Sun Heng, Zhang Xin, Chan Un In, Su Sek Man, Guo Sen, Xu Xiaoling, Deng Chuxia
Faculty of Health Sciences, University of Macau, Macau SAR, China.
Bio Protoc. 2020 Jul 5;10(13):e3667. doi: 10.21769/BioProtoc.3667.
For years, the mammary gland serves as a perfect example to study the self-renew and differentiation of adult stem cells, and the regulatory mechanisms of these processes as well. To assess the function of given genes and/or other factors on stemness of mammary cells, several assays were developed, such as mammospheres formation assay, detection of stem cell markers by mRNA expression or flow cytometry and so on. However, the capacity of reconstruction of whole mount in the cleared fat pad of recipient female mice is a golden standard to estimate the stemness of the cells. Here we described a step-by-step protocol for mammary gland formation assay, including preparation of "cleared" recipients and mammary cells for implantation, the surgery process and how to assess the experimental results. Combined with manipulation of mammary cells via gene editing and /or drug treatment, this protocol could be very useful in the researches of mammary stem cells and mammary development.
多年来，乳腺一直是研究成体干细胞自我更新与分化以及这些过程调控机制的完美范例。为评估特定基因和/或其他因素对乳腺细胞干性的作用，人们开发了多种检测方法，如乳腺球形成检测、通过mRNA表达或流式细胞术检测干细胞标志物等。然而，在受体雌性小鼠清除脂肪垫中进行全乳重建的能力是评估细胞干性的金标准。在此，我们描述了一种乳腺形成检测的分步方案，包括制备用于植入的“清除”受体和乳腺细胞、手术过程以及如何评估实验结果。结合通过基因编辑和/或药物处理对乳腺细胞进行的操作，该方案在乳腺干细胞和乳腺发育研究中可能非常有用。
