This article details a method for transfecting mouse oocytes using a lentiviral vector. The process involves preparing the oocytes, injecting the viral vector, and facilitating genetic transfer into the host genome.
To perform transfection, take a specialized chamber containing freshly fertilized mouse eggs suspended in a drop of appropriate media. At this stage, the egg contains a large central oocyte containing both the paternal and maternal pronuclei.
The oocyte is covered by a glycoprotein-rich zona pellucida, surrounded by closely arranged clusters of cumulus cells embedded within the hyaluronic acid-rich extracellular matrix. Now, treat the eggs with the enzyme hyaluronidase, which digests the hyaluronic acid. This dissociates the cumulus cells, eventually exposing the zona pellucida surrounding the oocyte.
Position a single treated oocyte near the tip of a holding pipette and apply suction pressure to restrain its position. Take a narrow-bore microinjection needle containing an engineered lentiviral vector suspension carrying a transgene encapsulated within the viral envelope. These retroviruses have the inherent capacity to integrate into the host genome.
Next, pierce the needle through the zona pellucida to reach the perivitelline space - a space between the outmost zona pellucida and the innermost plasma membrane. Introduce the lentiviral vector suspension into the perivitelline space and incubate to facilitate transduction - a process of virus-mediated genetic transfer.
The viral vector fuses with the oocyte membrane, allowing the release of the transgenic RNA into the cytoplasm of the oocytes, which reverse transcribes into double-stranded DNA. Eventually, the DNA integrates at specific loci in the host genome, resulting in the transgene expression.
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.
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