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 female mice (outbred strain CD-1 or ICR) using CO2 narcosis, followed by cervical dislocation and harvest strings of mouse embryos at embryonic day 13 (E13, 3-5 epithelial buds) following procedures approved by the institutional IACUC committee. The day of vaginal plug discovery is designated as E0. Salivary glands can also be harvested and cultured at the E12 stage when there is a single primary bud and stalk with clefts just starting to initiate. SMGs prior to this stage require different culture conditions than those indicated here.</li>
 <li>Transfer embryo strings into sterile 10 cm tissue culture dishes filled with 25 ml of DMEM/Ham's F12 Medium (F12) lacking phenol red (Life Technologies) and supplemented with 100 U/ml penicillin, and 100 &mu;g/ml streptomycin (pen-strep, Life Technologies).</li>
 <li>Using a sterile scalpel (#11 blades) and forceps (#5, Fine Science Tools, 11252-20), remove the embryos from the sacs into a separate 10 cm dish containing 25 ml of DMEM/F12 + pen-strep.</li>
 <li>Sever the embryo heads with an angled cut just below the lower jaw.</li>
 <li>Isolate the lower mandibles from the heads under a stereo dissecting microscope with a transmitted light base (Nikon SMZ645 or equivalent) using a cut below the upper jaw. Place the tissues on top of an extra piece of glass rather than directly on the glass component of the microscope base.</li>
 <li>Collect the lower mandibles into 3 ml of DMEM/F12 + pen-strep in a sterile 35 mm tissue culture dish.</li>
 <li>Place a mandible slice on the dissecting microscope stage so that the tongue is on the bottom of the slice but is pointing towards the 12:00 o'clock position.</li>
 <li>Using one side of two crossed forceps in a scissoring motion, remove and discard the lower 1/3rd of the mandible slice.</li>
 <li>Turn the slice 90 &deg; to the right (if right-handed) and, using a scissoring cut with the forceps, cut the top portion of the mandible slice (without cutting the tongue) halfway into the slice.</li>
 <li>Peel the excess tissue away and remove it to expose the SMGs underneath. There will be one on each side near the base of the tongue.</li>
 <li>Using one pair of forceps to hold down the tissue by the tongue, use the other forceps to tease the SMG away from the tongue. Repeat for the other gland.</li>
 <li>Collect the microdissected salivary glands into 3 ml of DMEM/F12 + pen-strep into a new sterile 35 mm tissue culture dish. Note: the larger submandibular glands (3-5 epithelial buds) along with the connected smaller sublingual gland (1 bud) can be cultured intact by skipping to step 4 and performing steps 4.1, 4.3, and 4.4, as described previously 2,8.</li>
</ol>
2. SMG Epithelial Rudiment Separation
<ol>
 <li>Place 5 (or more) salivary glands into a glass-bottomed, 50 mm diameter microwell dish (MatTek Corporation, P50G-1.5-14-F) containing 200 &mu;l of Hank's balanced salt solution (HBSS lacking Ca++or Mg++, Life Technologies) containing 0.4% (v/v) dispase (Life Technologies, cat. no. 17105-041) and incubate for 15 min at 37 &deg;C.</li>
 <li>After the incubation, using a sterile 200 &mu;l pipette tip, carefully aspirate out the dispase solution without disturbing the glands. Immediately add 200 &mu;l of DMEM/F12 media containing 5% (w/v) BSA (DMEM/F12-BSA, pre-sterilized with a 0.22 &mu;m filter) to neutralize the dispase.</li>
 <li>Under a dissecting microscope, using a pair of forceps with fine tips (#5 Dumostar, Fine Science Tools, 11295-20), gently separate the loosened mesenchyme from around the SMG epithelial buds and ducts, taking care to leave the epithelium intact.</li>
 <li>Pre-wet a sterile 200 &mu;l pipette tip with DMEM/F12-BSA media and gently pipette out the epithelial rudiments into a new 50 mm diameter microwell dish containing 200 &mu;l of DMEM/F12 + pen-strep. Use a high quality 200 &mu;l pipette tip with a smooth edge.</li>
 <li>In the dish that contains the mesenchyme pieces, discard any non-mesenchymal tissue including the sublingual glands and any detached submandibular gland buds.</li>
</ol>
3. Adenoviral Infection of Epithelial Rudiments
<ol>
 <li>Make 200 &mu;l of viral infection media by diluting the adenovirus of interest in DMEM/F12 + pen-strep in a microcentrifuge tube so that the titer of the resulting solution is 1x108-109 PFUs. The optimal titer required for different viruses may vary. It is important to use cesium chloride (CsCl)-purified viral preparations for optimal uptake efficiency.</li>
