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