An oval electroporation begins with opening the act to expose the cheek embryo. At hamburger Hamilton, stages nine to 11 DNA is injected into the midbrain region. Then electrodes are placed left and right of the midbrain and an electrical voltage is applied.
This causes a mural progenitor cells at the ventricular zone to take up the DNA For ventral Transfection. One electrode. The anode is positioned under the membrane covering the A below the midbrain area.
The cathode is placed lateral dorsally at the opposite side of the midbrain before an electrical voltage is applied. Please take care that the electrodes do not contact the neural tube. 24 hours later at Hamburger Hamilton stage 16 or 17, the embryos are removed from the egg fixed and analyzed by antibodies, staining, or in situ hybridization.
GFP positive cells indicate the area of gene transfect. Electroporation of microRNA precursors molecules, or a microRNA duplex provides an immediate but short effect to assure a longer expression of microRNAs. We electro operate an expression vector that contains sequences coding from micro RNA together with a vector expressing GFP.
To visualize transected cells, MicroRNA expressed from a vector will be present earliest four hours after electroporation. Please see our written protocol for detailed instructions regarding sterilization of tools and preparing solutions and constructs we used. Hello everybody.
I am an MD student in the lab of Andrea Mann in the Department of Experimental Embryology at the Institute of Anatomy at the University of Tubingen. In the following movie, I want to show you how to transfect in oval specific areas of the mid brain with DNA or RNA. This method can be applied to different brain areas.
We use the method to overexpress micro RNA in vent Midbrain Fine tipped Micropipet should be prepared before beginning using a micropipet puller using a long gel loader tip, fill the micropipet with approximately six microliters, DNA solution instead of a backfill. The DNA can also be absorbed with an injector after breaking the tip of the micropipet to receive chick embryos at the required embryonic stage. The XR incubated at 37 degrees Celsius and 65%humidity after hamburger and Hamilton instructions XR laid on their side.
The top where the embryo settles is marked with a pen line. After the required time of incubation, the XR removed from the incubator such that the pen line is still pointing upwards. The XR disinfected with 70%ethanol at the broad side of the SSON air chamber until which a hole is applied.
We use a commercially available egg pincher, but a big needle will do the chop too. A 10 milliliter syringe is inserted into the hole in a rather vertical angle to avoid a damage of the Eggo and one to two milliliters of egg white are removed. This helps to detach the embryo from the eggshell to avoid any eggshell dripping on the embryo.
Attach a small stripe of saddle tape to the top of the egg, then cut a small window into the eggshell to reveal the embryo. To visualize the embryo. A small amount of inus injected underneath.
Then shake the egg a bit to Distribute the ink. Another, the stereo microscope. The embryo is easy to recognize.
Adding thyroids, detach the embryo a bit from the vilin membrane with a sharp tungsten wire. Pierce the vilin Membrane and wrap a gap.Gap. Pierce the wall of the neuro Epithelium posterior or anterior of the midbrain with a micropipet and inject the DNA into the lumen of the neural tube.
At midbrain level To electro operate, we use an intra cell dual pulse electro radar with a foot pedal to electro operate Broad areas of the midbrain. We use flat wire electrodes with a diameter of O 0.5 millimeters fastened into a self-made holder. The electrodes show a distance of around five millimeters from each other to electrolyte vent brain.
We use plate wire with a diameter of oh 0.2 millimeters as anode and oh point 25 millimeters for the cathode place. The electrodes parallel to the embryo midbrain level to achieve a broad inspection of midbrain cells. Then press the foot pedal three to five times to the liver, current pulses to the embryo.
The bubbles that format the negatively judged electrode shows successful electroporation To Achieve a transfection of ventral mid brain. The anode is pushed below the membrane of the area, lucida below the ventral mid brain to avoid scorching the neural tube. The cathode is positioned also laterally at the opposite side of the midbrain and the current pulses are applied.
The XR sealed with a tape to protect them from dehydration and infections and are incubated for another 20 to 24 hours. After the required incubation, the X are removed from the incubator and the tape is opened with a pair of scissors with a fine pair of scissors, cut the area around the embryo generously, and moved the embryo to a dish. With PBS, The Remaining membranes, amni, and extra embryonic tissue are removed.
A cut in the four brain ensures a good access of solutions to the ventricular area of the neural tube. Then transfer the embryo to 4%PFA and fix overnight at four decreased celsius. I have just shown You how to transfect micro RNA into ventric midbrain using electroporation.
We investigate the influence of micro RNA on differentiation and proliferation of specific midbrain areas. However, this method can also be very helpful to study mis expression of genes or gene knockdowns by S-I-R-N-A in other specific regions of the nervous system of the chick embryo. I hope this was helpful And good luck with your experiments.