 <li>Remove the media from the dish containing the five epithelial rudiments. Quickly add 200 &mu;l of the infection media, keeping the rudiments wet at all times. Take necessary precautions when handling the virus and disposing of contaminated pipet tips. Disinfect any virus-contaminated surfaces with 10% bleach solution.</li>
 <li>Move the epithelial rudiments far away from each other with forceps to prevent them from sticking together and allow them to settle to the bottom of the plate. Incubate rudiments in viral media for 1 hr at room temperature. During the incubation, separate the rudiments, if they move close together. Excessive rocking or movement will allow the glands to clump together.</li>
 <li>After incubation, gently remove the viral-containing media and discard appropriately, taking care not to disrupt the rudiments. Add 200 &mu;l of DMEM/F12 + pen-strep. Repeat this wash at least two more times and replace with 200 &mu;l of DMEM/F12 + pen-strep media. These washes are critical to remove any virus that is not strongly attached to the epithelium and TO prevent infection of the mesenchyme.</li>
 <li>Incubate epithelial rudiments with 200 ml of DMEM/F12 containing 2% (v/v) Matrigel (BD Biosciences, 356231) for 20 min. We find that this short incubation in Matrigel minimizes the regression of existing clefts during the initial culture period, but this step can be dispensed with if exposure to Matrigel is not desired.</li>
</ol>
4. Ex vivo Culture of Recombined SMGs
<ol>
 <li>Place each rudiment, one at a time, using a pre-wet 200 &mu;l pipette tip, on top of a notched (small cut) 13 mm, 0.1 &mu;m Nuclepore Track-Etch membrane filter (Whatman) floating over 200 &mu;l of culture media (5 rudiments /filter) in a glass-bottomed microwell plate. The culture media is: DMEM/F12 + pen-strep containing 50 &mu;g/ml transferrin and 150 &mu;g/ml ascorbic acid, as previously described2,8. Transferrin is known to stimulate survival and branching morphogenesis of salivary gland and other organ explants9. Addition of ascorbic acid has been shown to stimulate SMG branching.10 It is known to act as a cofactor in many hydroxylation reactions of metabolic pathways (such as proline hydroxylation during collagen fiber formation11) and also stimulates fibronectin and laminin production in other organ explants12.</li>
 <li>Remove as much media on top of the filter as possible after adding each epithelial rudiment. Too much media on top of the filter may cause the filter to sink, while less media makes placement of the rudiments and mesenchyme much easier. Excess media can be removed either with a pipette tip or by using forceps to grip the media between the tips.</li>
 <li>Place mesenchyme pieces derived from three to four glands on top and/or around each epithelial rudiment. Remove any extra media that is surrounding the glands to avoid movement of the mesenchyme away from the rudiments. Mesenchyme derived from more glands is not beneficial but less can be detrimental to epithelial rudiment growth.</li>
 <li>Incubate recombined SMGs in a humidified incubator (95% air/5% CO2) at 37 &deg;C for 48 -72 hr, as desired.</li>
 <li>Acquire brightfield and/or fluorescent images of live glands at 0 hr (if desired), 2 hr after recombination and every 24 hr thereafter on an inverted, upright, or stereo light microscope fitted with a digital camera using a 4X or 5X magnification objective to capture the entire recombined gland in a single frame of view. Images of the salivary gland show the best contrast using either the brightfield setting or the appropriate phase ring placed part way into the light path (which is not possible on fully-automated microscopes). Standard phase contrast is not necessary since the gland is thick and creates contrast.</li>
 <li>At the desired time-points, recombined SMGs can be fixed for 20 minutes with fixative solution made of 2% paraformaldehyde (PFA) in 1X PBS containing 5% (w/v) sucrose (to better preserve internal cellular structure by keeping the cells in an isosmotic state). Unlike 4% PFA, 2% PFA will preserve a significant amount of the GFP signal if the fixative is completely removed after fixation. Glands can be stored for up to 1 week in 1X PBS in the dark at 4 &deg;C until whole mount immunocytochemistry and confocal imaging is performed, as reported previously3,6,13-15. Ideally, immunocytochemistry and imaging should be performed soon after fixation to obtain the best quality images. Glands may also be lysed in RIPA buffer for SDS-PAGE followed by Western blotting analysis.</li>
</ol>

