between injections by placing it into the test droplets located on the paraffin film. Repeat these steps until all pups are injected to increase conductance and prevent burning of the skin.
Cover three millimeter platinum tweezer TROs with electroporation gel. Next, adjust the electro settings for injections into the cortex. Place the negatively charged probe over the injected area and the positively charged probe around the contralateral region Below the eye.
The electrode orientation should be at a 45 to 60 degree angle relative to the midline. Use the foot pedal to trigger the current and hold the tweezer TROs in place for three to five pulses. Depending on the pup's age and weight, the pulses should target the DNA into the underlying peel surface cells.
The negative pole can be swept over the injection area. In between pulses or a larger electrode can be employed to increase the electro area immediately after electroporation, carefully clean off the gel from pups and place them under a heat lamp for approximately five minutes. Pups will acutely lose their natural reddish pinky in the minutes after electroporation due to cyanosis.
Observe the pups for the recovery of natural color and the initiation of normal movement before returning them to their cages or injections into the superior colus. Locate the target region just posterior and lateral to Lambda and roughly at the midline, followed the same steps just demonstrated. With successful injections, the plasmid mix naturally fills the contours of the superior colus.
When electro operating after superior colus injections place the negatively charged probe of the electrode over the injected area and the positively charged probe around the eye, snout or chin. In this case, the electrode orientation is at a 25 to 40 degree angle relative to the midline. This image shows the dorsal lateral cortex after electroporation of fluorescent reporter plasmids expressing membrane tagged clover in green and tag bfp.
Two nuclear protein shown here in red pseudocolor. Extensive dendrites are found in this cortical inter neuron roughly two months post electroporation. EGFP positive cells are BRDU positive indicating that they were proliferating during the electroporation procedure.
When attempting this technique, it's important to focus on proper targeting of the peel surface in order to avoid disruption of the underlying cortex and or vasculature.
