The overall goal of this procedure is to investigate the tissue specific functions of signaling pathways that are critical for hair follicle formation. This is accomplished by first isolating primary dermal and epidermal cells from newborn mice. Next, the gene expression is altered by infecting primary cells with lentivirus.
The third step is to combine the epidermal and dermal cell populations into chambers secured to a wound bed on a nude mouse. The graft is then monitored for hair growth. Ultimately, the effect of gene manipulation on the hair follicle formation can be assessed visually and by histological staining of the graft site.
The main advantage of this technique over existing methods, like a mouse nacal model, is that graft can be completed in three weeks. This method can answer key questions in the developmental biology field, such as how factors in one tissue can impact signaling and function in another. We first had the idea for this method when we wanted to dissect tissue specific functions of ceiling molecules in the hair follicle formation After washing a mouse that had been euthanized by asphyxiation.
Cut off each limb and tail at the base of the torso with sterile scissors. Then grasp the body firmly between a pair of curved forceps and make an incision along the dorsal skin from the head to the tail using a scalpel. Do not penetrate the underlying fascia.
Now, carefully peel away the skin from the midline of the mouse. Grasp the exposed mouse firmly with the long side of the curved forceps and insert another pair of curved forceps underneath the skin at the posterior end of the mouse. Now carefully peel the skin completely off the mouse in one smooth motion and discard the carcass.
Place the skin in the first dish of wash solution with the dermis side down. Spread the skin out in the solution and agitate it with the forceps. Then leave the skin in the wash solution while dissecting the next skin.
After placing the next dissected skin in the first dish of wash solution, transfer the prior skin to the second dish of wash solution. Keep the skins in the second wash solution until all skins are collected. After final wash, transfer up to five skins to a dish containing cold disbe solution.
Flatten out the skins dermo side down and allow them to float for eight to 16 hours at four degrees Celsius. Transfer one skin from cold dis bays to a new dish. Flatten it dermis side down.
Grasp the epidermis with forceps and slowly peel it away from the dermis. The epidermis should peel away in one piece, it will be white and thin, and the dermis should be brownish, thicker, and gelatinous. Sort the two tissues into separate culture dishes to begin cell isolation.
To isolate dermal cells, mince the dermis into very small pieces. With two scalpels. Incubate the minced dermal tissue with freshly made collagenase solution At 37 degrees Celsius for an hour every 10 minutes, gently agitate the cell mixture when the digestion is complete.
The suspension should be mostly single cells, and the solution should have changed from clear to cloudy. Now, add a 10%volume of FBS to the cell suspension. Pass them through a 70 micron cell, strainer into 50 milliliter tubes.
Collect up to 30 milliliters of flow through per tube. Continue with dermal cell isolation as per the written protocol. To isolate the keratinocytes slice each epidermis into six to eight smaller pieces.
Incubate the sliced tissue with freshly thawed 0.05%tripsin EDTA at 37 degrees Celsius for 15 minutes. Continue with keratinocyte isolation as per the written protocol, anesthetize a seven to 12 week old female nude mouse using ketamine and xylazine or a preferred method. Make sure to apply eye lubricant and place the mouse on a heating pad.
Now disinfect the back skin with iodine, followed by alcohol swabs. Using forceps, pinch the skin at the base of the neck and cut a one centimeter diameter hole in the pinched skin. Place the chamber onto the wound bed and cover the chamber's edges with surrounding skin.
Secure the chamber with about six sutures Along its edges only attach one chamber per mouse. Once the chamber is secured, check the cell slurry. If it is condensed at the bottom, gently resuspend it with a one milliliter pipette tip.
Now gently swirl the cell slurry and draw it into a 23 gauge needle attached to a one milliliter syringe. Puncture each chamber with the needle and slowly drip PBS suspended cells onto the wound. When the mice recover from the anesthesia, place them in clean autoclave cages with two mice per cage.
Make sure to deliver antibiotics and Tylenol via the drinking water. After seven to nine days, anesthetize the treated nude mouse as done before. Disinfect the skin around the chamber with iodine and clean it with alcohol swabs.
Carefully cut off the stitches and remove them from the chamber. Gently remove the chamber if the new skin sticks to it. Gently separate the graft from the chamber.
Using forceps, hold the graft down. During removal of the chamber, the graft should look dull and opaque. Apply a non-adherent pad with a thin film of antibiotics to the open wound and suture it into place Using two stitches.
Wrap a bandage around the mouse to secure the pad and protect the wound. Suture the bandage into place After three days, remove the bandages and pad. The mouse does not need to be anesthetized during this step.
The graft should be dry and opaque to brown in color. Check the graft periodically for hair growth, which should begin to show 16 days after the graft. A dermal specific knockdown of the smoothened gene, which is a critical sonic hedgehog signaling component, was made in isolated dermal cells.
These cells were applied to hair reconstitution grafts using the outlined protocol. After three weeks of growth, positive control grafts with lentiviral vector alone showed robust hair growth. However, the smoothened gene knockdown resulted in a severe reduction in hair growth.
Negative control grafts, omitting either primary dermal cells or keratinocytes, resulted in a recovered wound site without any hair. Likewise, cell death or cell loss by one of the cell types in the graft also resulted in no hair growth. The lentivirus vector was determined to have good infection efficiency prior to the graft experiment, and it was also designed to produce GFP along with this Smoothened, S-H-R-N-A.
So viewing the dermal papilla of hair follicles under fluorescent optics made a useful control in confirming the per dur of lentiviral expression After three weeks, H and d staining is used to depict the growth stage of the hair follicles in the control graft and their delayed stage of development when smoothened expression was knocked down. Once mastered, this technique can be performed in 14 hours over the course of three days if performed properly. This technique can be extended to explore the relationship between tumor and stroma during cancer progression in the field of cancer biology.
After watching this video, you should have a good understanding of how to study gene function in hair follicle formation. Use our rapid reconstitution hair assay as a, an alternative to traditional genetic knockouts.
