Characterization of Thymic Settling Progenitors in the Mouse Embryo Using In Vivo and In Vitro Assays

The overall goal of this procedure is to analyze the phenotype kinetics of generation and numbers of T cells produced by E 13 or E 18 thymic settling progenitors. This is accomplished by first isolating the thymic lobes from E 13, E 14 and E 18 mouse embryos. In the second step, the E 14 thymic lobes are irradiated and then colonized with flow sorted E 13 or E 18 cells for 48 hours in a hanging drop.

In the final step, the colonized lobes are grafted under the kidney capsule of adult CD three knockout mice. Ultimately, flow cytometry can be used to assess the presence of the E 13 and E 18 thymic settling progenitor progeny in the blood of the chimeric animals. The main advantage of this technique of our existing methods, like in vitro T cell culture on ine so cells, is that this technique combines the methods that allows full T-cell maturation from defined progenitors in a three dimensional assay with the grafting of thymic organ organ cultures in intact recipient mice, allowing the evaluation of the differentiation and function of the thymic slic progenitors and the progeny.

As a research engineer with rodent surgery expertise, I will be demonstrating the procedure Two days before the grafting experiment in a laminar flow hood, what the abdomen of a pregnant female mouse with 70%ethanol and make a longitudinal incision in the skin at the midline, pull the skin apart to open the incision and then use forceps and scissors to open the peritoneum without touching the digestive tract. Next, pull out the bifid uterus and use scissors to separate it from the vagina. Then place the uterus in a 90 by 15 millimeter Petri dish containing 40 to 50 milliliters of DPBS and cut transversally between the decidua of each embryo.

Use a pair of fine tip scissors and fine forceps to remove the muscle membrane of the uterus cutting between the placenta and the yolk sac to take out each embryo. Then remove the amnio retaining the embryos, and place the embryos in a new Petri dish containing HBSS plus 1%FCS. To isolate the thymus, place the embryos one at a time in the supine position under a binocular magnifying lens.

Then insert one forceps longitudinally along the cartilage of the sternum and pinch and open the thoracic grid using curved forceps. Pull the thoracic wall aside to visualize the thymic lobes on each side of the trachea. Then pinch underneath the thymus with a fine forceps to carefully remove each lobe and place them in a Petri dish containing one milliliter of HBSS plus 1%FCS.

Next, use two fine forceps to remove the surrounding connective tissue without damaging the lobes. Then wash the lobes in three milliliters of HBSS plus 1%FCS to eliminate the red blood cells to begin the fetal thymic organ culture irradiate. E 14 thymic lobes isolated from CD 45.2 embryos as just demonstrated.

Let the lobes rest for about three hours. Then spin down freshly sorted E 13 or E 18 thymic settling progenitors. Re suspend the pellet in culture medium to a final concentration of 500 cells per 35 microliters of medium.

Then place a 35 microliter drop of cell suspension in every other well of a 60 well terasaki plate and plate one irradiated lobe on the surface of each drop. Close the plate and turn it upside down to let the cells reach the apical pole of the drop by gravity, hitting the upper side of the inverted plate gently to force the lobes to slide to the apical edge of the drop. Then incubate the inverted plate in a humidified incubator for 48 hours at 37 degrees Celsius and 5%carbon dioxide.

After the thymic lobes have been colonized, verify the level of anesthesia of the recipient animal by a lack of reflex response. Then place a drop of eye ointment on each eye and place the mouse on its right side under a laminar flow hood. Then shave the surgical area just above the joint of the hind leg.

Next, done a pair of sterile gloves and sterilize the surgical field with 70%ethanol and a 10%IOD DY DER solution. Use a scalpel to make a 1.5 centimeter incision through the cutaneous tissue, followed by an incision through the muscle tissue. Now apply a slight pressure to both sides of the incision to force the kidney out of the abdominal cavity and keep the kidney moist with saline solution.

Maintain the kidney with a cotton bud outside of the retroperitoneal cavity, and use two fine forceps to make a two to three millimeter hole in the capsule. Then holding the wall of the capsule open carefully slide up to six thymic lobe grafts under the towards the edge of the kidney pole and place the kidney back in the retroperitoneal cavity. Suture the muscle a neurosis followed by skin suture with individual surgeon knots.

Then wet the wound with 10%iodide povidone solution, and place the mouse on a 37 degree Celsius heating pad, monitoring the animal and surgical wound until they are both fully recovered to determine the best method for depleting endogenous thymocytes for the development of colonizing progenitors. In this experiment, the levels of T-cell reconstitution in a colonized thymic lobe after a radiation or a five day deoxy guine treatment were compared. While no difference in the number of colonizing lymphocytes was evident at day nine of culture, the irradiated lobes contained more T cells than those treated with deoxy guine on day 12, making irradiation the preferred treatment for facilitating T-cell development after thymic colonization to study the developmental potential of E 13 and E 18 thymic settling progenitors here, E 14 irradiated thymic lobes were colonized with an equal number of the two types of progenitors as observed, the E 13 thymic settling progenitors gave rise to fewer thymocytes and double positive T cells than the E 18 thymic settling progenitors.

The frequency of mature CD three positive cells, however, was increased and the production of dendritic epithelial T cells was detected only in the progeny of the E 13 thymic settling progenitors to analyze the in vivo potential of E 13 and E 18 thymic settling progenitors thymic lobes colonized with each type of progenitors were grafted under the kidney capsule of CD three knockout recipients. As just demonstrated as expected. The E 13 thymic settling progenitors gave rise to T cells more quickly than the E 18 progenitors, although the number of circulating T cells was significantly lower in the E 13 thymic settling progenitor colonized animals overall After its development, this technique paved the way for researchers in the field of developmental immunology to explore the properties of T-cell subset, some of which are strict embryonic origin in a mammalian model.

After watching this video, you should have a good understanding on how to trace the differential and functional pathways of Timex settling progenitors and their progeny in a natural environment even, and a competitive conditions.