<ol>
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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. 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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. 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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>

No conflicts of interest declared.

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 viral purity. We have found that it is necessary to use CsCl gradient-purified viruses for efficient epithelial transduction, the purification of which is not described here. Since we have also been able to infect the mesenchyme cells prior to recombination (data not shown), independent genetic manipulation of the mesenchymal population is also possible. We recently used this adenoviral transfection/tissue recombination method to identify a role for the polarity protein, PAR-1b, in the control of the placement of basement membrane by the epithelium in developing salivary glands. Kinase-dead PAR-1b adenovirus and wild-type PAR-1b adenovirus were both used in this study13 to infect epithelial rudiments and recombined with E13 mesenchymes. The ability to use mutant viruses to interrogate functions of specific molecules greatly enhances the utility of this method.
There is currently a lack of effective tools to target specific molecules within the epithelial cell population in intact embryonic salivary glands without affecting the mesenchymal compartment. Although multiple studies have used pharmacological inhibitors to manipulate signal transduction in intact organ cultures, these inhibitors affect both the epithelium and mesenchyme. To directly assess effects of inhibitors on the epithelium, epithelial rudiments must be cultured in the absence of mesenchyme in either Matrigel or laminin-111 gels6,7. Small inhibitory RNAs (siRNAs) are an effective method to down-regulate epithelial gene expression since siRNAs are preferentially taken up by the epithelial cells in the presence of most lipid carriers13,14,17,18. However, neither of these methods for interfering with protein levels or function allow overexpression of a wild-type or mutant gene. Attempts at traditional (non-viral) transfection of embryonic whole SMG cultures have met with limited success in that only a small number of epithelial cells can be targeted (M.L., unpublished data). In comparison, adenoviral transduction represents an effective method for specifically transfecting salivary epithelial cells.
Parasympathetic nerves have recently been shown to be critical for normal salivary gland branching by maintaining a keratin 5-positive progenitor cell population19. An advantage of this tissue recombination method over the mesenchyme-free epithelial rudiment culture method is that the parasympathetic ganglion can be maintained in the recombination culture system. By keeping careful track of the approximate location of the parasympathetic ganglion, which is located in the mesenchyme adjacent to the primary epithelial duct19, it is possible to ensure that each rudiment ends up being associated with a ganglion. However, we find that by combining 3-4 glands worth of mesenchymes with every epithelial rudiment, each rudiment is associated with a ganglion sufficient to innervate the buds to some extent (Figure 6), although not always to the same as in the intact unmodified glands.
There are alternate methods for infecting the epithelium in the context of the intact gland. We previously reported that microinjection can be used to introduce adenovirus into epithelial cells in intact glands20,21. However, microinjection is difficult to control, may physically damage the glands, and typically leads to infection of only a localized subset of cells20. Adeno-associated viruses (AAV) have been shown to be useful for transfection of distinct cell populations within the context of intact SMG organ explants22. The serotype scAAV2 was shown to be substantially more efficient at specific transduction of the epithelium of intact SMGs over other recombinant AAV vectors23. Since AAVs are not widely commercially available, nor does the expertise to prepare AAV exist in most laboratories, the adenoviral transduction and tissue recombination protocol provided here is more generally accessible to most laboratories than is the use of AAV vectors.
While this tissue recombination method recapitulates epithelial-mesenchymal interactions to some extent, it is also possible to infect the epithelium with adenovirus and then grow the epithelium outside of its native context. As previously mentioned, we infected intact salivary epithelial rudiments with adenovirus and then grew the cultures in Matrigel with exogenously added growth factors17,20,21. Salivary gland organ explants can also be cultured on nanofiber scaffolds, although these scaffolds do not recapitulate the full function of the mesenchyme in their current form15. While neither of these methods recapitulates the native mesenchyme, such methods allow for imitation of specific properties of the mesenchyme in ex vivo culture.
Each ex vivo culture method has its own advantages and disadvantages but is applicable for addressing specific scientific questions. While the recombined glands do not undergo as much branching morphogenesis as do intact salivary gland organ explants, organ explants, in general, only recapitulate in vivo branching morphogenesis for a few days. A solution to this problem is of course to create transgenic animals containing targeted gene expression within the epithelial compartment. Since there is a lack of known promoters that are activated specifically in early developing SMG epithelium, and transgenic technology remains significantly more expensive than the described methods, the tissue recombination experiments described here constitute a viable model system to study both epithelial and mesenchymal cell signaling in early developing salivary gland cells within their tissue context.

