This article demonstrates the procedure developed by DeOme KB et al. (1959) and the sparing procedure developed by Brill B et al. (2008) for clearing the 4th inguinal mammary fat pad of a pubescent mouse in preparation for transplantation of mammary fragments, mammary epithelial cells, or mammary tumor cells.
This article describes and compares the fat pad clearance procedure developed by DeOme KB et al.1 and the sparing procedure developed by Brill B et al.2, followed by the mammary epithelial transplant procedure. The mammary transplant procedure is widely used by mammary biologists because it takes advantage of the fact that significant development of the mammary epithelium doesn’t occur until after puberty. At 3 weeks of age, growth of the mammary epithelial tree is confined to the vicinity of the nipple and the fat pad is largely devoid of mammary epithelium, but by 7 weeks of age the epithelial ductal tree extends throughout the entire fat pad. Therefore, if this small portion of the fat pad containing epithelium, the region between the nipple and the lymph node, is removed at 3 weeks of age, the endogenous epithelium will never populate the mammary fat pad and the fat pad is described as “cleared”. At this time, mammary epithelium from another source can be transplanted in the cleared fat pad where it has the potential to extend mammary ductal trees through out the fat pad. This procedure has been utilized in many experimental models including the examination of tumor phenotype in transgenic mammary epithelial tissue without the confounding effects of genotype on the entire animal3, in the identification of mammary stem cells by transplanting cells in limited dilution4,5, determining if hyperplastic nodules proceed to mammary tumors6, and to assess the effect of prior hormone exposure on the behavior of the mammary epithelium7,8.
Three week old host mice are anesthetized, cleaned and restrained on a surgical stage. A mid-sagittal incision is made through the skin, but not the peritoneum, extending from the pubis to the sternum. Oblique cuts are made through the skin from the mid-sagittal incision across the pelvis toward each leg. The skin is pulled away from the peritoneum to expose the 4th inguinal mammary gland. The fat pad is cleared by removing the fat pad tissue anterior to the lymph node. Epithelium fragments or epithelial cells are transplanted into the remaining cleared fat pad and the mouse is closed.
1. Preparation of Transplant Epithelium
Fresh or frozen donor epithelial tissue can be transplanted into the cleared fat pad, but prior preparation of frozen donor epithelium is more convenient with a minimal decrease in transplantation efficiency (>85% efficiency). The protocol for the preparation of frozen donor epithelium is as follows:
Before preparing donor transplant epithelium, prepare freeze media for epithelium and aliquot about 1ml into cryovials. This can be stored at 4°C for up to 6 months until needed. Freeze media contains DMEM/F12 media buffered with HEPES and NaHCO3, 10% fetal bovine serum (FBS) and 10% dimethyl sulphoxide (DMSO).
Harvest mammary epithelium from a recently euthanized female mouse.
2. Preparation of Transplant Cells
Our lab has transplanted different cell types, including immortalized mammary epithelial cell lines, mammary tumor cell lines or primary mammary cells into a mouse cleared fat pad. The protocol for the preparation of Comma-D cells for transplantation is as follows:
For the transplantation of primary mammary cells, the primary mammary epithelial cells are isolated from mouse 5th mammary glands through mechanical and enzymatic dissociation. Cells are resuspended in DMEM:F12 media and adjusted to the desired concentration, usually 5 x 106/ml.
3. Standard Procedure for Clearing the Mouse Mammary Fat Pad
4. Alternative “Sparing Procedure for Clearing the Fat Pad
After one has become competent with the standard procedure for clearing the mouse mammary fat pad, they may prefer to attempt the less invasive sparing procedure developed by Brill et al. (2008)2.
5. Tissue/Cell Transplantation
6. Closing/Revival of Mouse
Figure 1. Demonstration of incision pattern.
Figure 2. Location of lymph node, blood vessels, nipple and endogenous epithelium. Cauterize at locations indicated.
Figure 3. Whole mount demonstrates that endogenous epithelium is restricted to the area within the circle and therefore margins are “clear’.
7. Representative Results
Figure 4. Whole mount of removed fat pad from 3 week old mouse showing cleared margins.
Figure 5. Whole mount from a 10 week old mouse showing fat pad cleared of endogenous mammary epithelium.
Figure 6. Hematoxylin and eosin stained section of a cleared fat pad from a 10 week old mouse.
Figure 7. Whole mount from a 10 week old mouse showing normal mammary ductal tree developed from transplanted epithelium.
Figure 8. Hematoxylin and eosin stained section of mammary ducts developed from transplanted epithelium.
Figure 9. Hematoxylin and eosin stained section of a mammary tumor developed from transplanted epithelium.
In this report, we present two alternative methods for clearing the mouse mammary fat pad in preparation for mammary epithelial transplants. The standard procedure has been in use since its development in 1959 by DeOme et al.1 While more invasive, the standard method is the preferred method for training the novice because the structure and orientation of the mammary gland are readily viewed. However, the sparing procedure may be preferred by the more experienced surgeon because of several advantages2. Most importantly, the sparing procedure is less invasive, requires less time and can be performed using a brief period of inhalation anesthesia. This reduces the recovery period for the mouse. The sparing procedure can also be performed on younger mice (<21 days) ensuring successful clearance of epithelium. In addition, there is a reduction of tissue adhesion after surgery which may be advantageous if experiments require repeat surgery to remove primary tumors, yet allow for continued long-term observation of the mouse for metastasis.
The transplant portion of the procedure takes advantage of the fact that full development of the mammary gland does not occur until after puberty. Prior removal of the small portion of the fat pad containing the endogenous epithelium “clears” the remaining fat pad of the young mouse and eliminates the potential for the development of epithelial ductal trees. Experimental epithelium can be transplanted into the cleared fat pad of the host mouse and assessment of the resulting outgrowths can be made without the confounding effects of the endogenous host epithelium. Therefore, this procedure is useful for assessing the capacity of mammary epithelial cells to develop in vivo.