The overall goal of the following experiment is to examine developmental effects of knockdown or overexpression of genes in specific subsets of neuronal progenitor cells in the neocortex. This is achieved by in vivo electroporation of DNA into rat embryos to transfect neocortical progenitor cells. As a second step, the electroporated region is dissected in order to enrich for transfected cells.
Next, these cells are dissociated and plated in chamber slides and cultured. In order to examine the effects of both intrinsic genetic alteration and application of exogenous factors, results were obtained that show effects on neurite outgrowth and branching based on quantitative measurements using avision software. The main advantage of this technique over others, such as lipid-based transfection of primary neuronal cultures, is that we can target specific subsets of neuronal progenitor cells.
For example, only glutamatergic neurons will be transfected using this method. In addition, we can electro pray at earlier embryonic stages and target early born deeper layer neurons, or we can electrolyte at later stages of development and target later born neurons that are destined for more superficial layers of the cortex. This method can help answer key questions in the field of neurodevelopment.
For instance, how do intrinsic and extrinsic factors interact to affect neuro outgrowth and branching? Although this method can provide insight into neuronal process outgrowth, it can also be applied to other neurodevelopmental processes such as synaptogenesis, cell proliferation and sulfate determination. Prepare an injection needle as described in the accompanying text.
Avoiding bubbles backfill the needle with DNA expressing GFP and the gene of interest include fast green To visualize the injections, load the remaining space in the needle with corn oil. Attach a pico spritzer to the loaded needle to control the injections. Expose the uterine horns of an anesthetized pregnant spray doorly rat as described previously.
And in the text accompanying this video injection of E 15 embryos is shown here. Identify the injection site by illuminating the embryos with a gooseneck lamp. Gently manipulate one embryo to find where the head is and locate the midline suture.
Use this as a landmark to find the lateral ventricle. Inject DNA through the uterine wall and into the lateral ventricle. Inject multiple pulses into the ventricle until this is filled with DNA.
The vector determined by the placement of the electrodes is critical to determining which region of the neocortex is electroporated. Here the positive electrode is placed near the dorsal medial positions across the cerebrum to target this region of the forebrain. Alternate placement of the electrodes will target other regions of the cortex, including the lateral cortical stream.
Inject and electroporated each embryo. In turn, return the uterine horns to the body cavity and close the incision. Monitor the animals as they recover from anesthesia.
Here animals are harvested for culture 24 hours after electroporation, so that GFP is detectable. Remove embryos from the uterus of the euthanized rat and place them in a Petri dish filled with ice cold HBSS with divalent cation in a lamina flow hood. And under a fluorescent dissecting microscope, use dumont number five forceps.
To dissect out the cortices. Use dumont size number three forceps to remove the meninges turn on the fluorescence using a pair of val scissors. Cut out the GFP positive regions of tissue.
Transfer these pieces to a 50 milliliter conical tube filled with ice cold HBSS without divalent cation. Collect all the GFP positive pieces in the tube. Remove the HBSS from the tube and replace it with one milliliter of 0.25%tripsin EDTA solution mixed gently by inversion incubator 37 degrees Celsius for five minutes.
Remove the tripsin solution. Replace it with between one and five milliliters of plating medium to depending on the amount of GFP positive tissue. Using a two milliliter stripette pipette ate five to seven times to dissociate the cells.
Count the cells using a hemo cytometer dilute with plating medium to a concentration of two to 3.5 times 10 to the five cells per 1.5 milliliter of medium plate 1.5 milliliters in each chamber of a CC two coated chamber.Slide. Incubate the cells at 37 degrees Celsius for four hours to allow the cells to adhere to the slides. Aspirate the plating medium had 1.5 milliliters of neuronal medium per chamber culture.
The cells at 37 degrees Celsius to the desired stage before proceeding with immuno staining. For measurements of neuronal process. Outgrowth timing culture varies between 24 hours and seven days.
In a fume hood, aspirate the medium from each culture chamber. Fix the cells with 4%formaldehyde for 15 minutes. Wash twice quickly with one times PBS before blocking and immuno seen in the cultures as detailed in the accompanying text mount the cultures with fluorescent mounting medium and a cover slip.
Visualize the cultures at 20 x under a fluorescent microscope. Record images of GFP positive neurons for analysis. We have found that with this technique, approximately 75%of electroporated brains are targeted to the desired region of the cortex, whether that be dorsal medial cortex or ventral lateral cortex.
We have found early electroporation at E 13 to 14 target deep layer neurons such as TBR one positive layer six neurons while later electroporation target CT IP two positive TBR one negative layer five cells still later electroporation target BRN two positive layer two slash three cells. Here we show coronal sections of brains electroporated at either embryonic day 15.5 or 17.5 and harvested at postnatal day five. In culture, the percent of cells that A GFP positive can range widely depending on how conservative you are when dissecting out the GFP positive region.
However, even when we are very conservative and dissect out only the GFP positive patch of cells, the highest percentage that we observe is five to 10%This low transfection efficiency is helpful in identifying which processes belong to the electroporated cell that you are analyzing. Plating cells at this higher density contributes to having healthier cultures. However, it is difficult to discern which process belongs to which cell body in the GFP negative cells.
Following this procedure, immuno staining can be performed for several different markers in order to answer questions regarding proliferation, synaptogenesis, and sulfate determination. While attempting this procedure, it's important to remember to use sterile reagents and tools to prevent contamination and infection Once mastered. Each aspect of this technique can be performed in two to three hours if performed properly.
So that's it. Thanks for watching. Good luck with your experiments.
