br Experimental Procedures br Acknowledgments We thank
Acknowledgments We thank A. Voigt, N. Perrimon, S.X. Hou, X. Zeng, H. Sun, G.H. Beag, and X. Yang for reagents; the Iowa Developmental Studies Hybridoma Bank for antibodies; the Bloomington Stock Center and Drosophila Genetic Resource Center for Drosophila stocks; J.M. Kofron for help with confocal images; and James Wells, Tatyana Belenkaya, and Lorraine Ray for comments on the manuscript. This work was supported by grants from the Strategic Priority Research Program of the Chinese Academy of Sciences (XDA01010101), Nature Sciences foundation of China (31030049), National Basic Research Program of China (2011CB943901 and 2011 CB943902), Mizutani Foundation (120146), NIH (2R01 GM063891 and 1R01GM087517), and Wenzhou Medical University (XNK07005).
Introduction Lineage tracing is a powerful tool for studying tissue development, homeostasis, and disease, and has provided unprecedented insights into stem cell biology (Kretzschmar and Watt, 2012). Previous lineage-tracing studies mostly relied on inducible Cre-estrogen receptor fusion protein (CreER)-expressing transgenic mice upon induction by tamoxifen. This inducible system was recently used for fate-mapping studies of mammary epithelial gnrh agonists (MECs) under the physiological setting (Lafkas et al., 2013; Rios et al., 2014; Šale et al., 2013; van Amerongen et al., 2012; Van Keymeulen et al., 2011). However, wider application of this approach is limited by several factors. First, the choice of specific inducible CreER-expressing lines is often limited, and generating new mouse lines for this purpose can be time consuming. Second, most CreER mice do not target MECs exclusively, and for breast cancer modeling studies, their activities outside of the mammary gland (MG) may lead to systematic deficiency or unwanted tumor induction in other tissues, which could limit their use for studying MECs. Third, administration of tamoxifen may interfere with development of hormone-dependent tumors (e.g., mammary tumors), as well as normal MG development (Rios et al., 2014). Lastly, recent studies showed that the tamoxifen doses commonly used to induce Cre/lox recombination in mice might continue to label significant numbers of cells for weeks after tamoxifen treatment (Reinert et al., 2012) and that tamoxifen could change the behavior of stem cells (Zhu et al., 2013), both of which could affect interpretation of results from lineage-tracing experiments. Adenovirus is a DNA virus, and it does not integrate into the host genome. It can infect both dividing and nondividing cells, leading to transient high-level protein expression (Anderson et al., 2000). Intraductal injection of adenovirus was previously shown to be an efficient way to transduce genes in MECs (Russell et al., 2003). Cre-expressing adenovirus (Ad-Cre) under the control of the constitutive CMV promoter (Ad-CMV-Cre) has been successfully used to induce cancer development in multiple organs (e.g., lung, ovary, and bladder; [DuPage et al., 2009; Flesken-Nikitin et al., 2003; Puzio-Kuter et al., 2009]). Recently, Sutherland et al. (2011) used cell-type-specific promoters and Ad-Cre to initiate small cell lung cancer development from different subsets of lung cells. We hypothesized that, similarly to the inducible CreER system, transient expression of Cre from adenoviral vectors could offer a temporal and spatial genetic-marking system for pulse-chase lineage-tracing studies in adult cells. In this study, we tested this approach in the MG by generating MEC lineage-specific Ad-Cre lines, and demonstrated that they can be used for MEC fate-mapping, gene loss-of-function, and cancer-induction studies in the native environment. This approach should also be suitable for lineage-tracing studies in other systems in which introduction of Ad-Cre is feasible.
Results and Discussion
Acknowledgments We are grateful to Grigoriy Losyev and Yiling Qiu for expert technical assistance with FACS sorting, Dr. Beverly Davidson and the University of Iowa Gene Transfer Vector Core for pacAd5 K-NpA, Dr. Bogi Andersen for K14-luciferase plasmid, and Drs. Morris White and Xiaocheng Dong for Irs1/2 conditional knockout mice. This research was supported by a K99/R00 grant from the NCI (CA126980), a Seed Grant and Cancer Program Pilot Grant from Harvard Stem Cell Institute, a Milton Fund Award from Harvard University, a Hearst Foundation Young Investigator Award from Brigham and Women’s Hospital (BWH), startup funds from BWH, and NIH grant R01 HL107663 to Z.L.