This video demonstrates procedures to harvest memory. Epithelium, clear the memory fat pad and transplant exogenous epithelium to study memory development in three week old mice, growth of the memory epithelial tree is confined to the vicinity of the nipple, and the fat pad is largely devoid of memory epithelium. As the mouse matures, the epithelial tree grows through the fat pad.
By seven weeks of age, the epithelial ductal tree extends throughout the entire fat pad. If the small portion of the fat pad containing epithelium is removed at three weeks of age, the endogenous epithelium will never populate the memory fat pad and the fat pad is described as cleared and would remain cleared at maturity. Transplanted donor memory, epithelium or epithelial cells, however, have the potential to extend memory ductal trees throughout the cleared host.
Fat pad. Histology can then be used to monitor the progression of memory development in transplanted tissues from transgenic or test animals. Hello, my name's Joseph Jerry, and I'm in the Department of Veterinary and Animal Sciences at the University of Massachusetts.
Hi, my name is Karen Dunphy. I work in the Jerry Lab. Today we're gonna do a mammary epithelial transplant.
We use this procedure to study the molecular and cellular basis for memory tumor genesis and a model for breast cancer. So let's get started. The first step of the transplantation procedure is to harvest memory epithelial tissue from the donor mouse.
Begin by washing a recently euthanized female mouse with 70%ethanol without piercing the SRUs membranes of the ventral body cavity. Make a mid sagittal cut through the skin extending from the pubis to the sternum. Then make an oblique cut from the initial incision at the pubis toward each hind leg.
Using curved forceps, grasp the SRUs membranes. Then using straight serrated forceps. Grasp the cut edge of the skin and carefully peel the body wall away from the S cs membranes.
To expose the fourth inguinal memory gland attached to the underside of the body wall, the lymph node within the memory gland is located at the intersection of three prominent blood vessels in the memory gland and can be felt as a dense nodule within the fat pad. With the tip of a pair of eye scissors, nip the adipose tissue surrounding the lymph node. Then with a forefinger on the epidermal side of the skin, apply pressure to raise the node out of the fat pad and pinch it off.
With a pair of curved forceps, discard the lymph node. Now while holding the margins of the skin. Grasp the memory fat pad with the curved edge of a pair of curved forceps and gradually peel the fat pad away from the body wall Following the direction of the fat pad towards the dorsal surface of the mouse, remove the entire memory gland in one piece.
Put the memory gland on a sterile plastic cutting block at a small amount of media, about 200 microliters to the surface of the gland to keep it moist. Then mince the gland into one millimeter pieces with a fresh razor blade. Divide the minced gland into four portions.
Then using forceps, distribute among four cryo vials of freeze media. Place the cryo vials in an Nalgene cell freezer with 250 milliliters of isopropyl alcohol in the outer compartment. Then place the cell freezer at minus 70 degrees Celsius for 24 hours.
After 24 hours, transfer the frozen vials of memory fragments to liquid nitrogen until required for transplantation. Each cryo vial contains enough memory gland fragments to transplant into the cleared fat pads of four to eight host mice. After verifying the depth of anesthesia of a three week old host mouse by toe pinch firmly restrain the mouse ventral side up limbs outstretched on a surgical stage so that its abdomen is taut.
Preoperative analgesic, bupivacaine or buprenorphine should be administered.Subcutaneously. Disinfect the surgical area with alcohol and an iodine based scrub using forceps. Grasp the skin a few millimeters above the pelvis and lift it up from the abdomen.
Carefully nip the skin, ensuring that the initial incision is not too close to the urethra, where placement of wound clips may interfere with the mouse's ability to urinate. Next, taking care not to pierce the SRUs membranes. Make a midsagittal cut through the skin extending from the pubis to the sternum.
Now from the initial incision, make oblique cuts through the skin from the midline across the pelvis towards each hind leg. Using curved forceps, gently grasp the S ceros membranes of the ventral body wall. Then use tissue forceps to grasp the cut margin of the skin and pull the body wall away from the ceros membranes.
To expose the fourth inguinal memory gland, identify the fourth memory gland. Identify the fifth memory gland, cauterize the nipple node and two arteries leading to the node. Separate the fourth and fifth glands, then cauterize the margin of the fifth gland.
Next, using scissors, cut across the fat pad just ventral of the node to divide the fat pad of the fourth memory gland. Using curved forceps firmly grasp the ventral portion of the fad pad and pull it away from the body wall. In one piece, it is advisable to press the excise tissue between two labeled slides and fix in car noise fixative.
Later the tissue can be stained, mounted and assessed for cleared margins. Using forceps, remove all traces of the memory gland around the region of the removed portion of the fat pad. The animal is now ready for tissue or cell transplantation.
Fresh or flash frozen donor epithelial tissue can be transplanted into the cleared fat pad. Alternatively, immortalized memory, epithelial cell lines, memory tumor, cell lines, or primary cells can be transplanted. For details on cell preparation, please see the accompanying written protocol for transplantation of the epithelium fragments.Prepared.
This video begin by defrosting a cryo vial of epithelium and transferring the fragments to a Petri dish containing fresh media. To dilute out the DMSO using scissors, make a pocket in the cleared fat pad epithelium of the recipient mouse. Then using needle forceps, place a one millimeter piece of donor epithelium into the pocket.
If necessary, hold the pocket closed with tissue forceps and withdraw the needle forceps so the tissue remains in place. Grasp opposing margins of skin. Hold them together with forceps.
Lift them away from the ceros membranes of the ventral body cavity and close with wound clips. Use two clips to close the midsagittal incision and one clip for each oblique incision. Avoid obstructing the urethra if needed.
Close gaps in the skin at the intersection of the midsagittal and oblique incisions with a suture. Place the mouse in her cage and keep the cage under heating lamp until the mouse has revived. The wound clips should be removed after two weeks.
The following slides show representative images of what one can expect to find in the host memory gland after clearing the fed pad and transplanting epithelium, this image shows the endogenous memory epithelium removed from a three week old mouse. In the gland clearing procedure, note the clear margins at the cut edge. A whole mount like this one should be prepared following each procedure to assess for cleared margins.
This image shows the cleared fat pad of the mouse seven weeks after the clearing procedure. Note that the entire fat pad is devoid of memory, epithelium, hematin, and asin. Staining of a cleared fat pad seven weeks after the clearing procedure also demonstrates that the gland is devoid of memory epithelium.
A representative whole mount of a cleared fat pad transplanted with donor memory epithelium from a 10 week old mouse is shown. Note that the transplanted epithelium produced normal epithelial ductal trees, hematin and asin. Staining of a cleared fat pad and memory.
Epithelial transplant at the same age reveals the normal production of epithelial ducts from the exogenous epithelium. Here, hematin and asin, staining of a cleared fat pad shows an epithelial transplant that developed into a memory tumor. We have just shown you how to harvest mammary epithelium and do a mammary epithelial transplant.
When performing this procedure, it's important to remember to sever the connections between the fourth and fifth gland to cauterize the node and the nipple, and to remove all traces of endogenous epithelium. You want to ensure that there is no possibility that endogenous epithelium can grow into the cleared fat pad. In addition, it is advisable to make a whole amount of the removed portion of the mammary gland to check for cleared margins.
So that's it. Thanks for watching and good luck with your experiments.