<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 recombined SMGs fixed and imaged after 72 hr in culture followed by immunocytochemistry, are shown in Figure 5. The epithelial marker, E-cadherin, demarcates the GFP-expressing epithelial cell population from the surrounding mesenchyme. Detection of the basement membrane protein, perlecan, localized at the periphery of the epithelium demonstrates at least partial reconstitution of the basement membrane in recombined glands. Figure 6. Parasympathetic submandibular ganglia undergo neurite outgrowth and innervate the glands in recombination cultures as demonstrated previously.19
<img alt="Figure 1" src="/files/ftp_upload/50060/50060fig1.jpg" /> 
 Figure 1. (A) Flowchart and (B) Schematic depicting the major experimental steps.
<img alt="Figure 2" fo:content-width="4.5in" fo:src="/files/ftp_upload/50060/50060fig2highres.jpg" src="/files/ftp_upload/50060/50060fig2.jpg" /> 
 Figure 2. Brightfield images of (A) a whole embryonic day 13 (E13) salivary gland showing the sublingual gland epithelium (SL Epi) and the submandibular gland epithelium (SMG Epi) surrounded by mesenchyme (mes). (B) Isolated epithelial rudiment, and (C) Separated Mesenchyme. Scale bars = 100 &mu;m.
<img alt="Figure 3" src="/files/ftp_upload/50060/50060fig3.jpg" /> 
 Figure 3. Brightfield images of epithelial rudiments (outlined by white dashed lines) surrounded by mesenchyme pieces grown in ex vivo culture for the indicated times. Epithelial cells undergo branching morphogenesis and mesenchymal condensation is evident as early as 24 hr in culture. Scale bars = 100 &mu;m
<img alt="Figure 4" src="/files/ftp_upload/50060/50060fig4.jpg" /> 
 Figure 4. Brightfield (A), fluorescent (B) and overlaid (C) images of recombined SMGs in which only the epithelial cells (outlined in white dashed lines) were infected with GFP-expressing adenovirus and cultured ex vivo for 48 hr. Scale bars = 100 &mu;m.
<img alt="Figure 5" fo:content-width="6in" fo:src="/files/ftp_upload/50060/50060fig5highres.jpg" src="/files/ftp_upload/50060/50060fig5.jpg" /> 
 Figure 5. Confocal images or brightfield images (BF) of recombined SMGs labeled with (A) nuclei (DAPI, blue), adenoviral GFP (green), the epithelial marker. E-cadherin (red), scale bars = 250 &mu;m , or, (B) nuclei (DAPI, blue), adenoviral GFP (green), the epithelial marker, E-cadherin (red), and the basement membrane marker, Perlecan (cyan), scale bars = 50 &mu;m. Dashed white lines outline epithelium. <a href="https://www.jove.com/files/ftp_upload/50060/50060fig5large.jpg" target="_blank"> Click here to view larger figure</a>.
<img alt="Figure 6" src="/files/ftp_upload/50060/50060fig6.jpg" /> 
 Figure 6. Parasympathetic submandibular ganglia undergo neurite outgrowth and innervate the glands in recombination cultures, scale bars = 250 &mu;m.

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 ...

genetic-modification-recombination-salivary-gland-organ

Universidad San Sebastian

Research

JoVE Journal

Biology

12.8K Views.  University at Albany, SUNY. 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.

Video: Genetic Modification and Recombination of Salivary Gland Organ Cultures

Dissection of Midgut and Salivary Glands from Ae. aegypti Mosquitoes

The mosquito midgut and salivary glands are key entry and exit points for pathogens such as Plasmodium parasites and Dengue viruses. This video protocol demonstrates dissection techniques for removal of the midgut and salivary glands from Aedes aegypti mosquitoes. 



