Fat Pad Clearing and Transplantation

Mammary Gland Clearing

MATERIALS

1. Dissecting microscope
2. Electric cautery, cautery pencil and eye cautery tip or disposable cautery pencil
3. Cork boards, 3 x 4 x 1/4 inch
4. Straight pins, 1 inch
5. Operating scissors, 4 1/2 to 5 inches, S/S
6. Dressing forceps, 4 1/2 inch (skin forceps, tissue forceps
7. Forceps, angle, 4 to 4 1/2 inch
8. Forceps, jewelers, 45^o angle, 4 1/2 inch
9. Iris scissors, angle, 4 1/2 inch
10. Autoclips, Clay-Adams, 9 mm (wound clips)
11. Autoclip applicator
12. Autoclip remover
13. Cotton balls
14. Cotton swabs
15. 70% EtOH
16. Hamilton syringe, 100 microliter
17. Nembutal, 6 mg/ml
18. 1 ml syringe
19. Syringe needles, 25 and 30 gauge
20. 3 week old female mice
21. Appropriate donor(s) and/or cells
22. Microscope lamps

METHOD

1. Swab operating table surface with 70% EtOH
2. Inject 3 week old female mice IP with Nembutal, 1microliter per gram body weight; mice will be anesthetized in 30 to 60 seconds and will remain so for 45 to 60 minutes.
3. Pin mice to corkboard through webbing of paws, ventral side up.
4. Liberally wipe inguinal area of mice with 70% EtOH.
5. Locate #4 and #5 nipples.
6. Make a 1 to 1.5 cm midline incision beginning at a point between the #4 nipples; hold scissors so that blades are at a vertical and not at a horizontal plane or at an angle; cut towards the sternum, using care not to injure the abdominal musculature.
7. Make angled lateral incisions from the midline point between the #4 nipples and ending at a point between the #4 and #5 nipples so that the incision looks like an inverted "Y" .
8. Use fingers or cotton swab and dressing forceps to loosen skin from body wall; pin one skin flap back to expose #4 and #5 fat pads (Fig. A).
9. Cauterize #4 nipple (Fig. B-F), blood vessel near junction by lymph node, and blood vessel at a point on the fat pad bridge between the #4 and #5 fat pads (Fig. B-D).
10. 
11. Use iris scissors to carefully excise and discard the triangular area defined by the cautery points (Fig. B). Developing mammary gland (Fig. B-E) has not grown past the lymph node (Fig. B-C) into the #4 mammary pads in 3 to 4 week old female mice. Removal of the above triangular area will result in a "cleared" #4 mammary fat pad into which tissues can be transplanted without interference from the host's mammary gland.
12. Make sure incisions on the pad are precise and clean.
13. Repeat gland clearing operation on contralateral side.

Mammary Gland Transplantation

1. Obtain donor animal and/or cells for implantation; size of tissue for transplantation should be 1-2 mm³; implantation of cells should be anywhere between 10³ to 10⁷ cells per 10 microliter, depending on cells and/or cell line.
2. Use 45^o angle jeweler's forceps for implanting tissue; use 10 microliter Hamilton syringe for cells.
3. Prepare a pocket in the middle of the fat pad for tissue implantation by holding points of jeweler's forceps together and carefully inserting the points into the fat pad such that they do not rupture the surrounding connective tissue layer on the underside; tension on the fat pad can be produced by holding fat pad near lymph node with another forceps, making it easier to insert jeweler's forceps.
4. Remove forceps points from fat pad, and insert tissue to be transplanted into the prepared pocket; remove forceps by releasing points; this allows the transplant tissue to slip into the pocket more snugly.
5. Cells are implanted in 10 microliter volume using 100 microliter Hamilton syringe and 30 gauge needle.
6. Repeat transplantation on contralateral side.
7. Suture skin flaps using wound clips; generally only 4 wound clips are necessary (Fig. 4); care should be taken that the abdominal wall is not attached to skin; and cut edges of skin should be aligned as closely to each other as possible.
8. Ear tag mice and place in clean cage.
9. If mice are too deeply anesthetized, e.g., shallow breathing, place in clean cage and keep mice warm with a heat lamp. Be sure that heat lamp is not too close. Make sure that mice are shaded with a paper towel.
10. Give feed and water after mice awake.

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Click here for reference illustrating the use of the transplant technique.

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Lawrence JT Young
Department of Medical Pathology
University of California, Davis, CA 95616
Phone: 916-734-3858
Fax: 916-752-4548
Pager: 916-762-6635
EMail: ljyoung@ucdavis.edu

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LAST UPDATED OCTOBER 25, 1996