Laboratory of Cell and Developmental Biology, National Institute of Dental and Craniofacial Research, National Institutes of Health, Building 30, Room 426, 30 Convent Drive, MSC 4370, Bethesda, MD 20892-4370, USA.
J Dent Res. 2012 Feb;91(2):197-202. doi: 10.1177/0022034511429346. Epub 2011 Nov 17.
Branching morphogenesis is essential for the formation of salivary glands, kidneys, lungs, and many other organs during development, but the mechanisms underlying this process are not adequately understood. Microarray and other gene expression methods have been powerful approaches for identifying candidate genes that potentially regulate branching morphogenesis. However, functional validation of the proposed roles for these genes has been severely hampered by the absence of efficient techniques to genetically manipulate cells within embryonic organs. Using ex vivo cultured embryonic mouse submandibular glands (SMGs) as models to study branching morphogenesis, we have identified new vectors for viral gene transfer with high efficiency and cell-type specificity to developing SMGs. We screened adenovirus, lentivirus, and 11 types of adeno-associated viruses (AAV) for their ability to transduce embryonic day 12 or 13 SMGs. We identified two AAV types, AAV2 and bovine AAV (BAAV), that are selective in targeting expression differentially to SMG epithelial and mesenchymal cell populations, respectively. Transduction of SMG epithelia with self-complementary (sc) AAV2 expressing fibroblast growth factor 7 (Fgf7) supported gland survival and enhanced SMG branching morphogenesis. Our findings represent, to our knowledge, the first successful selective gene targeting to epithelial vs. mesenchymal cells in an organ undergoing branching morphogenesis.
分支形态发生对于唾液腺、肾脏、肺和许多其他器官在发育过程中的形成是必不可少的，但这一过程的机制尚未得到充分理解。微阵列和其他基因表达方法是鉴定潜在调节分支形态发生的候选基因的有力方法。然而，由于缺乏有效技术来遗传操作胚胎器官内的细胞，这些基因的提出作用的功能验证受到了严重阻碍。我们使用体外培养的胚胎鼠下颌下腺（SMG）作为模型来研究分支形态发生，已经鉴定出了用于病毒基因转移的新型载体，这些载体具有高效和细胞类型特异性，可用于发育中的 SMG。我们筛选了腺病毒、慢病毒和 11 种腺相关病毒（AAV），以确定它们将外源性基因递送至胚胎第 12 或 13 天 SMG 的能力。我们鉴定出两种 AAV 类型，AAV2 和牛 AAV（BAAV），它们分别选择性地靶向 SMG 上皮细胞和间充质细胞群体进行表达。用自我互补（sc）AAV2 转导 SMG 上皮细胞，表达成纤维细胞生长因子 7（Fgf7），可支持腺体存活并增强 SMG 分支形态发生。据我们所知，我们的发现代表了在经历分支形态发生的器官中，上皮细胞与间充质细胞的首次成功的选择性基因靶向。
