Glioblastoma Induction Using SB Mediated Transposition: A Technique for Generating Novel Mouse Model Using Transposon Mediated Integration of SB Plasmid into Neonatal Mouse Brain
成績單
3 to 4 weeks prior to the experiment, set up a mouse breeding cage with one male and one female. Include a colored plastic igloo to enrich the environment and increase the chances of mating. Remove the male once a pregnancy has been confirmed, and 18 days after the mating day, start monitoring the female for delivery. Make sure that a surrogate mother is available around the time of delivery.
On the first postnatal day, perform the intraventricular injections. Prepare the injection solution by first aliquoting 20 microliters of the DNA mix containing the transposon plasmids at a final concentration of 0.5 micrograms of DNA per microliter. Next, add a 20-microliter aliquot of PEI solution to the DNA solution and let them incubate at room temperature for 20 minutes to an hour after which the mix should be stored on ice.
For the injection needle, attach a 10-microliter syringe to a 30-gauge hypodermic needle beveled at 12.5 degrees. Then, attach the syringe to a pump with automatic injection. Test the setup with 10 microliters of water and be sure to empty the syringe. Now, cool down the neonatal stereotaxic stage with a slurry of dry ice and alcohol. When it is cooled to between 2 and 8 degrees Celsius, proceed with the injections.
Anesthetize a pup by placing it on ice for 2 minutes. While the pup is on ice, load the syringe with the injection solution. Once anesthetized, transfer the pup to the frame and immobilize its head between the gauze-covered ear bars. Check that the dorsal side of the skull is horizontal and parallel to the frame surface. Also, the cranial sutures should be clearly visible.
If the head of the pup is not immobilized firmly enough, it will move during the injection, whereas if it is squeezed too firmly between the ear bars of the frame, the lambda will not be visible and the pressure within the head will force the injected solution to leak out.
With the pup gripped firmly by the frame, wipe the head off with 70% ethanol. Then, lower the needle and adjust its position so it contacts the lambda. Next, raise the needle and move it 0.8 millimeters laterally and 1.5 millimeters rostrally. Then, lower it again until it slightly dimples the skin. Record the coordinates of this position. Then, lower the needle 1.5 millimeters through the skin and skull and into the cortex at the lateral ventricle.
There, inject 0.75 microliters over 90 seconds and wait a minute to let the solution disperse. While waiting, the next pup to be injected can be anesthetized. After the wait, slowly raise the needle. Then, transfer the pup to a recovery location under a heating lamp. The time from anesthetization to warming must be less than 10 minutes; the quicker the better.
Monitor its breathing, and if necessary, gently rub the limbs to stimulate breathing. Once the pup is warmed up, has a rosy color and steady breath, return it to its mother.
