In Utero Electroporation Based Gene Delivery: A Technique to Inject Plasmid DNA into Embryonic Cerebellar Cells of Murine Embryos
DEŞİFRE METNİ
Begin by taking a pregnant anesthetized female mouse model placed in a supine position. Prep the mouse by removing hair from the abdominal region. Incise the skin and underlying muscle layers to expose the uterine horns – the sites that host viable embryos.
Now, pull the uterine horns out of the abdominal cavity. Take a sharp tip glass capillary containing the desired plasmid DNA solution. Locate the brain of the embryo and inject the plasmid DNA solution through the uterus wall into the fourth ventricle. This position is anterior to the cerebellar primordium – the desired location for eventual DNA delivery.
After injection, retract the empty capillary. Next, for electroporation of the DNA injected embryo, position the two electrodes over the uterine wall such that the positive electrode covers the cerebellar primordium and the negative electrode covers the region around the ears.
Apply electric pulses for the desired duration. These pulses make the cerebellar neuronal cells permeable, allowing the plasmid DNA to enter the targeted cells. Finally, place the uterine horns back inside the abdominal cavity at their natural position. Suture the cuts, transfer the mouse to its cage, and allow it to recover.
