Cas9 Mediated in Vitro Gene Editing in Primary Mammary Epithelial Cells

乳腺癌研究中面临的最大问题之一是疾病的异质性。乳腺癌亚型特异性治疗的下一步是了解正常乳腺上皮细胞的发育等级。最近已经有可能分离和培养原代乳腺上皮细胞，而不会失去它们在体内的分化潜力。这允许通过使用CRISPRCas9基因组编辑进行操作。在该项目期间，将开发用于研究基因功能的靶向缺失和快速基因靶向以研究候选癌症或原代乳腺上皮细胞中的细胞命运调节因子的方案。该项目将专注于开发一种新的报告结构，它将允许对体内和体外的细胞群体进行积极和消极的选择。因此，这将允许使用CRISPR操纵细胞，并将目标细胞送回小鼠清除的乳房脂肪垫。之后，可以评估诱导突变的可能影响，以确定它们在疾病中的作用。通过这种方式，该项目将有助于理解干细胞和祖细胞在正常乳腺发育和乳腺癌中的命运。

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.