The mosquito midgut and salivary glands are key entry and exit points for vector pathogens like Plasmodium falciparum and the dengue virus. This video demonstrates the dissection techniques for removing the midgut and salivary glands from Aedes aegypti mosquitoes.

The mosquito midgut and salivary glands are key entry and exit points for pathogens such as Plasmodium parasites and Dengue viruses. This video protocol ...

PDMS Device Fabrication and Surface Modification

Method for Culture of Early Chick Embryos ex vivo (New Culture)

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying  the formation and patterning of brain, neural tube, somite and heart primordia. Applications of chick embryo culture include electroporation of DNA or RNA constructs in order to analyze gene function, grafts of growth factor coated beads such as FGFs and BMPs , as well as whole mount in situ hybridization and immunohistochemistry. This video demonstrates the different steps in chick embryo culture; First, the embryo is explanted in saline. Then, the embryo is centered on a glass ring. The membranes surrounding the embryo are lifted along the walls of the ring. The ring is then placed in a culture dish containing a pool of albumine. The culture dish is sealed and placed in a humid chamber, where the embryo is cultured for up to 24 hrs. Finally, the embryo is removed from the ring, fixed and processed for further applications. A troubleshooting guide is also presented.

Method for Culture of Early Chick Embryos ex vivo New Culture

This video demonstrates New culture, a method by which chick embryos are cultured outside the egg for up to 24 hr. This method enables one to study early development (primitive streak to 14 som.), a period corresponding to E7-9 in mouse. Applications of this technique include electroporation, in situ hybridization and immunohistochemistry.

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

Double Whole Mount in situ Hybridization of Early Chick Embryos

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying  gene expression in brain, neural tube, somite and heart primordia formation. This video demonstrates the different steps in 2-color whole mount in situ hybridization; First, the embryo is dissected from the egg and fixed in paraformaldehyde. Second, the embryo is processed for prehybridization. The embryo is then hybridized with two different probes, one coupled to DIG, and one coupled to FITC.  Following overnight hybridization, the embryo is incubated with DIG coupled antibody. Color reaction for DIG substrate is performed, and the region of interest appears blue. The embryo is then incubated with FITC coupled antibody. The embryo is processed for color reaction with FITC, and the region of interest appears red. Finally, the embryo is fixed and processed for phtograph and sectioning. A troubleshooting guide is also presented.

This video demonstrates 2-color whole mount in situ hybridization, a method by which the spatial and temporal expression pattern of 2 different genes can be visualized in young chick embryos. This method was originally introduced by David Wilkinson, Domingos Henrique, Phil Ingham and David Ish -Horowicz.

Preparation of 2-dGuo-Treated Thymus Organ Cultures

In the thymus, interactions between developing T-cell precursors and stromal cells that include cortical and medullary epithelial cells are known to play a key role in the development of a functionally competent T-cell pool. However, the complexity of T-cell development in the thymus in vivo can limit analysis of individual cellular components and particular stages of development. In vitro culture systems provide a readily accessible means to study multiple complex cellular processes. Thymus organ culture systems represent a widely used approach to study intrathymic development of T-cells under defined conditions in vitro. Here we describe a system in which mouse embryonic thymus lobes can be depleted of endogenous haemopoeitic elements by prior organ culture in 2-deoxyguanosine, a compound that is selectively toxic to haemopoeitic cells. As well as providing a readily accessible source of thymic stromal cells to investigate the role of thymic microenvironments in the development and selection of T-cells, this technique also underpins further experimental approaches that include the reconstitution of alymphoid thymus lobes in vitro with defined haemopoietic elements, the transplantation of alymphoid thymuses into recipient mice, and the formation of reaggregate thymus organ cultures. (This article is based on work first reported Methods in Molecular Biology 2007, Vol. 380 pages 185-196).

This video demonstrates the dissection and removal of the fetal thymus as well the preparation of ex vivo cultures of 2-dGuo-treated thymus.

In the thymus, interactions between developing T-cell precursors and stromal cells that include cortical and medullary epithelial cells are known to play ...

