The overall goal of this procedure is to alter the expression of a desired protein in the epithelium of the mouse, embryonic submandibular salivary gland using adenoviral constructs. This is accomplished by harvesting embryos at Embryonic day 13 and dissecting out the submandibular salivary glands. Next, the epithelial rudiments are separated from the surrounding mesenchyme and soaked in virus containing culture.
Media after washing the rudiment is incubated in matrigel containing media, and we combined with three of the mesenchymes for ex vivo culturing changes in the targeted proteins levels and its effect on branching morphogenesis may be analyzed using immunochemistry and standard microscopy or western blood analysis. This technique allows us to genetically modify only one tissue compartment in a complex organ and monitor changes in a reassembled whole organ explan without making a transgenic organism. The main advantage of this technique over existing methods is that it makes possible the expression of a wild type or dominant negative protein within the epithelial compartment.
Following standard procedures first harvest a 13 embryos into 10 centimeter dishes with 25 milliliters of media. Next, using forceps, remove the embryos from their sacks and transfer the embryos to a 10 centimeter dish containing 25 milliliters of D-M-E-M-F 12 with pen strep under a stereoscope with a transmitted light base, position the embryo on top of a clean piece of glass. Now separate the embryo's heads with an angled cut just below the lower jaw or mandible and isolate the lower mandibles using a cut below the upper jaw.
Then transfer the lower mandibles into three milliliters of D-M-E-M-F 12 with pen strep in a 35 millimeter tissue culture dish. Now return one mandible to the glass plate on the stage and position it so that the tongue is touching the glass and pointing towards the 12 o'clock position. Using one side of two crossed forceps in a scissoring motion, remove and discard the lower third of the mandible.
Next, turn the mandible 90 degrees towards your dominant hand, and with a scissoring motion cut through the top portion of the mandible. Now peel the excess tissue away and expose the SMGs beneath the tissue. There will be one on each side near the base of the tongue on each side.
Use one pair of forceps to hold down the tissue next to the tongue and the other pair to tease the SMG away from the tongue. At this point, the larger SMG and adjacent sublingual gland can be cultured intact with the surrounding mesenchyme. Otherwise, proceed to the next section.
Begin by placing at least five salivary gland plants into a glass bottomed 50 millimeter microwell dish with 200 microliters of HBSS plus diss paste. Incubate the glands for 15 minutes at 37 degrees Celsius. Then using a 200 microliter pipette tip carefully aspr out the diss paste solution without disturbing the glands immediately.
Add 200 microliters of D-M-E-M-F 12 media with BSA To neutralize the dispa, use a pair of number five forceps with fine tips to gently separate the loosened mesenchyme tissue with the sublingual gland from the epithelial buds and ducts of the larger submandibular gland. Be careful to leave the epithelium as intact as possible. Next, select a high quality 200 microliter pipette tip with a smooth edge pre at the tip in D-M-E-M-F 12 BSA media.
Then gently pipette out the mechy free epithelial rudiments into a new 50 millimeter diameter microwell dish containing 200 microliters of D-M-E-M-F 12 plus pen strep. Finally, in the dish containing the mesenchyme pieces, discard any non mesenchymal tissue. This includes the sublingual glands and any detached submandibular gland buds.
Set aside the remaining mesenchyme. First, prepare 200 microliters of infection media at the optimal titer concentration from a caesium chloride gradient purified viral preparation. Now under the dissecting microscope, remove the media from the dish containing the five epithelial rudiments.
Then quickly add 200 microliters of the infection media, keeping the rudiments wet at all times using forceps. Separate the epithelial rudiments far from each other to prevent them from sticking together and to allow them to settle to the bottom of the dish. Then allow the rudiments to incubate in the viral media for one hour at room temperature with minimal disturbance.
Check the rudiments every 15 minutes to ensure they have not moved close together. If necessary, separate them with forceps. After the incubation under the dissecting microscope, gently remove the viral media and discarded appropriately taking care not to disrupt the rudiments.
Next, wash the rudiments with 200 microliters of fresh media twice. These washes are critical to remove any virus that is weak attached to the epithelium and to prevent infection of the mesenchyme. Following the second wash with media at 200 microliters of media, continuing 2%matrigel, again, taking care not to allow the rudiments to clump together.
Then allow the rudiments to incubate for 20 minutes at room temperature using a Prewitt 200 microliter pipette tip, place five rudiments on top of a notched membrane filter, which is floating on 200 microliters of culture media in a glass bottomed microwell plate. Using a pipette tip or forceps, remove excess media from the top of the filter so that the filter does not sink and the epithelial rudiments stay in place. Next place all the meine pieces derived from three to four glands around each epithelial rudiment, ensuring that they are in direct contact act Recombination of the mesenchyme from three to four glands with the single virally infected epithelial rudiment is critical to the growth and development of the recombined submandibular salivary gland.
Then remove excess media surrounding the glands so that the mesenchyme does not move away from the rudiments. Now incubate the recombined glands in a humidified incubator for two to three full days using the outlined procedure. Intact embryonic SMGs were isolated, and after DYS pace treatment, the epithelial rudiment was removed from the surrounding mesenchyme.
Brightfield imaging revealed that the recombined SMGs continued to undergo branching morphogenesis. When cultured ex vivo. For the indicated times, we combined glands that were grown for 48 hours after treatment with A GFP.
Adenovirus expressed GFP only in the epithelium. We combined SMGs were fixed, stained and imaged. After 72 hours, several markers were used.
The nuclei are stained by dpi, the epithelial marker eco herein in red demarcates the epithelial cell population from the surrounding mesenchyme. The epithelial cells are expressing adenoviral GFP detection of the basement membrane protein peran in cyan localized at the periphery of the epithelium demonstrates that there is at least a partial reconstitution of the basement membrane in recombined glands. Confocal projections for DPI in blue and beta three tubulin in red show that the parasympathetic ganglia within the mesenchyme undergo URI outgrowth and innervate the glands in recombination cultures.
After watching this video, you'll have a good understanding of embryonic submandibular salivary gland culture techniques. Removing the mesenchyme from the epithelium as well as the recombination of the mesenchyme with the virally infected epithelium can both prove to be difficult steps. However, this procedure allows the alteration of protein expression using a viral construct that is applied to the epithelial cells.
Only adenoviral infection allows us to add constitutively active or dominant negative proteins to the epithelium to determine how manipulation of a specific protein will alter the growth and development of the embryonic submandibular salivary gland.
