Genetic Modification and Recombination of Salivary Gland Organ Cultures

Abstract

Branching morphogenesis occurs during the development of many organs, and the embryonic mouse submandibular gland (SMG) is a classical model for the study of branching morphogenesis. In the developing SMG, this process involves iterative steps of epithelial bud and duct formation, to ultimately give rise to a complex branched network of acini and ducts, which serve to produce and modify/transport the saliva, respectively, into the oral cavity [1][2][3] . The epithelial-associated basement membrane and aspects of the mesenchymal compartment, including the mesenchyme cells, growth factors and the extracellular matrix, produced by these cells, are critical to the branching mechanism, although how the cellular and molecular events are coordinated remains poorly understood 4 . The study of the molecular mechanisms driving epithelial morphogenesis advances our understanding of developmental mechanisms and provides insight into possible regenerative medicine approaches. Such studies have been hampered due to the lack of effective methods for genetic manipulation of the salivary epithelium. Currently, adenoviral transduction represents the most effective method for targeting epithelial cells in adult glands in vivo 5 . However, in embryonic explants, dense mesenchyme and the basement membrane surrounding the epithelial cells impedes viral access to the epithelial cells. If the mesenchyme is removed, the epithelium can be transfected using adenoviruses, and epithelial rudiments can resume branching morphogenesis in the presence of Matrigel or laminin-111 6,7 . Mesenchyme-free epithelial rudiment growth also requires additional supplementation with soluble growth factors and does not fully recapitulate branching morphogenesis as it occurs in intact glands 8 . Here we describe a technique which facilitates adenoviral transduction of epithelial cells and culture of the transfected epithelium with associated mesenchyme. Following microdissection of the embryonic SMGs, removal of the mesenchyme, and viral infection of the epithelium with a GFPcontaining adenovirus, we show that the epithelium spontaneously recombines with uninfected mesenchyme, recapitulating intact SMG glandular structure and branching morphogenesis. The genetically modified epithelial cell population can be easily monitored using standard fluorescence microscopy methods, if fluorescently-tagged adenoviral constructs are used. The tissue recombination method described here is currently the most effective and accessible method for transfection of epithelial cells with a wild-type or mutant vector within a complex 3D tissue construct that does not require generation of transgenic animals. Video LinkThe video component of this article can be found at http://www.jove.com/video/50060/ ProtocolThe protocol contains four major steps, as depicted in Figure 1. All steps are described in full detail. Adenovirus construction and viral purification should be performed in advance of the organ harvesting for use in the genetic transduction of dissected epithelial ...

Three-dimensional organotypic culture: experimental models of mammalian biology and disease

“…Howeve r, these molecular interactions can differ in a 3D tissue context; for example, the molecular composition and phosphorylation status of focal adhesions within fibroblasts are different on 2D surfaces compared with 3D ECM gels 98 . RNAi, Cre– lox -based recombination and lentiviral short hairpin RNA (shRNA) approaches have all recently been adapted to 3D culture, which has enabled the evaluation of single genes and genome-scale screening in tissues, including mammary 27 , intestinal 28 and salivary 99,100 epithelium. The application of CRISPR–Cas9 (clustered, regularly interspaced short palindromic repeats– CRISPR-associated protein 9)-based genome editing 101 further provides the potential for the rapid introduction and functional correction of disease mutations 102 .…”

Integrins Promote Cytokinesis through the RSK Signaling Axis

“…We found it is difficult to quantify failed cytokinesis with any confidence by determining the presence of binucleated cells in the context of the 3D gland. Therefore, after inhibitor treatment, glands were dissociated into individual cells as previously described (Sequeira et al, 2013;Wei et al, 2007). These cells were plated in DMEM/F12 containing 10% FBS onto laminin-rich matrix, gently centrifuged to promote their association with the matrix and incubated for 2 hours at 37˚C to allow them to adhere and spread.…”

Endothelial cell regulation of salivary gland epithelial patterning

“…For mesenchyme-free, epithelial rudiment cultures, epithelial rudiments were physically separated from mesenchyme following a dispase digestion, as previously described (Sequeira et al, 2013), and were cultured ex vivo for 48 h in growth factor-reduced Matrigel (Corning,356231). FGF7 (200 ng/ml; Peprotech, 100-19) and epidermal growth factor (20 ng/ml; Peprotech, AF-100-15) were additionally supplemented into the complete DMEM/F12 medium.…”

“…To prepare unfractionated E12/E13 mesenchyme, dispase digestion of E12/ E13 whole SMGs was performed followed by manual separation of the mesenchyme from the epithelium, as previously described (Sequeira et al, 2013). To prepare E12/E13 primary mesenchyme cells, mesenchyme pieces were placed into a MatTek dish, mixed with 200 μl 0.3× collagenase/ hyaluronidase (7912, STEMCELL Technologies, Vancouver, Canada), and incubated for 5 min at 37°C.…”