This article describes a method for injecting a virus containing a desired gene into the spinal cord and brain ventricles of embryos in a pregnant mouse model. The technique includes the use of a tracking dye to visualize the injection sites and ensure accurate delivery of the viral particles.
Take a mixture of virus containing a desired gene for transduction and a tracking dye in a buffer.
Aspirate the virus-dye mixture into a fine-tip capillary.
Take an anesthetized pregnant mouse with a shaved abdominal region.
Disinfect the skin with ethanol, followed by a betadine solution.
Make a midline incision to expose the abdominal cavity, and place a paraffin film over it.
Expose the uterine horns and pour buffer over them to prevent dryness.
Note the order and position of the embryos. Adjust the embryo's position within the uterine sac to access the injection sites.
Inject the virus-dye mixture into the spinal cord and brain ventricles.
Ensure accurate injection by visualizing the dye distribution.
Place the uterine horns back into the abdominal cavity and pour some buffer.
Suture the abdominal cavity, followed by the skin.
The injected virus enters the host cell, releasing the target gene and enabling its expression.
Begin by loading a customized capillary tip containing four times 10 to the 11 viral particles of adeno-associated virus type 1 coating for the desired target, and labeled with Fast Green dye onto an aspirator tube. Add 15 microliters of the labeled adeno-associated virus particles into the capillary.
Next, confirm a lack of response to toe pinch in an anesthetized embryonic day 14.5 pregnant mouse, and disinfect the skin. Use curved serrated iris forceps and straight tungsten carbide scissors to make a skin incision along the linea mediana. Using straight Dumont tweezers to grip the peritoneal wall, continue along the linea alba with straight Vannas scissors and place a piece of fenestrated paraffin film over the abdominal opening and use a spoon-like device to expose the uterine horns without damaging the embryos inside the horns.
After applying a few drops of 37-degree-Celsius PBS onto the uterine horns, inspect the embryos for damage or malformations inside the uterine sac. Turn the embryos carefully inside their sacs until the desired position for each injection is achieved. When the embryos are in place, inject one to two microliters of the Fast Green labeled virus particle solution into each embryo.
After all of the embryos have been injected, place the uterine horns back into the abdominal cavity, and add a few drops of 37-degree-Celsius PBS into the abdomen. Then use polyamide 6-0 sized sutures to close the peritoneal wall and polyamide 3-0 sized sutures and Halsted's Mosquito hemostatic forceps to close the skin.
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