NEPTUNE allows targeting and genetic manipulation of the mouse embryonic nervous system from embryonic day 7.5 and enables the generation of knockdown or lineage tracing models in days. This protocol is adaptable, flexible, and fast. We can deliver shRNA, cdRNA, or barcoded viral libraries to address different questions in days rather than months or years.

We expect that NEPTUNE will provide insights in neuroscience but it can also be adapted to other organ systems. Identifying the amniotic cavity can be quite challenging, so take your time to look at the ultrasound image and maybe have a textbook or other anatomy resource at hand. To begin loading the needle, slide the glass needle onto the metal plunger, keeping the collett attached but slightly loosened.

Then, push the capillary needle along with the metal plunger through the front gasket until it reaches the spacer. Press Empty to push the plunger into the needle and expel the oil from the needle tip. Then press Fill to create an air bubble, dividing the oil and the solution.

Finally, lower the needle, immerse the tip into the solution, and press Fill again to load the needle. After fixating an anesthetized female mouse with ideal-size amniotic cavities on the heating table, apply eye gel to both eyes to prevent corneal desiccation and inject a painkiller subcutaneously. Then, sterilize the lower abdomen by wiping with a cloth soaked with 0.5 milligrams per milliliter chlorhexidine solution and dry the skin.

Next, using surgical scissors, make a 1.5-to 2-centimeter vertical midline skin incision in the lower abdomen. Then lift the skin gently and free it from the underlying muscle layer approximately one centimeter around the incision point to facilitate suturing after the surgery. Next, make a one-centimeter vertical midline incision into the muscle layer.

Using one pair of forceps, lift one side of the skin and muscle layer, and with another pair, look for the uterine horns. Then, 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. Move the stage to the next embryo and repeat the injection for the other two embryos as demonstrated. 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. Then, gently grasp the tissue adjacent to the third embryo and pull the uterus towards the upper end of the elastic incision. Gently pull up the fourth to the sixth embryos with forceps and allow the third embryo to reenter into the abdomen.

After all embryos with optimal amniotic cavities have been injected, gently push the embryos and uterus back into the mouse's abdomen. Successful transduction of the neural plate results in embryos with strong expression of a fluorescent reporter in the brain and other ectoderm-derived tissues, like skin. Injecting excessively-high volumes may result in neural tube defects, such as exencephaly.

Successful injections at embryonic day 7.5 result in uniform transduction from the forebrain to the hindbrain, including the choroid plexus. Injections of low titre result in the transduction of single-cell clones, while high titre virus transduces nearly 100%of the entire brain. Coronal sections through embryonic day 13.5 embryos showed high and uniform transduction of neural tissue of the eye, lens, cornea, and mesenchyme.

Following injection at embryonic day 9.5, salivary glands and ducts develop around embryonic day 11.5 from the oral epithelium. The lingual epithelium of the tongue is well transduced, whereas the underlying mesenchyme is negative. In addition, the positive clusters within the lingual epithelium are separated by negative sections, suggesting transduction of the papillar surface.

Injections at embryonic day 7.5 result in widespread transduction of the tongue mesenchyme at embryonic day 13.5, suggesting that early injections target neural crest cells, contributing to the mesenchyme in the tongue. Lentiviral injections also lead to widespread targeting of the central and peripheral nervous system, transducing both neurons and progenitors in the spinal cord, as well as the dorsal root ganglia. Good preparation is essential.

Ensure that there is no leakage and that the embryos are well fixed, so that you can focus entirely on the injections into the rat cavity. After NEPTUNE, analysis can be done by various methods, such as immunofluorescent analysis of the brain, single-cell RNA sequencing, or allowing the injected embryos to be born for behavioral analysis.

Summary