Pubertal Ductal Morphogenesis: Isolation and Transcriptome Analysis of the Terminal End Bud.. Mus musculus

The terminal end buds (TEB) are the growing part of the ductal mammary epithelium during puberty, which enable the formation of the primary mammary epithelial network. These bulbous end structures are highly proliferative, and this allows the ductal expansion into the mammary fat pad. The TEB comprise of an outer cap cell layer, which include the progenitor cells of the myoepithelium, and the body cells, which are thought to be the progenitors from which the luminal epithelium is formed. Since TEBs make up only a minute part of the whole mammary tissue, associated factors can be easily missed in whole-tissue section analysis. We have therefore optimised a method to enzymatically separate TEB and ducts, respectively, from the surrounding stroma of pubertal mice and performed transcriptome analysis on the isolated structures to identify potential novel regulators of epithelial outgrowth. Overall design: TEBs and duct fragments were isolated through collagenase-II digestion and microdissection from pubertal female C57/BL6 mice (for detailed protocol see Stein and Morris, Methods Mol Biol. 2017;1501:131-148.). Samples were analysed in technical replicates from pooled samples, representing ~300-400 isolated TEBs or ducts each.