The overall goal of this procedure is to obtain efficient gene transfection in the embryonic mouse central nervous system at a desired developmental time point and area of the brain. This is accomplished first by preparing precision capillaries and needle type electrodes to prevent uterine damage. The second step of the procedure is to learn a unique holding method of embryos using a fiber optic light cable to visualize small embryos for targeted DNA injection.
The third step of the procedure is to perform electroporation with stick type electrodes on the uterine surface in order to transfect DNA into superficial parts of the brain, the final step of the procedure is to perform electroporation with needle type electrodes into deeper structures of the brain, such as the thalamus and hypothalamus. Ultimately, results can be obtained that show cortical layer, specific transfection or efficient transfection in the thalamus. The main advantage of this technique over existing messes like regular, in neutral lation, is in having greater visibility and control of lation site of embryos in the uterus.
We use a fiber optic like cable, which makes it possible to visualize embryos from E 9.5 and needle hypos to transmit plasmid DNA into deeper part of the brain, such as thalamus and hypothalamus. This method can help answer key questions about the developing brain, such as neuronal differentiation, brain patterning, neural connectivity. Generally, Individuals new to this method will struggle because it's difficult to shape needle, electro road and to collective position.
The uter and embryos Visual demonstration of this method is critical to see how to make precise needle electrodes and how to position embryos to electro free. Since these two steps are key for successful results. To prepare capillaries use a micropipet puller according to manufacturer's instructions.
In this procedure, we use one millimeter glass capillary tubes without an internal fiber. Pulled needles should measure 800 micrometers to one millimeter from the tip with an external diameter less than 60 micrometers using a dissection microscope and forceps pinch off the needle tips to a diameter of 20 to 30 micrometers. Measure each needle using a micrometer.
Next, prepare micro electrodes by coiling. One end of a tungsten wire around a gold plated pin. Use tungsten wire for negative electrodes and platinum wire for positive electrodes.
Fix the wire to the pin with param. Then insert the wire into the plastic stem of a cotton swab and wrap both ends with phim evenly sharpen the tip of the wire with fine sandpaper until it reaches 20 to 30 micrometers at the tip and 60 micrometers between 800 micrometers and one millimeter from the tip. Again, check the final dimensions with a micrometer.
Next, submerge the sharpened wire into a drop of nail polish to apply a thin coat onto the entire wire for insulation. After the nail polish is dried, use an acetone so to cotton swab to remove it from the last 200 micrometers of the tip stick. Platinum electrodes are purchased pre-made and need no preparation.
In this procedure, we use the CUY six 10 P four dash one nepo gene electrode Purify plasma DNA with a maxi prep or equivalent method, eloquent 100 micrograms of DNA into a new tube, and add 10 microliters of 1%fast green dye. Use TE to bring the volume up to 100 microliters for a final one microgram per microliter plasma DNA concentration. Mix the solution gently by pipetting heading, then spin the DNA solution for five minutes at 14, 000 RPM room temperature to remove impurities and salts.
Transfer the SNAT to a new tube. The DNA solution can be kept at room temperature in the tube during the entire procedure. Next, connect a pulled needle to a micro manipulator according to the manufacturer's instructions, submerge the tip of the needle into the DNA solution to fill the needle.
Carefully set the micro manipulator aside and prepare for surgery prior to surgery. Weigh a pregnant mouse at embryonic days. 9.5 to 15.5 to determine body weight.
Then intraperitoneal. Inject pharmaceutical grade sodium pentobarbital at 50 micrograms per gram body weight and weight for five minutes. Alternatives to sodium pentobarbital could include ketamine, xylazine, ejection, or gas anesthetics.
After anesthetization, use a razor blade and 50%ethanol to shave the hair from the abdomen. Prepare the skin with alternating scrubs of Betadine and 70%alcohol. Make an incision at the abdominal midline with fine scissors.
Pull out all uterine horns carefully onto A one x phosphate buffered saline or PBS moist and cotton gauze placed around the wound. Keep the uterus moist with PBS all of the time. Using a flexible optic fiber cable.
Place the cable under the uterine horn. Position the uterus between the optic fiber light and the thumb and squeeze gently to push up the embryo closer to the uterine wall. When the embryo is positioned with the head on the left and facing up, insert the glass capillary carefully into the target ventricle and inject approximately one microliter of DNA solution for electroporation of the cortical layer four At embryonic day 13.5.
Place the stick platinum electrodes outside of the uterus and apply square wave current pulses five times using 38 volts for 50 milliseconds. If using micro electroporation to reach the thalamus or hypothalamus at embryonic day 10.5, insert the fine tungsten negative electrode into the DNA injected ventricle and the platinum positive electrode into the uterus. Place the target region between the ends of both electrodes and apply suggested square wave current pulses three times at seven volts for 100 milliseconds.
After electroporation is complete, return the uterine horn to its original location and add 500 microliters of PBS. Suture the incision inner layer with a surgical suture. Then close the outer layer with a nine millimeter auto clip.
Place the mouse on a heating pad for two hours to allow recovery from surgery and anesthesia stick. Platinum electroporation of the cortical region is seen here applied in embryonic days, 13.5, 14.5, and 15.5. Brains were harvested at postnatal.
Day six. The layer of cortical cells electroporated is clearly different in each experiment. Suggesting that time dependent electroporation makes cortical layers specific gain of function and loss of function possible.
As shown here, electroporation into a deep tissue such as the thalamus and hypothalamus. Using stick platinum electrodes often gives low efficiency using micro electrodes to electro operate. These deep tissues offers better efficiency and viability.
We injected plasma DNA solution into the third ventricle of mouse embryos. At embryonic day 11.5, we applied needle electroporation restricted to the thalamus most of the thalamus exons project into the cortex by postnatal day six. Here we show P-C-A-G-E-Y-F-P micro electroporation into the developing cephalon.
The region of the thalamus is shown by RORC, which labels all thalamic post mitotic neurons. Axon projections shown with white arrows into layer four of the cortex can also be visualized by EYFP, which fills up the entire neuron. The position of layer four in the cortex is also labeled by the RORC antibody.
Once MA starts, this technique can be done in 15 minutes per liter if it is performed properly. While attempting this procedure, it's important to remember to handle the you test very carefully to prevent any damage Following this procedure. Other methods like incorporating a specific chromo into MDNA can be performed in order to understand the molecular mechanism of cell specification after its development.
This technique, tap wave for researchers in the field of central nervous system development to explore the molecular mechanisms in patterning, axonal growth and circuit formation.
