Cas9 Mediated in Vitro Gene Editing in Primary Mammary Epithelial Cells

One of the biggest problems faced in breast cancer research is the disease heterogeneity.The next steps for the development of subtype specific treatment
in breast cancer involve understanding the developmental hierarchy of normal mammary epithelial cells. It has recently become possible to isolate and
culture primary mammary epithelial cells without them loosing their in vivo differentiation potential. This allows for manipulation through the use of CRISPR
Cas9 genome editing. During the project protocols will be developed for both targeted deletion to study gene function and to also quick gene targeting to
study candidate cancer or cell fate regulators in primary mammary epithelial cells. The project will focus on developing a novel reporter construct which will
allow for both positive and negative selection of cell populations in vivo and in vitro. This will therefore allow for manipulation of cells using CRISPR and
delivery of targeted cells back to the cleared mammary fat pad of the mouse. After which, possible effects of the induced mutations may be assessed in
order to characterise their roles within the disease. In this way the project will contribute to the understanding of stem and progenitor cell fate in normal
mammary gland development and in breast cancer.