The overall goal of the following experiment is to conduct a rapid assessment of gene function in cerebral cortex development using organotypic slices. This is achieved by introducing plasmids, coex, expressing and inhibitory RNA and A GFP marker into the murn cerebral cortex. Short electrical bursts are applied to the brain, allowing the plasmid DNA to transfect the neuronal progenitor cells.
Next, the brains are dissected, embedded, and sectioned to produce organotypic slices that can undergo neuronal morphogenesis ex vivo. The organotypic slices can be searched for cortical neuron subpopulations with migratory or morphological defects by directly observing the GFP labeled cells. Hi, welcome to my lab at Brown University in the molecular biology department today, Dr.Liza and two students, Kat Zer and Mark Seba will demonstrate the ex vivo electroporation technique and organotypic slice culture, which is a very useful technique for neurodevelopmental studies.
This method can help answer key questions in various aspects of neurodevelopment, including neurogenesis, neuronal migration, and neuronal morphogenesis. Because this analysis is done in an near approximation of the IBU environment, it provides a low cost and rapid alternative to the generation of transgenic or knockout animals for genes of unknown function. Finally, in comparison with in vivo electro operation technology, the success of these vivo electro operation experiments is not dependent upon proficient surgery, skill development, and can be performed with a shorter training time and a skill.
After euthanizing a pregnant female mouse, dissect the embryos out into ice cold, complete HBSS, keeping them in their individual placental sacks. Next in the cold HBSS solution, release an embryo from the sack and cut off its head after the first vertebra for an injection, place the head on a piece of paraform paper on top of a Petri dish using a custom made Hamilton syringe. Inject six to eight microliters of DNA fast green mix through the third ventricle.
From there, the mix will fill both lateral ventricles in the cortical vesicles. For electroporation, BTX tweezer platinum electrodes are utilized. Place the positive electrode towards the side of the cortex that will be electroporated.
For example, the electrode should be on top of the head For dorsal cortex. For high transfection efficiency, always adjust the concentration of DNA to one microgram per microliter and use DNA isolated from a Maxi prep. If necessary, use an endotoxin free kit to purify the DNA After the electroporation.
Incubate the head on ice for at least five minutes before dissecting it. It is very important to use the right amount of electrical pulses and current when doing the electroporation to avoid damaging the younger embryonic brains. After the electroporation, the tissue becomes very fragile.
It's important to do the dissections very carefully as the skin and the school can pull on the meninges of the cortical vesicles, and this could damage the cortical vesicles when being removed from the skull. After five minutes, dissect out the brain in cold. HBSS begin by making a small incision on the side of the head and peeling the skin off the sides of the head.
Next, with fine forceps, gently peel away the pia from the brain. Remove the intact brain from the skull. When finished, keep the brain in ice cold HBSS and prepare to embed the tissue.
Begin filling the molds with 3%low melting point.Agros. Place the molds on ice so the agros will solidify near the bottom of the mold. First, ensuring that the brains remain supported and centered.
Now with fine forceps, gently remove a brain from the HBSS, absorb excess buffer with a kim wipe or filter paper, and set the brain into the low melting point.Agros. Then use a pipette tip to swirl the brain inside the mold to maximize contact between agros and tissue. With all the brains transferred, position each brain in approximately the same orientation and depth within its mold.
The aros will solidify in about five minutes. So work quickly. Allow the aros to completely solidify by incubating an additional five minutes on ice.
Trim excess aros before mounting the blocks. Then attach the agros blocks to each other using a fast bonding plastic adhesive, each brain in a stack should be positioned with its olfactory bulbs upward. Once the blocks are attached, trim the aros to ensure each brain will be sliced by the vibrato.
Then immediately submerge them in ice cold HBSS. To slice the blocks, set the vibratos velocity to a low speed, about half the maximum, and set the blade vibration frequency at the highest setting. Then generate 250 micron thick coronal slices, retrieve slices using a bent fine spatula and to transfer them to tissue wells with a fine brush or forceps.
If the tissue falls out of the agros, the tissue agros contact was not sufficiently formed in a tissue culture hood, prepare a six well plate with 1.8 milliliters of slice culture media per well, and place organotypic inserts code of laminin polity lysine in each. Well add 500 microliters of slice culture media to the top of the organotypic slice inserts. Then transfer up to five slices to each insert.
Next, remove any excess media from the top of the slices To ensure that the slices preserve their morphology as much as possible and have minimal cell death. It is important that a healthy liquid air interface is maintained and the slices do not float in the media. Finally, incubate the slices at 37 degrees Celsius in a humidified incubator.
Replace half the volume of media under the membrane every other day. The slices should not be floating on the media neurons expressing electroporated constructs can be visualized as early as eight hours Post electroporation after the desired days of culturing. Fix the slices in the membrane for analysis.
Wash each organic typic insert with warm PBS for five minutes. Perform three PBS washes in total, and then fix the tissue with 4%paraform aldehyde for one hour at four degrees celsius. After fixation, the tissues can be analyzed with cellular markers or simply stained with hooks stain After an hour, wash the slices with three 10 minute washes in one XPBS.
Then permeable the slices for two hours at room temperature in a 10%goat serum solution in one XPBS with gentle shaking. Then stain the tissues with hooks for one hour at room temperature with gentle shaking. Remove the stain with three 10 minute washes in one XPBS with gentle shaking while waiting.
Place frosted glass slides in a water chamber that is deep enough to bathe the tissues after they're placed on the slides. To mount the slices. First, cut the membrane free with a scalpel.
Second, using fine forceps, transfer up to five membrane bound slices to each slide submerged in water. Third, remove any excess water from each slide. Fourth, apply a drop of floor mount solution to each brain slice.
And lastly, gently place a cover slip on top of the slices and remove any air bubbles. The slices are then ready for microscopy depending on the amount of DNA electroporated and the embryonic stage. At electroporation, the transfection efficiency was found to vary.
Eight hours after transfection with a P silencer, GFP vector cells started to express GFP. During a longer culturing period, cells undergo a normal sequence of neurogenic events including proliferation, migration, and early neuronal differentiation. These slices kept their morphology for five days, provided the media air interface was well maintained While attempting this procedure.
It's important to conduct the whole experiment from the moment the animal is euthanized to the moment the slices are placed in the membrane inserts under two hours to minimize cell death following this procedure. Other like addition of exogenous factors to the organotypic lysis can be performed to answer questions like, what are the role of different growth factors or pharmacological agents in the various neurodevelopmental mechanisms?
