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

Laser-assisted lentiviral gene delivery is a technique to transfer a transgene into a host system.

To produce transgenic mice, begin with isolated mouse fertilized eggs in a suitable culture medium. Place the fertilized egg-containing culture dish on a microscope stage pre-installed with a laser apparatus and attached to a controlling software. Observe at suitable magnification.

Fertilized eggs are surrounded by zona pellucida, a protective glycoprotein layer that barriers lentiviral particles. To permeate this barrier, turn on the laser apparatus and ensure the laser LED is visible over the zona pellucida. Using the controlling software, fire the laser. Adjust the firing duration according to the desired perforation diameter.

Place the eggs in an incubator for recovery. After brief incubation, remove the eggs and add lentivirus particles carrying the gene of interest and a fluorescent selectable marker.

With zona pellucida perforated, the lentivirus easily diffuses toward the egg cell membrane and enters via endocytosis. The lentivirus disassembles, releasing its RNA, which, when sequentially processed into a double-stranded DNA, enters the host nucleus, integrating the foreign genes into the host genome.

Incubate the eggs and allow them to develop into blastocysts. Implant the blastocysts into mated female mice. Once gestation is complete, recover the pups and analyze them for transgene presence.

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.