Hi, I'm RI Ov.And I'm Alexia Ying. We are post-grad students of Cent University. In this video, we'll present a method of in vivo electroporation for a target gene delivery to certain area of a leading brain tissue.
So let's see. Let's see. The method Of in vivo electroporation offers a rapid and efficient way of delivering genes of interest or small interfering RNA to living brain tissue.
It provides a low-cost alternative to creation of transgenic animals and to the viral gene delivery approach. In this video, we will demonstrate a detailed procedure of postnatal gene delivery to the neonatal rad cortex. We'll use the plasmid encoding for the enhanced green fluorescent protein and the chicken beta actin promoter with a CMV enhancer.
We perform all surgical manipulations in the laminar flow cabinet to maintain sterility and to minimize the risk of bacterial or fungal contamination of the animal. For plasmid delivery to the cortex, we use a WPI micro injector with electronic controller and a 10 microliter Hamilton syringe attached to a stereotactic manipulator. The red pup is anesthetized using an anesthesia unit consisting of a box And a mask.
The pup is then fixed using ear bars on the heating table attached to the stereotactic manipulator. For surgical manipulation and monitoring of microinjection, we use a binocular microscope. For electroporation, we use the CYV 21 in vivo electro operator with forcep shaped electrodes.
For the pup recovery. After anesthesia, a heated Chamber is used, We usually prepare 10 microliters of the plasmid injection solution in a 200 microliter thin wall tube. This solution is sufficient for several experiments.
We mix eight microliters of the Plasmid solution with one microliter of the 10 XPBS and one Microliter of 0.1%water solution of fast green. The final concentration of the plasmid in the injection solution should be between one and three micrograms per microliter. Use of lower concentrations of the plasmid DNA will reduce the transfection efficiency, while the higher DNA concentration will be too viscous for injection through the thin tip of the glass capillary.
For the glass pipette preparation, we use boa silicate glass capillary with a filament and the vertical electrode glass pooler on maximum pooling and heating parameters. Then we break the tip of the pipette using a piece of paper to achieve a tip of 10 to 20 micrometer in diameter. We check the tip, diameter and shape under the microscope Objective.
Using A thin polymer capillary, we fill approximately one third of the volume of a 10 microliter Hamilton syringe. With mineral oil, we make sure to avoid air bubbles. Then we fill the glass pipette with mineral oil and insert the pipette into the Hamilton syringe.
Again, avoiding air bubbles. The syringe with glass pipet is then fixed on a micro injector connected to a controller. The microinjection setup fixed on a stereotactic instrument allows us to safely immerse the tip of the glass pipette into the tube with a plasmid injection solution.
When the tip touches the surface of the solution, we dip it further by one to two millimeters. Then we use the micro injector controller to fill the pipee with approximately one microliter of the Injection mix. All of the experiments presented here are performed according to the University of Helsinki regulations for animal experiments.
For each experiment, we fill a gast tight syringe with isof fluorine. We fix the syringe on the anesthesia unit connected to the airflow source, the animal walks and the mask fixed on stereotactic setup. On the anesthesia unit, we adjust the airflow to approximately 250 milliliters minute and the iso fluorine level to 4%We place the red pop into the animal box for two to five minutes.
When the P stops moving, we put it on the heating pad attached to the stereotactic setup. We position the rostral part of the P'S head into the mask. It usually takes five to 10 minutes for the pup to enter the deep anesthesia.
When that is achieved, we decrease isoflurane in flow to approximately 1.5 to 2%We treat the skin on the P'S head With the 70%ethanol using small cassars and thin forceps. We cut the skin from the scruff of the pup to its forehead, bend the skin pieces sideways and pull ear bars slightly into the oral orifices of the skull for head and skin fixation. Using a binocular microscope will locate the rema point on the skull to identify the region of interest using the stereotactic coordinates, the position the injection pipet above this region and marked by dropping 25 to 50 nanoliters of the injection solution onto the skull surface.
We drill the skull gently and carefully at the marked point using a high speed surgical drill under the microscope until liquid appears in the Drilled area. We fix the electroporation forceps, electrodes on the sides of the skull. We apply a conductive gel to achieve better conductivity.
We remove the liquid drop from the drilled area using small tampon and dip the glass needle to the hole. According to the coordinates of the injection site, we infused 25 to a hundred nanoliters of the injection solution at the rate of five to 20 nanoliter per Second. We Then Quickly remove the pipette and proceed with electroporation immediately.
Electroporation is done by applying five pulses with the duration of 50 milliseconds at the pulse amplitude of 99 volts and the frequency of one hertz. We remove the electrodes and the ear bars. We stitched the cut skin using a combination of dumb and sharp forceps and The surgical threads.
We placed the pup into a warm chamber for 15 to 30 minutes to aid its recovery after anesthesia. Then we put the pop back into its mother's Cage. The Expression of the transgene in the successfully transfected cells will appear about 10 hours after the electroporation and it will remain stable for several weeks.
So that's all. Thanks for watching and good luck with your experiments.Bye.
