The aim of this procedure is to genetically label and manipulate the molecular constituents of sub ventricular zone neural stem cells. This is accomplished by first inserting a glass pipette loaded with DNA into the lateral ventricle of a P zero P.The second step is to expel DNA into the cerebral spinal fluid of the lateral ventricle. Next current is applied to the P.This allows for transfer of DNA to neural stem cells lining the lateral ventricles.
Ultimately, a combination of molecular techniques, including immunohistochemistry, molecular biology, microscopy, and electrophysiology, are used to visualize changes in fate, proliferation, migration, and maturation of subventricular zone neuro stem cells. Neonatal subventricular zone electroporation can help answer key questions in the field of neurogenesis, such as which molecular pathways are responsible for regulating the proliferation, differentiation, migration, and maturation of neuronal stem cells and their progeny. The main advantage of this technique over existing methods, such as transgenic mice, is that it allows for the rapid and robust labeling of neuron stem cells of the SVZ in a time efficient and cost-effective manner.
First, ensure that the DNA to electro purate is of high purity and concentration and is endotoxin free. Pull 10 centimeter fire polished BO silica glass capillary tubes into pipettes. A PP eight 30 ghhi PC 10 pipette puller set to pull a one step weighted pull at 70.5 degrees Celsius is used here After pulling the pipette, use circular forceps to break the tip.
Then inspect the pipette under a dissecting microscope to ensure that there are no jagged edges. If required, the tip can be beveled using a grindstone. Next place the poured pipettes under a UV lamp for 15 minutes.
Once the 15 minutes have elapsed, mark the pipette two millimeters from the edge of the tip with a fine tip marker. Finally, prepare a 0.1 weight per volume fast green solution by mixing sterile filtered 0.9%sterile saline with dry, fast green anesthetize. A zero to one day old pup, a glass Petri dish pre chilled to four degrees Celsius and placed on west ice After approximately five minutes, determine the state of anesthetization by utilizing the foot pinch response.
If no movement occurs, begin the intraventricular injection. Combine the fast green solution with the DNA solution and aliquot one to two microliters onto biofilm. It is important that this is done just prior to injection as the combination of fast green solution with DNA can result in rapid evaporation and crystallization with the subsequent blocking of glass pipettes, aspirate the entire aliquot volume with a microfilm syringe and load the glass pipette onto an air pressured injector.
Now, gently grasp and hold the head of the pup between the thumb and forefinger of the last dominant hand. Then turn on the lamp and hold the head of the pup just outside of the light beam. If the ventricles are not clearly visible, dampen the head with sterile saline to help reduce the amount of light reflected by the scalp.
Next, using the dominant hand, insert the needle into the near side lateral ventricle. The site of injection should be approximately equidistant from the OID suture and eye, and two millimeters lateral to the sagittal suture. Insert the pulled pipette to the two millimeter mark for a P zero pop, which should ensure penetration into the lateral ventricle.
Back filling of CSF into the pipette may occur due to the release of intracranial pressure. If this happens, loosen the grip on the head of the POP to reduce the pressure and assist in the injection of the plasmid. Once the pipette is optimally inserted, inject approximately 0.5 microliters of plasmid into the lateral ventricle.
Set the voltage of the electro operation generator for five square pulses, 50 milliseconds per pulse, and 100 volts with 50 millisecond intervals. The spatial specificity of plasma electro operation is given by the direction of the charge transfer, so spatial consideration should be given to the placement of the electrodes given how diverse the cells of the SVZ are. People have reported changes in terms of cell fate and proliferation in the ventral dorsal to CAU locations.
After injection of the plasmid dip, the tweezer electrodes into PBS to maximize charge transfer and help prevent burns caused by resistivity. Next, place the positive electrode on the dorsal lateral wall of the skull near the ear and dips the lateral to the cytoplasm injection. Then place the negative electrode on the contralateral hemisphere ventral lateral to the pop snout.
This figure shows the correct orientation of negative and positive electrodes with respect to the x, y, Z axis on the injected pup. Initiate current transfer by pressing the pulse foot switch pedal and sweep the electrodes from dorsal to lateral using around 25 degree angle intervals. Once the electroporation is complete, place all electroporated pups onto a heating pad for five minutes.
Gently rotate the pups every 30 seconds with mild stimulation. This image shows the lateral ventricle of a neonatal mouse 24 hours following electroporation of an EGFP encoding vector. The electro rated cells fluoresce green.
Here we see the lateral ventricle labeled LV 28 days after electroporation of Cree Recombinase and GFP encoding plasmids into mice carrying a stop code on upstream of TD tomato recombination induces expression of TD tomato seen in red, successive dilution of plasmid resulting few remaining EGFP positive SVZ cells or genomically expressed TD tomato is permanently expressed. Dilution of plasmid, DNA shown in green is apparent given that more cells in the granule cell layer of the olfactory bulb labeled OB, expressed the genomic marker TD tomato shown in red. After watching this video, you should have a good understanding of how to label and manipulate stem cells from the SVZ.
