Lentiviral Vector-Based Perivitelline Injection: A Method to Transduce the Gene of Interest Into Fertilized Single-Cell Mouse Oocytes

In preparation, centrifuge the prepared lentiviral vector suspension at 160 g for 2 minutes to pellet the debris often present in frozen lentiviral stock. In a Class II safety cabinet, transfer the supernatant to a new 0.5-milliliter tube. Next, load 1 microliter of supernatant into an injection pipette, then, attach the injection pipette to the right side micromanipulator, and attach the holding pipette to the left side micromanipulator.

Now, dispense 8 microliters of M2 medium in the center of the depression slide, and cover with light paraffin oil to avoid evaporation. Then, place 20 eggs into the shallow locations in the drop without creating bubbles. Next, make sure that the tip of the injection pipette is open. If not, tap the injection pipette with the holding pipette to open it, then, set the injector for 20-second injections.

Now, using the holding pipette and manual pressure, aspirate a fertilized egg that contains two pronuclei and two polar bodies. Then, using the injection pipette, inject the perivitelline space of the egg.

Do not touch the plasma membrane. Adjust the pressure so that the perivitelline space is filled with solution. The pressure should not exceed 600 hectopascals. Immediately after injecting the 20 eggs, transfer the batch into a bath of preheated M16 medium, and incubate them for at least 30 minutes before implanting them.