Lentiviral Vector-Based Transduction of Rat Fertilized Embryos

Take a microinjection dish with a drop of embryo culture medium in the center, covered with mineral oil to prevent evaporation.

Place the dish under an inverted microscope.

Position a microinjection capillary and a holding pipette onto a micromanipulator setup under the microscope.

The microinjection capillary is loaded with a suspension of lentiviral vectors carrying the transgene.

Then, transfer fertilized rat embryos into the medium.

Secure an embryo using the holding pipette by applying suction.

Under higher magnification, insert the microinjection capillary through the embryo’s extracellular glycoprotein coat, the zona pellucida, and inject the lentiviral suspension into the perivitelline space.

Then, transfer the injected embryos into a culture dish and incubate.

The virus enters the embryo and releases its genome containing the transgene into the cytoplasm.

Later, it is reverse transcribed into DNA and integrates into the embryo’s genome, incorporating the transgene.

Transduced embryos are now ready for processing.

For lentiviral vector microinjection under the zona pellucida of the one-cell stage embryos, use a pipette puller to prepare microinjection borosilicate glass capillaries wi