Mammary Epithelial Transplant Procedure

Summary

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.

Abstract

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 dilution^4,5, determining if hyperplastic nodules proceed to mammary tumors⁶, and to assess the effect of prior hormone exposure on the behavior of the mammary epithelium^7,8.

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.

Protocol

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 se....

Discussion

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 advantages