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 with a filament. After every time the filament is changed or a new glass capillary is used, run a Ramp test to set the optimal parameters.

Next, use a microloader tip to load approximately two microliters of freshly thawed lentiviral solution per microinjection pipette under a biosafety laminar flow hood. Add a 100 microliter drop of M2 medium to the center of the lid from a 60 millimeter Petri dish. Cover the medium with mineral oil and mount the holding pipette and the virus loaded microinjection capillary onto a micromanipulator.

Place the microinjection dish under an inverted microscope and transfer 15 to 20 one-cell stage embryos into the M2 drop in the microinjection dish. Capture one embryo with the holding pipette and use the 400 times magnification and the glass capillary to inject the lentiviral solution under the zona pellucida into the perivitelline space holding the capillary under the zona pellucida for a moment after the virus has been delivered. When all of the embryos have been injected, use a fine pipette to return the injected cells to the culture dish and return the dish to the cell culture incubator until their implantation.