Oral Delivery of Transformed Yeast Cells into the Mouse Intestinal Immune System

Begin with an oral tube containing a suspension of genetically transformed, auxotrophic yeast that are unable to produce uracil nucleobase.

Insert the oral tube into the mouth of a restrained mouse and guide it down to the stomach.

Administer the yeast suspension directly into the stomach, allowing it to eventually reach the small intestine.

The small intestine contains epithelial cells, with underlying Peyer's patches rich in immune cells.

Microfold cells, specialized intestinal epithelial cells near the Peyer's patches, engulf the yeast cells and internalize them into vesicles.

These vesicles move across the cells and release yeast cells into the underlying Peyer's patch.

Isolate the Peyer's patch from the sacrificed mouse intestine and obtain a uniform cell suspension.

Culture this cell suspension on a yeast-selective medium containing uracil.

Auxotrophic yeast utilizes the uracil and multiplies to form colonies, confirming the delivery of transformed yeast cells into the mouse intestinal immune system.

After preparing yeast cultures according to the text protocol, use a hemocytometer to determine the cell concentration, and adjust to 10 to the ninth cells per milliliter. Pellet the cells by centrifugation at 2,500 times g for three minutes. Then, pour off the supernatant, and resuspend the cells by adding the appropriate volume of sterile water, and gently pipetting up and down.

Fix an appropriate gauge gavage needle onto a 1-milliliter sterile syringe, and load the yeast sample, eliminating any air bubbles. Load an additional syringe with sterile water to gavage control mice. Using the non-dominant hand, pick up the mouse to be gavaged, and with the index finger and thumb, tightly grasp the skin around the neck. Tuck the tail under the small finger. Be sure the grip is secure and prohibits the mouse from moving its head.

Next, gently insert the gavage needle into the mouse esophagus by angling the needle along the roof of the mouth and back of the throat, keeping slightly to the left of center. Wait for the mouse to swallow the bulb of the needle. If resistance is felt or the mouse gasps, gently remove the needle and try again to find the esophagus.

After the mouse has swallowed the bulb of the gavaged needle, gently depress the syringe plunger to administer the yeast directly into the mouse stomach. Gently remove the gavage needle from the mouse stomach and esophagus and return the mouse to the cage. Check that the mouse is breathing and moving normally to ensure the gavage was performed properly.

After sacrificing the mouse according to the text protocol, position it with the abdomen fully exposed and use 70% ethanol to spray the abdominal area. With scissors, make a transverse incision through the fur and skin. Then, manually pry the incision open to further expose the peritoneum, the thin serosa lining that covers the abdominal organs. Gently lift the peritoneum and make a transverse incision to expose the intestines.

Then, use blunt forceps to carefully tease the small intestine away from the mesenteric arteries, fat, and other tissues. Expose the small intestine from the stomach in the upper left quadrant of the mouse abdomen to the cecum, the large pocket of intestinal tissue at the start of the large intestine.

Next, isolate Peyer's patches by looking for 1 to 2-millimeter roughly circular patches of opaque tissue along the small intestine. Then, using curved dissection scissors, cut away the dome of the Peyer's patch, leaving margins to ensure that none of the surrounding tissue is collected.

Transfer the dissected Peyer's patches into complete IMDM. Then, pour the solution with suspended Peyer's patches onto a 40-micrometer cell strainer. After washing the Peyer's patches with complete IMDM, use a plunger from a 1-milliliter syringe to gently break up the tissue and collect the cells in a 50-milliliter tube. Pellet the strained cells at 1,800 RPM for seven minutes. Then wait and analyze the cells according to the text protocol.