Laser-Assisted Lentiviral Gene Delivery: A Technique to Permeabilize Mouse Fertilized Eggs to Facilitate Lentiviral Gene Delivery

While the eggs are in the incubator, set up and calibrate the XYClone laser according to the manufacturer's recommendations. When the eggs are ready, place the first drop plate onto the laser microscope stage, and manually focus the zona pellucida. After confirming that the laser LED light is visible through the objective, move the microscope stage to target the zona with the LED laser, and use the computer software to set the XYClone laser to 250 microseconds.

Adjust the LED light size to the appropriate dimensions, and perforate the zona pellucida of each egg three times with the laser to produce three 10-micrometer holes.

It is critical to perform the perforation quickly but carefully, as the fertilized eggs cannot keep outside of the incubator for longer than 15 minutes.

Then, return the perforated eggs to the incubator for another two hours. At the end of the second incubation, treat the 50-microliter KSOM drop with 2 microliters of concentrated lentivirus without mixing, and return the eggs to the incubator for four days. When the eggs have developed into blastocysts, use a non-surgical embryo transfer device to deposit approximately 10 to 15 embryos in and about 2 microliters of KSOM.

17 days after the embryo transfer, allow the pregnant mouse to give birth naturally or collect the neonates by cesarean section as necessary.