Name: genetic modification and recombination of salivary gland organ cultures
Uploaded: 2013-01-28T12:00:00
Description: Watch this Scientific Journal Video about Genetic Modification and Recombination of Salivary Gland Organ Cultures at JoVE.com

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.

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
<ol>
 <li>Euthanize timed-pregnant f...

<ol>
	<li>Tucker, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Salivary+gland+development.">Salivary gland development.</a> Seminars in cell &amp; developmental biology. 18, 237-244 (2007).</li><li>Sakai, T., Onodera, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+organ+culture.">Embryonic organ culture.</a> Curr. Protoc. Cell Biol. Chapter 19, Unit 19 (2008).</li><li>Patel, V. N., Rebustini, I. T., Hoffman, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Salivary+gland+branching+morphogenesis.">Salivary gland branching morphogenesis.</a> Differentiation. 74, 349-364 (2006).</li><li>Sequeira, S. J., Larsen, M., DeVine, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+matrix+and+growth+factors+in+salivary+gland+development.">Extracellular matrix and growth factors in salivary gland development.</a> Frontiers of oral biology. 14, 48-77 (2010).</li><li>Zheng, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transient+detection+of+E1-containing+adenovirus+in+saliva+after+the+delivery+of+a+first-generation+adenoviral+vector+to+human+parotid+gland.">Transient detection of E1-containing adenovirus in saliva after the delivery of a first-generation adenoviral vector to human parotid gland.</a> J. Gene Med. 12, 3-10 (2010).</li><li>Larsen, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+PI+3-kinase+and+PIP3+in+submandibular+gland+branching+morphogenesis.">Role of PI 3-kinase and PIP3 in submandibular gland branching morphogenesis.</a> Developmental biology. 255, 178-191 (2003).</li><li>Hoffman, M. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+expression+profiles+of+mouse+submandibular+gland+development:+FGFR1+regulates+branching+morphogenesis+in+vitro+through+BMP-+and+FGF-dependent+mechanisms.">Gene expression profiles of mouse submandibular gland development: FGFR1 regulates branching morphogenesis in vitro through BMP- and FGF-dependent mechanisms.</a> Development. 129, 5767-5778 (2002).</li><li>Rebustini, I. T., Hoffman, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ECM+and+FGF-dependent+assay+of+embryonic+SMG+epithelial+morphogenesis:+investigating+growth+factor/matrix+regulation+of+gene+expression+during+submandibular+gland+development.">ECM and FGF-dependent assay of embryonic SMG epithelial morphogenesis: investigating growth factor/matrix regulation of gene expression during submandibular gland development.</a> Methods Mol. Biol. 522, 319-330 (2009).</li><li>Partanen, A. M., Thesleff, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transferrin+and+tooth+morphogenesis:+retention+of+transferrin+by+mouse+embryonic+teeth+in+organ+culture.">Transferrin and tooth morphogenesis: retention of transferrin by mouse embryonic teeth in organ culture.</a> Differentiation. 34, 25-31 (1987).</li><li>Naka, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+branching+morphogenesis+of+fetal+mouse+submandibular+gland+by+sodium+ascorbate+and+epigallocatechin+gallate.">Modulation of branching morphogenesis of fetal mouse submandibular gland by sodium ascorbate and epigallocatechin gallate.</a> In Vivo. 19, 883-888 (2005).</li><li>Bornstein, P., Traub, W., Neurath, H., Hill, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+chemistry+and+biology+of+collagen.">The chemistry and biology of collagen.</a> The Proteins. 4, 412-632 (1979).</li><li>Zhou, L., Higginbotham, E. J., Yue, B. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+ascorbic+acid+on+levels+of+fibronectin,+laminin+and+collagen+type+1+in+bovine+trabecular+meshwork+in+organ+culture.">Effects of ascorbic acid on levels of fibronectin, laminin and collagen type 1 in bovine trabecular meshwork in organ culture.</a> Curr. Eye Res. 17, 211-217 (1998).</li><li>Daley, W. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ROCK1-directed+basement+membrane+positioning+coordinates+epithelial+tissue+polarity.">ROCK1-directed basement membrane positioning coordinates epithelial tissue polarity.</a> Development. 139, 411-422 (2012).</li><li>Daley, W. P., Gulfo, K. M., Sequeira, S. J., Larsen, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+mechanochemical+checkpoint+and+negative+feedback+loop+regulating+branching+morphogenesis.">Identification of a mechanochemical checkpoint and negative feedback loop regulating branching morphogenesis.</a> Developmental biology. 336, 169-182 (2009).</li><li>Sequeira, S. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+regulation+of+focal+adhesion+complex+formation+and+salivary+gland+epithelial+cell+organization+by+nanofibrous+PLGA+scaffolds.">The regulation of focal adhesion complex formation and salivary gland epithelial cell organization by nanofibrous PLGA scaffolds.</a> Biomaterials. 33, 3175-3186 (2012).</li><li>Nogawa, H., Mizuno, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+control+over+elongating+and+branching+morphogenesis+in+salivary+gland+development.">Mesenchymal control over elongating and branching morphogenesis in salivary gland development.</a> Journal of embryology and. 66, 209-221 (1981).</li><li>Sakai, T., Larsen, M., Yamada, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibronectin+requirement+in+branching+morphogenesis.">Fibronectin requirement in branching morphogenesis.</a> Nature. 423, 876-881 (2003).</li><li>Daley, W. P., Kohn, J. M., Larsen, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+focal+adhesion+protein-based+mechanochemical+checkpoint+regulates+cleft+progression+during+branching+morphogenesis.">A focal adhesion protein-based mechanochemical checkpoint regulates cleft progression during branching morphogenesis.</a> Developmental dynamics : an official publication of the American Association of Anatomists. 240, 2069-2083 (2011).</li><li>Knox, S. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parasympathetic+innervation+maintains+epithelial+progenitor+cells+during+salivary+organogenesis.">Parasympathetic innervation maintains epithelial progenitor cells during salivary organogenesis.</a> Science. 329, 1645-1647 (2010).</li><li>Larsen, M., Wei, C., Yamada, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+and+fibronectin+dynamics+during+branching+morphogenesis.">Cell and fibronectin dynamics during branching morphogenesis.</a> J. Cell Sci. 119, 3376-3384 (2006).</li><li>Wei, C., Larsen, M., Hoffman, M. P., Yamada, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-organization+and+branching+morphogenesis+of+primary+salivary+epithelial+cells.">Self-organization and branching morphogenesis of primary salivary epithelial cells.</a> Tissue Eng. 13, 721-735 (2007).</li><li>Zheng, C., Baum, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+viral+and+mammalian+promoters+for+use+in+gene+delivery+to+salivary+glands.">Evaluation of viral and mammalian promoters for use in gene delivery to salivary glands.</a> Mol. Ther. 12, 528-536 (2005).</li><li>Hsu, J. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viral+gene+transfer+to+developing+mouse+salivary+glands.">Viral gene transfer to developing mouse salivary glands.</a> J. Dent. Res. 91, 197-202 (2012).</li></ol>

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...

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

genetic modification and recombination of salivary gland organ cultures

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 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...

Watch this Scientific Journal Video about Genetic Modification and Recombination of Salivary Gland Organ Cultures at JoVE.com

Genetic Modification and Recombination of Salivary Gland Organ Cultures

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 ...

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