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