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Biology

Genetic Modification and Recombination of Salivary Gland Organ Cultures

Published: January 28th, 2013

DOI:

10.3791/50060

1Department of Biological Sciences, University at Albany, SUNY
* These authors contributed equally

A technique to genetically manipulate epithelial cells within whole ex vivo cultured embryonic mouse submandibular glands (SMGs) using viral gene transfer is described. This method takes advantage of the innate ability of SMG epithelium and mesenchyme to spontaneously recombine after separation and infection of epithelial rudiments with adenoviral vectors.

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 cavity1-3. 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 vivo5. 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-1116,7. 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 glands8. 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 GFP-containing 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.

The 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 rudiments. All standard BSL-2 safety precautions should be followed when working with adenoviruses.

1. Mouse Embryonic Submandibular Gland (SMG) Harvesting and Microdissection

  1. Euthanize timed-pregnant f.......

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The flow of the major experimental steps is outlined in Figure 1. An example of an intact SMG, an isolated epithelial rudiment, and its corresponding mesenchyme are shown in Figure 2. Brightfield images of recombined SMGs, which continue to undergo branching morphogenesis when cultured ex vivo for the indicated times, are shown in Figure 3. Recombined glands grown for 48 hr expressing epithelial GFP are shown in Figure 4. Confocal images of reco.......

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The ex vivo epithelial-mesenchymal recombination technique was first published for submandibular salivary glands in 198116. In this protocol, we expand upon the original method, using adenoviral infection to manipulate epithelial cell gene expression within the context of a recombined gland. We show that a percentage of the epithelial cells are infected with the adenovirus, whereas the percentage of cells that are infected depends upon the properties of the viral promoter, viral titer, and vira.......

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The authors would like to thank Dr. Deirdre Nelson for helpful comments and for critical reading of the manuscript. This work was funded by NIH grants DE019244, DE019197, and DE021841 to M.L., F32DE02098001 to S.J.S, and C06 RR015464 to the University at Albany, SUNY.

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Name Company Catalog Number Comments
Name of the Reagent Company Catalog Number Comments
DMEM/Ham's F12 Medium without phenol red Life Technologies 21041-025  
Penicillin and Streptomycin Life Technologies 15070-163 10X stock
Dispase Life Technologies 17105-041 Freeze single use aliquots at -20C
BSA Sigma A2934-100G Fraction V, low endotoxin
Adeno-X-GFP BD Biosciences 8138-1 Should be high titer (1x1010 pfu/ml). CsCl purified viruses are more effective than column-purified viruses in this assay.
16% Paraformaldehyde Electron Microscopy Sciences 15710 Diluted to 2% in PBS with 5% sucrose (w/v)
1X Phosphate-buffered saline (PBS) Life Technologies 70011-044 Prepared from 10X stock
Hank's Balanced Salt Solution Life Technologies 14175095 no Calcium, no Magnesium, no Phenol Red
Transferrin Sigma T8158 25 mg/ml stock solution in DMEM/F12 media. Freeze single-use aliquots at -20C
L- Ascorbic acid (Vitamin C) Sigma A4403 75 mg/ml stock solution in DMEM/F12 media.Freeze single-use aliquots at -20C
      Table 1. List of reagents required for SMG recombination protocol.
       
10 cm sterile plastic dishes Corning 430167 Non-tissue culture-treated plates can also be used.
Stereo dissecting microscope with transmitted light base Nikon SMZ645 Any stereo dissecting microscope can be used that has a transmitted light base.
35 mm tissue culture dishes Falcon 353001 Non-tissue culture-treated plates can also be used.
50 mm diameter microwell dishes MatTek Corporation P50-G-1.5-14F  
Nuclepore Track-Etch membrane filters Whatman 110405 13 mm diameter, 0.1 mm pore size
Widefield fluorescence microscope Carl Zeiss, USA Axio Observer Z1 Any fluorescence microscope (upright, inverted or stereo dissecting microscope) can be used to monitor GFP expression at low magnification with an attached digital camera.
Confocal microscope Leica Microsystems TCS SP5 Confocal microscopy is necessary to see detailed cell structures. Any confocal microscope can be used.
Timed-pregnant female mice, strain CD-1 or ICR Charles River Labs   Embryos are harvested on day 13 (with day of plug discovery designated as day 0).
Scalpel blade #11 Fine Science Tools 10011-00  
Scalpel handle #3 Fine Science Tools 10003-12  
Dumont #5 forceps inox alloy, 0.05mm X 0.02mm Fine Science Tools 11252-20 Ideal for harvesting glands from embryos
Dumont #5 forceps dumostar alloy, 0.05mm X 0.01mm Fine Science Tools 11295-20 Fine tips are required for removing mesenchyme from epithelium. Tungsten needles can also be used.
      Table 2. Equipment used in SMG recombination protocol.

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