Isolation of Mouse Salivary Gland Stem Cells

Mature salivary glands of both human and mouse origin comprise a minimum of five cell types, each of which facilitates the production and excretion of saliva into the oral cavity. Serous and mucous acinar cells are the protein and mucous producing factories of the gland respectively, and represent the origin of saliva production. Once synthesised, the various enzymatic and other proteinaceous components of saliva are secreted through a series of ductal cells bearing epithelial-type morphology, until the eventual expulsion of the saliva through one major duct into the cavity of the mouth. The composition of saliva is also modified by the ductal cells during this process.
In the manifestation of diseases such as Sj&ouml;gren's syndrome, and in some clinical situations such as radiotherapy treatment for head and neck cancers, saliva production by the glands is dramatically reduced 1,2. The resulting xerostomia, a subjective feeling of dry mouth, affects not only the ability of the patient to swallow and speak, but also encourages the development of dental caries and can be socially debilitating for the sufferer.
The restoration of saliva production in the above-mentioned clinical conditions therefore represents an unmet clinical need, and as such several studies have demonstrated the regenerative capacity of the salivary glands 3-5. Further to the isolation of stem cell-like populations of cells from various tissues within the mouse and human bodies 6-8, we have shown using the described method that stem cells isolated from mouse salivary glands can be used to rescue saliva production in irradiated salivary glands 9,10. This discovery paves the way for the development of stem cell-based therapies for the treatment of xerostomic conditions in humans, and also for the exploration of the salivary gland as a microenvironment containing cells with multipotent self-renewing capabilities.

An optimized protocol for the isolation of stem cells from the mouse salivary gland is described. The method employs enzymatic and mechanical digestion, and permits isolation of salispheres containing cells with characteristics of stem cells.

Mature salivary glands of both human and mouse origin comprise a minimum of five cell types, each of which facilitates the production and excretion of ...

DNA Methylation: Bisulphite Modification and Analysis

Epigenetics describes the heritable changes in gene function that occur independently to the DNA sequence. The molecular basis of epigenetic gene regulation is complex, but essentially involves modifications to the DNA itself or the proteins with which DNA associates. The predominant epigenetic modification of DNA in mammalian genomes is methylation of cytosine nucleotides (5-MeC). DNA methylation provides instruction to gene expression machinery as to where and when the gene should be expressed. The primary target sequence for DNA methylation in mammals is 5'-CpG-3' dinucleotides (Figure 1). CpG dinucleotides are not uniformly distributed throughout the genome, but are concentrated in regions of repetitive genomic sequences and CpG "islands" commonly associated with gene promoters (Figure 1). DNA methylation patterns are established early in development, modulated during tissue specific differentiation and disrupted in many disease states including cancer. To understand the biological role of DNA methylation and its role in human disease, precise, efficient and reproducible methods are required to detect and quantify individual 5-MeCs.
This protocol for bisulphite conversion is the "gold standard" for DNA methylation analysis and facilitates identification and quantification of DNA methylation at single nucleotide resolution. The chemistry of cytosine deamination by sodium bisulphite involves three steps (Figure 2). (1) Sulphonation: The addition of bisulphite to the 5-6 double bond of cytosine (2) Hydrolic Deamination: hydrolytic deamination of the resulting cytosine-bisulphite derivative to give a uracil-bisulphite derivative (3) Alkali Desulphonation: Removal of the sulphonate group by an alkali treatment, to give uracil. Bisulphite preferentially deaminates cytosine to uracil in single stranded DNA, whereas 5-MeC, is refractory to bisulphite-mediated deamination. Upon PCR amplification, uracil is amplified as thymine while 5-MeC residues remain as cytosines, allowing methylated CpGs to be distinguished from unmethylated CpGs by presence of a cytosine "C" versus thymine "T" residue during sequencing.
DNA modification by bisulphite conversion is a well-established protocol that can be exploited for many methods of DNA methylation analysis. Since the detection of 5-MeC by bisulphite conversion was first demonstrated by Frommer et al.1 and Clark et al.2, methods based around bisulphite conversion of genomic DNA account for the majority of new data on DNA methylation. Different methods of post PCR analysis may be utilized, depending on the degree of specificity and resolution of methylation required. Cloning and sequencing is still the most readily available method that can give single nucleotide resolution for methylation across the DNA molecule.

The gold standard for DNA methylation analysis is genomic sequencing of bisulphite converted DNA. This method takes advantage of the increased sensitivity of cytosine compared with 5-methylcytosine (5-MeC) to bisulphite deamination under acidic conditions. Unmethylated cytosines can be distinguished from methylated cytosines after PCR amplification of the target genomic DNA.

