The overall goal of this protocol is to use loss-and gain-of-function manipulations to identify stage-specific molecules that control inner cell mass differentiation of mouse embryos during pre-implantation. This method can help answer the key question regarding the early cell fate liberation in the basic developmental biology, as well as in stem cell biology, animal science, and reproductive biotechnology, such as animal cloning. The main objective of this technique is that the stage-specific receptor, and there is ligands of using to enhance the expression of only embryonic stem cell-specific factor during the embryo development.

This protocol was developed in collaborate with multiple laboratories. So demonstrating the process will be my collaborate, Professor Sang Jin Lee, and his graduate student, Yong Il Cho. 18 to 20 hours after the last injection, sacrifice the pregnant females by carbon dioxide intoxication followed by cervical dislocation.

Then, open the backside, and cut through the inner layer with fine scissors. Find and pick up the uterine horns using fine forceps, and gently pull them out of the body cavity, along with the uterus, oviduct, ovary, and fat pads. Next, cut the area between the oviduct and the ovary using fine scissors.

Then, reposition the forceps and cut the uterus near the oviduct, leaving at least one centimeter of the upper part of the uterus attached. Now, transfer the oviductal ampulla into the mineral oil-covered M16 drop of media on a 35 millimeter Petri dish at room temperature. Pool the oviducts from several mice in the same dish.

After preparing the one CC syringe needle, as indicated in the text protocol, use fine forceps to hold and enter the end of the oviduct. Then gently puncture and squeeze out cumulus mass at the media drop phase. Under a stereomicroscope, recover the 2-PN embryos along with surrounding cumulus masses, using a sterile glass pipette, and transfer them into a new Petri dish.

Next, dissociate the cumulus cells by bathing the tissues in one milliliter of hyaluronidase solution. Now, incubate the dish at 37 degrees Celsius for five to 10 minutes. The next step is to denude the embryos.

Using the glass pipette, gently flow the bath solution to release the cumulus cells from the surrounding embryos. Next, wash the denuded embryos with 30 to 50 milliliters of fresh PBS per embryo. Perform this wash three times.

Now, transfer the embryos into a 35 millimeter plastic dish with M16 plus BSA, and keep them in a humidified incubator for up to four hours before the microinjection. Before the microinjection, briefly wash the denuded 2-PN embryos with one milliliter of fresh M16. Then, transfer the embryos in solution to a centrifuge tube, and spin them down at 1, 000 Gs for 10 minutes.

Now, transfer each embryo into a culture dish with 20 to 30 microliters of M16 media. This will make the pronuclei visible. Cover the media with a thin layer of mineral oil, and incubate the embryos at 37 degrees Celsius in 5%carbon dioxide for one to two hours.

Meanwhile, with a mechanical puller, prepare the injection pipettes and holding pipettes from borosilica glass capillary tubing. Next, using a microgrinder and a microforger, fabricate the injection pipettes with blunt tips, and polish them into two micron wide openings. Tip length should be less than 500 microns.

Similarly, fabricate holding pipettes by making blunt tips and polishing their openings to an inner diameter of 20 microns, and an outer diameter of 100 microns. Now, load the injection pipettes with at least 200 nanoliters of plasmid solution at a concentration of one microgram DNA per microliter. Next, mount the injection pipette in a motorized micromanipulator.

Then, set the injector to pulse bul-ih-ses of about 10 picoliters. For the microinjection, secure the embryos using negative pressure from the holding pipette. Then, advance and place the tip of the injection pipette adjacent to the cell membrane just above one of the pronuclei.

Then, spear the cell membrane, push further into the pronucleus, and make the 10 picoliter injection. After the microinjection, collect the injected embryos and wash them three times with fresh M16 media. Each wash can be less than a minute in duration.

Then, proceed with culturing the embryos by covering M16 media with a drop of mineral oil to prevent it from evaporating, and placing each embryo on a plastic culture dish on a drop of M16 media. Every 24 hours, observe the embryos using a confocal laser-scanning microscope with 488 nanometer or 555 nanometer DIC optics at 200x magnification. Neogenin is transiently expressed during the early developmental stages of preimplantation.

It appears as early as the 2-cell stage, and lasts until the early morula stage. Its expression is mainly spatially restricted to the outside of cells. Neogenin expression levels were manipulated by microinjecting 2-PN zygotes with neogenin cDNA vectors, or vectors harboring short hairpin RNA against neogenin.

RFP and GFP were coexpressed as indicators. The resulting neogenin expression level was confirmed both by immunofluorescence and by immunoblotting. There were no gross morphological differences in either the neogenin loss or the neogenin gain embryos, nor did they develop differently until the blastocyst stage.

However, ICM development was less pronounced in neogenin loss embryos, as gauged by Oct three and four positive ICM cells. Also, there were more Oct three and four positive ICM cells per blastocyst in the neogenin gain embryos. This observation was further confirmed by rtPCR analysis.

In neogenin gain embryos, expressions of Oct three and four, SOX2, and Nanog were dramatically increased, in contrast to a negligible expression change in Cdx2 and Tead4. Once mastered, this microinjection portion of this protocol can be completed in about one hour. While attempting this protocol, keep in mind that the 2-PN embryo are freezers optimized isolation microinjection are culturing of embryo to minimize the damage.

Complementing this procedural method, such as drug studies, can be performed to answer age-old question about the signalling pathway and so forth.