Regioselective Biolistic Targeting in Organotypic Brain Slices Using a Modified Gene Gun

The goal of this procedure is to rapidly and efficiently transfect or label distinct regions of organotypic slices using a gene gun. This is accomplished by first preparing viable organotypic slices of the brain or other tissues. The second step is to produce DNA or decoded gold particles for biotic delivery.

Next, the organotypic slice is placed under a stabilized gene gun mount and the coated particles a fired into the tissue or episomal transformation or labeling. The final step is to observe or analyze the effect of heterologous genes delivered to the isolated regions. Ultimately, this approach allows the user to precisely transfect distinct regions of the organ slices with various heterologous genes.

At the main advantage of this method over existing methods is that biologistics permits the transfect terminally differentiated cells, as well as limiting the radius of action to a small diameter. This method can help understand key neurobiological questions, such as understanding the regional contribution of a single gene. Begin this procedure by placing a fresh brain into a small recipient mold.

Cover it with the ARO solution, which has been called to slightly above room temperature. Then rapidly cool the mold to four degrees Celsius by placing it on ice. When the aros has set.

After five to 10 minutes, remove the aros embedded brain from the mold. After that, glue it to the fibro slicer platform. Subsequently transfer the platform to the Vibra slicer chamber containing sterile ice cold PBS.

After slicing, gently place the slices on the cell culture inserts in a six multi-well tray with culture media on the outside of the insert. Incubate them in a humidified incubator at 37 degrees Celsius with 5%carbon dioxide. In this procedure, prepare the nano projectiles using 40 nanometer diameter gold particles by first adding 50 microliters of 0.05 molar sperm aine, and 10 microliters of DNA at one milligram per milliliter to 10 milligrams of gold particles.

Then add calcium chloride in the solution mixture to aid the binding of DNA molecules to the gold nanoparticles. Afterwards, draw the solution into the tubing with a syringe. Place the tubing in the tubing preparation station.

Allow the gold particles to settle. Then remove the supinate by aspiration With a syringe. Rotate the tubing to evenly spread the gold particles.

Subsequently dry the particles under a constant nitrogen flow at five liters per minute. To create DNA bullets, cut the tubing into one centimeter pieces using a tubing cutter. Then either insert them immediately into the gene grown cartridge or keep them desiccated at four degrees Celsius until use in a sterile lamina flow hood, load the cartridge containing the DNA coated gold into the cartridge holder.

Then load the holder into the gene gun. Attach the modified gene gun barrel to it using sterile forceps. Place a filter insert containing an organotypic slice in a sterile plastic dish.

Remove the media from the organotypic slice. Next, set the gas pressure at 50 PSI using the mount. Place the gene gun at a distance of 10 millimeters above the desired region of the slice for genetic delivery.

Then carefully aim at the center of the barrel and fire the coated particles into the tissue. After the biotic transfection, wash the slice gently twice with PBS. Then fix the slice by incubating in 4%ice cold PFA in PBS for 20 minutes.

After that, wash the slice twice in PBS. Again, gently cut the membrane away from the insert using a scalpel without disturbing the slice. Then place the membrane support on a microscope slide with forceps and allow the organotypic slice to rest on it.

Next, add one drop of mounting media on the slice. Place a cover slip gently without any force directly in contact with the tissue. Afterwards, secure the cover slip with a thin layer of nail varnish.

This figure shows the biotic delivery of fluorescent protein encoded DNA into the organotypic slice of a six week old mouse. As can be seen here, a bikini neuron with a remarkable dendritic harbor found at the interface of the bikini layer and the molecular layer of the cerebellum shows pronounced labeling following transient biotic transfection with P-E-G-F-P-N one. Here is a higher magnification of the dendrites with the spines, and here is a parametal cell found in the ca one region of the hippocampus.

Following the biotic delivery of PDS Red FP coated gold nanoparticle Once mastered, this technique can be performed in only a few hours over the period of three to four days following this procedure. Other methods such as confocal microscopy can be used to answer additional questions such as how certain neurons are connected to neighboring regions.