Epigenetics describes the heritable changes in gene function that occur independently to the DNA sequence. The molecular basis of epigenetic gene ...

Alternative Cultures for Human Pluripotent Stem Cell Production, Maintenance, and Genetic Analysis

Human pluripotent stem cells (hPSCs) hold great promise for regenerative medicine and biopharmaceutical applications. Currently, optimal culture and efficient expansion of large amounts of clinical-grade hPSCs are critical issues in hPSC-based therapies. Conventionally, hPSCs are propagated as colonies on both feeder and feeder-free culture systems. However, these methods have several major limitations, including low cell yields and generation of heterogeneously differentiated cells. To improve current hPSC culture methods, we have recently developed a new method, which is based on non-colony type monolayer (NCM) culture of dissociated single cells. Here, we present detailed NCM protocols based on the Rho-associated kinase (ROCK) inhibitor Y-27632. We also provide new information regarding NCM culture with different small molecules such as Y-39983 (ROCK I inhibitor), phenylbenzodioxane (ROCK II inhibitor), and thiazovivin (a novel ROCK inhibitor). We further extend our basic protocol to cultivate hPSCs on defined extracellular proteins such as the laminin isoform 521 (LN-521) without the use of ROCK inhibitors. Moreover, based on NCM, we have demonstrated efficient transfection or transduction of plasmid DNAs, lentiviral particles, and oligonucleotide-based microRNAs into hPSCs in order to genetically modify these cells for molecular analyses and drug discovery. The NCM-based methods overcome the major shortcomings of colony-type culture, and thus may be suitable for producing large amounts of homogeneous hPSCs for future clinical therapies, stem cell research, and drug discovery.

Here, we present human pluripotent stem cell (hPSC) culture protocols, based on non-colony type monolayer (NCM) growth of dissociated single cells. This new method, utilizing Rho-associated kinase inhibitors or the laminin isoform 521 (LN-521), is suitable for producing large amounts of homogeneous hPSCs, genetic manipulation, and drug discovery.

Human pluripotent stem cells (hPSCs) hold great promise for regenerative medicine and biopharmaceutical applications. Currently, optimal culture and ...

DNA Electroporation, Isolation and Imaging of Myofibers

Mature muscle has a unique structure that is amenable to live cell imaging. Herein, we describe the experimental protocol for expressing fluorescently labeled proteins in the flexor digitorum brevis (FDB) muscle. Conditions have been optimized to provide a large number of high quality myofibers expressing the electroporated plasmid while minimizing muscle damage. The method employs fluorescent tags on various proteins. Combining this expression method with high resolution confocal microscopy permits live cell imaging, including imaging after laser-induced damage. Fluorescent dyes combined with imaging of fluorescently-tagged proteins provides information regarding the basic structure of muscle and its response to stimuli.

This protocol utilizes electroporation to introduce and express fluorescently labeled proteins in mouse muscle fibers. Following recovery after electroporation, fibers are isolated. Individual fibers are then imaged using high resolution confocal microscopy to visualize muscle structure.

Mature muscle has a unique structure that is amenable to live cell imaging. Herein, we describe the experimental protocol for expressing fluorescently ...

Genetic Modification of Cyanobacteria by Conjugation Using the CyanoGate Modular Cloning Toolkit

Cyanobacteria are a diverse group of prokaryotic photosynthetic organisms that can be genetically modified for the renewable production of useful industrial commodities. Recent advances in synthetic biology have led to development of several cloning toolkits such as CyanoGate, a standardized modular cloning system for building plasmid vectors for subsequent transformation or conjugal transfer into cyanobacteria. Here we outline a detailed method for assembling a self-replicating vector (e.g., carrying a fluorescent marker expression cassette) and conjugal transfer of the vector into the cyanobacterial strains Synechocystis sp. PCC 6803 or Synechococcus elongatus UTEX 2973. In addition, we outline how to characterize the performance of a genetic part (e.g., a promoter) using a plate reader or flow cytometry.

Here, we present a protocol describing how to i) assemble a self-replicating vector using the CyanoGate modular cloning toolkit, ii) introduce the vector into a cyanobacterial host by conjugation, and iii) characterize transgenic cyanobacteria strains using a plate reader or flow cytometry.