The overall goal of this procedure is to manipulate the gene expression in embryonic hind brain progenitors through the introduction of plasmid DNA by electroporation at earlier gestational time points. This is accomplished by first isolating embryos at 11.5 days of gestation and orienting them dorsal side up in sterile phosphate buffered saline. The second step is to inject about 50 microliters of the expression plasmid in 0.01%fast green.
In a successful injection, the entire ventricular system will be filled with the DNA fast green mixture. Next, the embryo is subjected to an electrical pulse. The side of the embryo closest to the positively charged electrode will take up the plasmid DNA.
The other side will serve as an internal negative control. The final step is the placement of the embryos into tissue culture for at least 24 hours. Ultimately, the electro rated gene will be expressed after 24 hours of culture and can be assessed by immunohistochemistry.
The advantage of this technique over existing methods is that manipulation of embryonic hind brainin progenitors is possible at earlier gestational time points. This method can help answer key questions in the field, such as how different ural genes can influence the fate of embryonic progenitors. Begin this procedure by sterilizing the dissecting tools and lamina flow hood according to the accompanying manuscript.
To harvest the embryos place a euthanized female mouse that is pregnant with pops at gestational day E 11.5 on the dissecting tray in the hood. Next, spray the abdomen of the mouse with 70%ethanol. Open the peritoneal cavity and pin the walls to the dissecting tray.
Then pull out the uterine horns so that it rests within the peritoneal cavity. Using tweezers, carefully open the wall of the uterine horn. Use a 20 millimeter spoon to gently remove the embryos in the yolk sac from the placenta.
After that, place the embryos in a 100 millimeter tissue culture dish. Prefilled with 10 milliliters of sterile preheated one times PBS. In this step, transfer the first embryo to a new 100 millimeter dish prefilled with 10 milliliters of sterile one times PBS that was preheated to 37 degrees Celsius.
Next, carefully remove and discard the yolk sack. Then position the embryo so that its dorsal side is facing up. Gently hold the embryo with the seven millimeter electrode paddles under a dissection microscope.
Place the electrodes one on each side of the neural tube at the level of the hind brain fourth ventricle. After that, use a one cc syringe to draw the mixture of plasmid DNA and 0.01%Fast screen in general, 500 microliters of the mixture should be sufficient for the electroporation of at least eight to 10 embryos. Next, gently puncture the roof plate overlying the fourth ventricle with a 25 gauge five eighth tuberculin needle attached to the one CC syringe.
Then inject about 50 microliters of the DNA DI mixture into the ventricle. Successful injections are characterized by the DNA DI mixture filling the entire ventricular system. An alternate means of delivering the DNA DI mixture will be through accessing the ventricular system by puncturing the vellum, overlying the midbrain, turn on the pulse generator, then deliver five square pulses using an electrode pulse generator and the seven millimeter electrode paddles the tissue closest to the positively charged electrode.
While then take up the plasmid in a lamina flow hood, fill the ulcer wells of a 12 well cultured dish with two milliliters of DME MF 12 media supplemented with 10%fetal bovine serum, 5%equine serum, 1%glutamine, 1%penicillin streptomycin that was preheated to 37 degrees Celsius. Then pinch the embryo at the midsection with a pair of forceps. Remove the posterior portion of the embryo afterward, place the anterior portion into one of the filled wells of the culture dish.
Repeat the procedure for all the embryos and fill only the outer wells of the 12 well plate. To avoid contamination of cultures culture, the embryos in a 37 degree Celsius incubator with 5%carbon dioxide for 24 hours to express the electroporated plasmid. If longer culture time is desired, fill the ser wells of a new 12 whale dish with two milliliters of the D-M-E-M-F 12 media.
Transfer the embryos with a sterilized spoon to the wells of a new plate. Then place them back in the 37 degrees Celsius incubator and culture them for up to 48 hours to fix the embryos for analysis. Fill a 12 well plate with one times PBS, then transfer the embryos and rinse at four degrees Celsius for five minutes.
Next, fix the embryos in 2%paraldehyde in one times PBS for two hours at four degrees Celsius. Then rinse them three times in one times PBS at four degrees Celsius for five minutes. Again, after filling a new 12, well plate with 30%sucrose in one times PBS.
Transfer the embryos to those wells and equilibrate overnight at four degrees Celsius. Then embed the embryos in the optimal cutting temperature compound using a dry ice ethanol bath or store them at minus 20 degrees Celsius. At the end section the embryos to 30 micron sections with a cryostat.
Mount them on slides and store them at minus 20 degrees Celsius from uni Histochemical. Study later shown here are the representative results of the immunohistochemistry for GFP on the transverse sections of an electroporated E 11.5 embryo after 24 hours of culture. The arrows shown here indicate the roof plate which traps the secondary antibody.
The results show that the electroporated area of the lower ROIC lip was restricted and did not extend to the entire anterior posterior axis of the neural tube. Here shows the results from two of the proteins analyzed MASH one and math one. We observed that the majority of the embryos retain normal levels of expression following electroporation and culture as compared to the control embryos at a 11.5 Once mastered, this technique can be completed in one to two hours if performed properly.
After watching this video, you should now have a good understanding of how to use the in vitro electroporation assay to manipulate gene expression in the progenitors of the mouse embryonic membrane.
