Cas9 Mediated in Vitro Gene Editing in Primary Mammary Epithelial Cells

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One of the biggest problems faced in breast cancer research is the disease heterogeneity.The next steps for the development of subtype specific treatmentin breast cancer involve understanding the developmental hierarchy of normal mammary epithelial cells. It has recently become possible to isolate andculture primary mammary epithelial cells without them loosing their in vivo differentiation potential. This allows for manipulation through the use of CRISPRCas9 genome editing. During the project protocols will be developed for both targeted deletion to study gene function and to also quick gene targeting tostudy candidate cancer or cell fate regulators in primary mammary epithelial cells. The project will focus on developing a novel reporter construct which willallow for both positive and negative selection of cell populations in vivo and in vitro. This will therefore allow for manipulation of cells using CRISPR anddelivery of targeted cells back to the cleared mammary fat pad of the mouse. After which, possible effects of the induced mutations may be assessed inorder to characterise their roles within the disease. In this way the project will contribute to the understanding of stem and progenitor cell fate in normalmammary gland development and in breast cancer.