Neural Plate Cell Manipulation Through In Utero Nanoinjection in an Embryonic Mouse Model

Secure an anesthetized pregnant mouse with its exposed uterus containing pre-neurulation embryos. At this stage, the neural plate is open and directly exposed to amniotic fluid.

Position the selected embryos on a petri dish with an elastic membrane to stabilize them. Secure the embryos and apply saline for hydration.

Align the ultrasound probe over the embryo and measure the amniotic cavity diameter to calculate the optimal injection volume.

Position a nano-injection needle containing a high-titer lentiviral solution above the target embryo at a near-perpendicular angle.

Inject a precise small volume into the amniotic cavity, gently retract the needle and remove the ultrasound probe.

Gently push the embryos back inside the mouse.

The injected lentivirus fuses with neural plate cells, releasing its genetic material, which undergoes reverse transcription into DNA and is integrated into the host genome.

This enables stable gene manipulation in the neural plate cells, supporting potential therapeutic applications.

Using forceps, hold the tissue between the embryos and carefully pull out both uterine horns from the abdomen. Count and number the embryos, either from the ovary to the cervix or from the cervix to the ovary, on both sides.

Next, with a moist cotton swab, gently push all embryos back into the abdominal cavity, except the first three to be injected. Then, place a drop of PBS onto an elastic membrane glued to the round central opening of a commercially-available modified Petri dish and hold it immediately above the embryos. Insert closed forceps into the incision of the elastic, then release the forceps to open the elastic incision, dropping the liquid onto the embryos.

Using forceps, pull the section of the uterus corresponding to the three embryos through the elastic and gently perch the Petri dish on the mouse's abdomen. Using four clay feet, secure and fasten the Petri dish above the abdomen to reduce pressure on the mouse, and also reduce the sensitivity of imaging to the mouse's breathing and heartbeat. Next, using forceps and a moist cotton swab, adjust the uterus and the elastic to ensure that the elastic is sealed around the mouse's skin, preventing PBS leakage.

Next, to fixate the uterus, press the modeling clay cylinder down to the right of the embryos or uterus and add PBS to the Petri dish until the embryos and uterus are entirely covered with the PBS. To facilitate the recording of the injections, dip the ultrasound probe into the PBS and adjust the mouse or surgical table so that the first embryo is aligned with the ultrasound probe. Scan through all three embryos and inspect the amniotic cavities.

Then, using the ultrasound machine, measure the diameter of the amniotic cavity to determine the appropriate injection volume. Using the injection controller, set the determined injection volumes and the injection speed to slow, with an injection rate of 23 nanoliters per second. After lowering the needle into the PBS, using the main wheels on the rail system, press Empty on the nanoinjector controller until the liquid reaches the needle tip.

Then, lift the needle out of the PBS and press Inject to verify that a drop of approximate desired volume is discharged. Lower the needle into the PBS again and move the nanoinjector with the Y-plane wheel on the micromanipulator to align the needle with the ultrasound probe and embryo. Adjust the needle angle with the inject angle wheel to ensure a near-perpendicular injection angle relative to the uterine wall.

Then, using the inject wheel on the micromanipulator, insert the needle into the amniotic cavity in one motion. If the needle tip disappears from the ultrasound image, move the probe forward or backward to bring the needle back in focus. Then, inject the desired volume by pressing Inject for the appropriate number of times.

After the required volume has been injected, wait for 5 to 10 seconds before retracting the needle in one gentle movement.

Next, lift the ultrasound probe and needle out of the PBS using the micromanipulator, turning the needle away from the operator to avoid damage and injury.

Using forceps, place the first and second embryos back into the abdomen by gently pushing them through the elastic.

After all embryos with optimal amniotic cavities have been injected, gently push the embryos and uterus back into the mouse's abdomen.